Monitoring the battleground: exploring antimicrobial resistance and virulence factors in wound bacterial isolates

  1. Silas Onyango Awuor
  2. Eric O. Omwenga
  3. Richard M. Mariita
  4. Jonathan M. Musila
  5. Stanslaus Musyoki

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Abstract

Antibiotic resistance poses a grave global public health threat, exacerbated by widespread and often inappropriate antibiotic usage. Vigilant surveillance of antibiotic utilization and emergence of antimicrobial resistance (AMR) is essential. Of particular concern in the era of AMR is the persistent issue of chronic wound infections. To address this, we conducted a comprehensive evaluation of wound isolates from chronic wounds at Jaramogi Oginga Odinga Teaching and Referral Hospital (JOOTRH) in Kenya, to identify relevant bacteria and assess their drug resistance patterns.Wound samples were collected and processed using standard microbiological methods. Bacterial isolates were identified and assessed for their susceptibility to a panel of antibiotics using the Kirby-Bauer disk diffusion method. A total of 103 bacterial isolates were obtained from the wound samples, with a higher prevalence in male patients (59%). Staphylococcus aureus (20.7 %) emerged as the most predominant pathogen, followed by Klebsiella spp. (14.8 %), Pseudomonas aeruginosa spp. (14.8 %) and Escherichia coli (4.4 %) in wound samples. High levels of antibiotic resistance were observed among the isolates, with the highest resistance rates reported for cotrimoxazole (48.1 %), clindamycin (25.9 %) and erythromycin (25.9 %). Furthermore, among the isolates, 75 % produced haemolysin and protease, while 50 % produced lipase and phospholipase, factors that enhance virulence and survival. The findings of this study highlight the alarmingly high prevalence of antibiotic resistance among bacterial pathogens isolated from chronic wounds in Kenya. This poses a major challenge to the effective management of chronic wound infections. There is an urgent need to implement effective antimicrobial stewardship programs and develop new antibiotics to combat the growing threat of antibiotic resistance.

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  1. Version published to 10.1099/acmi.0.000613.v6 on Access Microbiology
  3. Version published to 10.1099/acmi.0.000613.v5 on Access Microbiology
  4. Access Microbiology

    Thank you for adding clarification to the biofilm methodology. The additions mean that others would be able to replicate the work. There are still two outstanding issues. 1) The AMR data with respect to the description of strain 13642. Please amend this to remove ambiguity. The sentence should read "out of 10 P. aeruginosa isolates, 4 strains were MDR, with 1 strain resistant to 3 classes of antibiotics (12583), 2 strains resistant to 5 classes of antibiotics (13642 and 14421), and 1 strain resistant to 6 classes of antibiotics (11985)". Table 4 is also redundant- please remove it. 2) The representation of the biofilm data are also problematic. The new combined graphs look great, thank you for resolving this issue. However, the comparison to an unrelated PC strain is not methodologically sound ("The P. aeruginosa isolates showed that the biofilm formation inhibitory effects of the various concentrations (0.5, 0.25, 0.125, 0.0625 and 0.03125 mg ml−1) were significantly lower than that of the positive control, an indication that biofilm formation was inhibited at these concentrations"). I can see from the methods section that you describe how Pa% antibiofilm activity is calculated ("Abf A (%) = (1-(ODTest sample - ODBlank)/ (OD Untreated sample - OD Blank) × 100"), and there is nothing wrong with that. The problems come in when calculating the statistical significance in relation to PC strain P. aeruginosa ATCC 12934. I understand the inclusion of this strain into your biofilm assays to ensure biofilm is formed, but the comparison of the antibiofilm activity against each strain should be compared to the untreated sample. Please recalculate these P values making the comparison between untreated samples vs treated samples, not treated samples vs control strain. The paragraph describing these results (lines 246-260) need to be rewritten as they are currently not as comprehensible as the rest of the manuscript.

  5. Version published to 10.1099/acmi.0.000613.v4 on Access Microbiology
  6. Access Microbiology

    Thank you for attempting to address the most recent comments on this manuscript. However, there are still several issues that are not fully resolved, as detailed below; Point 1) Thank you for adding clarification to the statistical analysis section. Point 2) The text in manuscript reads- “Then a colony was identified, picked and inoculated in 10 ml of LB broth and incubated at 37 ˚C 48 overnight while shaking at 100 r.p.m. for 18 h. By use of a parafilm the flat-bottomed polystyrene tissue culture microplate was sealed for purposes of preventing medium evaporation.” Information is still missing from this method. There is a step missing in between taking a single colony and culturing it in 10 ml broth overnight, to then sealing the plate with parafilm. What volume of culture do you place in each well? And what is the culture density- do you normalise the cultures to a particular OD or CFU before putting it into the wells of the plate and sealing it with parafilm? If you don’t normalise the starting inoculum, then how do you compare across plate replicates and in between strains? Point 3) I’m afraid I still don’t understand the MDR data presented in figure 1. Can you clarify why strain 13642 gets grouped with strains that show resistance to 3, 4, and 5 classes of antibiotics. Surely, they should just be grouped into strains which are resistant to 5 classes of antibiotics as it is therefore a given that they show resistance to 2, 3, and 4 classes of antibiotics because they are resistant to 5 classes in total. So out of 10 P. aeruginosa isolates, 4 strains were MDR with 1 strain resistant to 3 classes of antibiotics (12583), 2 strains resistant to 5 classes of antibiotics (13642 and 14421), and 1 strain resistant to 6 classes of antibiotics (11985). (with no need for figure 1 to be presented). Point 4) Thank you for attempting to put together the biofilm data into one single figure instead of 3 separate ones. However, the figure looks like it has been cut and pasted from the original graphs rather that being replotted which makes the figure messy and the resolution and scale of each plot doesn’t match. Before publication these will need to be replotted. Additionally, upon looking at this biofilm data to check the plots I have noticed something important that wasn’t flagged in the peer review reports. The use of strain ATCC 12934 at the “positive control” and comparative strain in your anti-biofilm treatment graphs is inappropriate. This strain has been used as the benchmark to test the ability of 6 different antibiotics to disrupt/prevent biofilm formation by strains 12583, 14421, and 11985, but the true comparative assay should be each of those strains with no antibiotic added. This would give you the baseline biofilm formation by these strains and allow you to see how well each antibiotic disrupts the biofilm of each strain. For instance, your data make it look like strain 11985 is more sensitive to the antibiofilm activity of all antibiotics tested compared to the other two strains, but how do you know it isn’t just a weaker biofilm producer in the first place? These antibiofilm experiments need to be completely redone to include each strain with no antibiotic added. If you are not able to redo these experiments, I would suggest removing all of the phenotypic characterisation of P. aeruginosa data completely as it is not clear why you have chosen these strains for characterisation out of all 135 bacteria you isolated from wounds to begin with. What was interesting about the 10 P. aeruginosa strains that led you to do AMR, antibiofilm, and virulence phenotyping- this was a question raised by reviewer 1 in their original peer review (result point 4) but hasn’t been made clearer in your revisions.

  7. Version published to 10.1099/acmi.0.000613.v3 on Access Microbiology
  8. Access Microbiology

    I have taken a detailed look at the changes you have made, which wasn't a straight forward task as the track changes document doesn't contain any changes beyond the abstract. However, I am afraid I am going to have to send this manuscript back to you as several points have not been sufficiently addressed, and a few of the tables have now been changed into figures which are poorly labelled. Key points from the original review that need to be addressed; 1) Please specify what statistical tests have been used through out. This question was asked by reviewer 1 but was not addressed. 2) Point 7 raised by reviewer 1 has not been fully met- please specify the inoculum used to seed the microtiter plates in the biofilm assays. 3) Point 3 raised by reviewer 2 has not been addressed- "Paragraph from lines 202 to 206. It states that only 4 isolates are MDR but describes 6 isolates since isolate 13642 was included in three categories (resistant to 3, 4 and 5 antimicrobial classes). This mistake is then translated into Figure 1." (which is now figure 5). How can the same isolated been included in the category of resistant to 2 classes, 3 classes, and 4 classes of antibiotics? 4) Point 5 raised by reviewer 2 about merging the biofilm data into a single figure has not been actioned- "Figures 2-4 should be converted into a single summary figure using colours to distinguish the 4 isolates, and in this way facilitate the comparison between isolates. The text that describes those figures is also very hard to understand, with sentences that don't make sense (for instance lines 229 to 231). Since the authors compare results of the biofilm formation inhibition assay with the antimicrobial sensitivities for the 4 P. aeruginosa MDR isolates, the specific resistances for these isolates should be presented in a table alongside the summary figure (including data now in Figures 2-4) as to facilitate interpretation". This was something I specifically drew attention to in my original decision letter and something which would greatly improve the presentation on the results. See these manuscripts for good examples of what it could look like https://journals.asm.org/doi/pdf/10.1128/AAC.02885-15 , https://www.microbiologyresearch.org/docserver/fulltext/jmm/66/3/377_jmm000446.pdf?expires=1689773745&id=id&accname=sgid025019&checksum=54782BB94A5B25C934FDF7C6DC6C7E70 Additionally, I am not sure why tables 1 and 2 have now become figures? They don't convey the information as well as the tables did, and the text in the legend and the key do not match- "Gram +ve and Gram -ve cases are represented in light-blue and dark-blue, respectively while cases where neither bacteria strains were observed are shown in light grey". Please revert back to the original tables which were much more informative and easier to extract information from. Lastly, there is talk throughout the manuscript of growth rates to mean incidences of positive growth of test samples (for example- Among total growth, the highest growth rate was found in age groups >45 years in both Gram-positive and Gram-negative at 14 (26.9%) and 12 (23.1%) respectively. Least growth was found in an age group of 45 years....... etc)

  9. Version published to 10.1099/acmi.0.000613.v2 on Access Microbiology
  10. Access Microbiology

    Please address all reviewers comments below paying careful attention to discrepancies in data in the manuscript, and methodologies that need to be clarified. Both reviewers have made good suggestions to improve the data presentation in the manuscript. Please do merge the data from figures 2-4 into one figure as suggested by reviewer 2. I appreciate that reproducing the plate images in the supplementary figures may not be possible if you no longer have the bacterial strains, but if it is possible better photographs would be appreciated, especially for the zone of clearance plates. Additionally, both reviewers highlight issues with structuring and legibility of the text that need to be thoroughly addressed during revision.

  11. Access Microbiology

    Comments to Author

    This manuscript gives an overview of AMR persistence in patients with wounds in a Kenya hospital. The study appears to be well designed, but the manuscript needs to be improved. The Introduction section needs an in-depth revision to be up to the standards of the journal. For instance, lines 61-65 would benefit from being re-written and important review papers (like https://pubmed.ncbi.nlm.nih.gov/35065702/) should be cited. Additionally, lines 67-70 need to be made clearer. The last two paragraphs are particularly confusing and one of the papers cited (19) cannot be accessed, which obfuscates the point the authors try to make. The Methods section is thorough and allows for good reproducibility, with good description of the protocols and controls used. Note that at the end of line 120 the word "well" is missing. The Results section is a bit confusing mainly due to the structure of the sentences and the use of percentages when it doesn't add extra information, as well as several mistakes (see below). I would also like the authors to clarify if any of the samples analysed had both gram positive and gram negative bacteria. 1) In Table 1 the row for "Median Age" is not formatted correctly and in my opinion doesn't add any relevant information so it should be removed (or its relevance should be made apparent). 2) The text and Table 2 refer to 135 samples, but Table 1 to 134 samples, which has implications in the accuracy of all percentages calculated in the text and tables (currently they don't match, and this should be corrected). 3) Paragraph from lines 202 to 206. It states that only 4 isolates are MDR but describes 6 isolates since isolate 13642 was included in three categories (resistant to 3, 4 and 5 antimicrobial classes). This mistake is then translated into Figure 1. 4) Table 4 is confusing as it has +/- but also p/n for positive/negative. Authors should choose only one nomenclature (usually it's +/-) and rewrite paragraph from lines 210 to 218 since it is confusing and particularly lines 213/214 don't make sense. 5) Figures 2-4 should be converted into a single summary figure using colours to distinguish the 4 isolates, and in this way facilitate the comparison between isolates. The text that describes those figures is also very hard to understand, with sentences that don't make sense (for instance lines 229 to 231). Since the authors compare results of the biofilm formation inhibition assay with the antimicrobial sensitivities for the 4 P. aeruginosa MDR isolates, the specific resistances for these isolates should be presented in a table alongside the summary figure (including data now in Figures 2-4) as to facilitate interpretation. Lines 236/237 should be included in the discussion, not in results. The Discussion section is written in a very convoluted way, particularly the initial paragraphs. It doesn't help that the citations appear not to match what the authors wanted to cite (for instance, references 22, 25 and 26 refer to methods and not studies, or reference 29, that is referred in text as a study from Nigeria, is actually a book chapter about Vibrio). I suggest the authors re-write this section and go straight to the point (for instance, in the first paragraph the information could be summed as: "In our study, X% of the samples tested were positive, in line with the results from studies Z and W that showed z% and w% respectively"). Furthermore, from line 348 authors focus on the production of different enzymes by the 4 MDR isolates, and compare these with other studies, but with such a small number of samples, conclusions such as "finding didn't conform with previous studies" cannot be made. Authors should refrain from using colloquial names for effects, and in my opinion the sentence on lines 365/366 should be removed. The Conclusion section includes good points, but a careful revision of the text should be done to allow better readability.

    Please rate the manuscript for methodological rigour

    Good

    Please rate the quality of the presentation and structure of the manuscript

    Poor

    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

  12. Access Microbiology

    Comments to Author

    The author's present a good manuscript detailing the use of antibiotics for bacterial infections, focussing on bacteria associated with wound discharge such as P. aeruginosa and S. aureus. This work will add to the literature to provide insight into the phenotypic characteristics of wound-associated bacteria and antibiotic use for chronic wound infections. Whilst this is a good start, I do feel there are some things that the author's could improve on in the manuscript which are described below. Methods: 1) Check for correct spellings throughout manuscript. For instance, a dash in Mac-conkey is not needed, as it should read MacConkey 2) On line 98, the author's describe the plates to be incubated between 24-48 hours growth, which is a huge difference. Could this be more defined as to which plates were incubated to what specific time point. 3) Were any specific bacteria targeted during the screening? It is confusing when the methods jump straight to saying "Isolated S. aureus was screened". Could a table be used to describe the bacteria isolated and the number/percentages of these? 4) Could the author's further define what statistical analyses were done i.e. was it parametric/non-parametric. I did notice that the statistical analysis sections also contradict each other - in the main methods section it says that SPSS/GraphPad was used, but in the statistical analysis section it says that Stata was used. Could the author's please make it clear which one was used/if different statistical analysis packages were used for different experiments? 5) Line 111 - what size microtiter plate was used? 6) For antibiotics tested on each bacteria, could the names and concentrations be added to the manuscript to make it clearer. This would also be useful for cross-examining when writing the discussion. 7) The starting concentrations of the P. aeruginosa strains was not clear for the biofilm formation as it jumps from saying about inoculation of 10 ml LB from a colony to create the overnight culture to incubation for 48-hours in the microtiter plates. A sentence in the middle of this describing the CFU/ml of P. aeruginosa added to the microtiter plate from the overnight culture would be advantageous. 8) Would the use of parafilm on the P. aeruginosa biofilm formation cause a microaerophilic/more anaerobic environment? How does the author feel this may affect the results compared to without the use of parafilm? 9) Line 112: what percentage ethanol was used? E.g. 100% (v/v) 10) What was the autoclaving time and temperature for the skimmed milk? Did the media caramalise? 11) Line 127: should be a colon at the end of the sentence rather than a ',' 12) Can the authors give final w/v or v/v percentages for the protease agar plates rather than just the grams added per 100 ml 13) In terms of protease production, could the author's add to the methods how the amount of protease production was quantified. It is mentioned that the clear hydrolysis zone indicates a positive test, but it does not mention how this was defined. Was it just a positive/negative score per plate, or was it measured in some way? 14) In terms of lipase/phospholipase production, can the author's note at what time points lipase production was scored? The methods note 1-5 days and 1-3 incubation, respectively, but this is a large range. Like with the protease, was there a scoring method for lipase/phospholipase production? 15) Controls were mentioned for the antibiotic screening but it wasn't clear for the other methods what controls were used. Could the author make it clearer in each section what controls there were per experiment. Results: 1) Table 1 is very nice and clear - it helps to explain the results well 2) The results are very clear on the bacteria which were isolated from the infections - this could be linked better to the methods 3) S. aureus is part of Staphylococcus so how do the authors define that S. aureus was the predominant bacteria isolated compared to Staphylococcus spp? Do we know what the other species are? Could further testing be done to find out what these are or would it be worth grouping all together as Staphylococcus predominant then branching to say "of these, 20.7% were S. aureus" 4) As Klebsiella was the most dominant Gram negative bacteria isolated, why was P. aeruginosa chosen for phenotypic testing? Is this because of the MDR phenotype? If the author's could comment/explain further this would be useful within the manuscript 5) Could the author's discuss that there was one E. coli which had resistance to Imipenem and why this may be 6) Line 211: should read MDR not MRD Discussion: 1) Line 269 'Similar' should not have a capitalised S 2) Discussion is good in comparing findings of author's findings to previously published literature and giving examples of why it might/might not agree - very well done 3) Line 282 long sentence is quite hard to understand - could this be broken into smaller sentences? 4) Line 287 - 'to' does not need to be italic 5) Please can the author's elaborate on the reasons they do not think their results match what has been previously seen in other studies? The author's do well in listing the findings and reasons for the reference studies, but could then compare their own findings more thoroughly. Could it be down to isolation techniques? The authors do well in listing the virulence factors of the predominant pathogens - but how does this link with isolation? Whilst Gram-negatives were more commonly isolated in this study, it was a relatively even split overall 6) Line 309 - it should read that S. aureus is resistant to Erythromycin whereas it currently reads that the drug is resistant to S. aureus 7) Line 348: which isolates are the author's talking about? S. aureus? Please add in the bacterial name to make it clearer 8) Lines 348-350: I would be more inclined to argue about the fact that hemolysin is produced the majority of isolates, like the study compared. This is because, even though only 75% of the isolates were hemolysin producing, it is a small sample size and when comparing to numbers, it was actually only one which was the outlier. I would focus on more why this was the outlier with possible explanation rather than saying they do not conform. 9) The authors mention about the organisation of a genetic element which may have affect production of protease/lipase/phospholipases. Do the authors have access to the genomes and could do some work on investigating about this arrangement? This is not compulsory for the manuscript, but could be interesting future work as it is an interesting discussion point. Pathogenicity Island (PAI) Viewer would be an easy way to see whether these elements are on a PAI. Overall comments: 1) Some sentences hard to read so might be worth re-wording some of these and also splitting into shorter sentences which are easier to digest 2) Try to adjust tables so that headings are on one line and easier to read. For example, the antibiotic Table 2. If the names are too long, use the abbreviations and make a legend at the bottom of the table explaining them. 3) Some figures could be improved in upload quality. For instance, the legends on figure 1 are readable, but slightly pixelated 4) Make sure all antibiotic names have capitals (example line 231 Ampicillin needs a capital A) 5) Make sure the word 'Gram' for Gram positive/negative always has a capital G (noticed some places in discussion that it was not capitalised) 6) Make sure all bacterial names are italicised as the start of some abbreviations are not 7) When making comparisons in the discussion, the authors could make note about the small sample size they are working with meaning that some conclusions could be biased/more research needed with a larger sample size. This does not mean the results are invalid, just something worthy of recognising. 8) Make sure to give full names before abbreviations e.g. confusing what AM stands for on line 305, which I can assume is 'antimicrobial'. The abbreviations were done well for the agar types in the methods so make sure this is consistent throughout.

    Please rate the manuscript for methodological rigour

    Good

    Please rate the quality of the presentation and structure of the manuscript

    Good

    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

  13. Version published to 10.1099/acmi.0.000613.v1 on Access Microbiology