Bacterial profile of wound site infections and evaluation of risk factors for sepsis among road traffic accident patients from Apex Trauma Centre, Northern India

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Abstract

Background. Among the most significant yet often ignored health issues worldwide are trauma and accidental injuries. India accounts for 11% of global deaths in road accidents, the highest in the world, according to the World Bank report. There are limited data about the bacterial contamination of road traffic accident (RTA) wounds and their antibiotic susceptibility patterns.

Materials and Methods. This prospective study was conducted in a tertiary care centre in northern India from January 2023 to January 2024. Wound deep swabs or aspirates were collected from RTA patients with traumatic injuries at different time intervals. Gram stain and culture were performed, and positive aerobic culture was subjected to antibiotic susceptibility testing. Organism identification was done using MALDI-TOF MS and routine biochemical tests. Blood samples were also collected to rule out bloodstream infections during follow-up if the patient became febrile or showed symptoms of systemic infection. Sepsis was defined in those patients who had two or more scores in the systemic inflammatory response syndrome criteria with a positive microbiological culture. Risk factors were evaluated for sepsis on the basis of the patient’s vitals, injury characteristics, procalcitonin, Glasgow Coma Scale (GCS) score, need for mechanical ventilation and complete blood count, which were obtained from the patient’s admission file.

Results. A total of 189 wound samples were collected, of which 99 (52.38%) samples showed the growth of microorganisms. The aerobic isolates included 69 (69.69%) Gram-negative bacilli, of which the majority were Klebsiella pneumoniae , 28 (28.28%) Gram-positive cocci, of which the majority were Staphylococcus aureus and 2 (2.02%) anaerobic isolates. Among the Gram-negative isolates, none of the isolates were resistant to colistin. All S. aureus isolates were susceptible to vancomycin, teicoplanin and levonadifloxacin. Sepsis developed in 50 (26.45 %) patients. Significant risk factors evaluated for sepsis were a raised procalcitonin level, a low GCS score, a higher injury severity score, the need for mechanical ventilation and a raised quick sequential organ failure assessment score.

Conclusion. It is essential to ascertain the profile of microorganisms isolated from RTA wounds in order to reduce antibiotic resistance and deliver efficient treatment.

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  1. gram should be Gram throughout Line 330- "Carbapenems were resistant among 52.17% (36/69) gram negative isolates"- edit to "carbapenem resistance among Gram negative isolates was......." Lines 365-366- incorrect capitalisation used in naming medical conditions (Diabetes for example)- please correct. Line 388- remove full stop after "obtained" Line 409- Non fermenter should be non-fermenter Figure 2 has no axis and inappropriate axis labels Figure 3 has no Y axis. The % of top of the columns are merging into each other. If the data can't be clearly represented by a graph without the use of numbers on the top of the bars, then a table should be used. Either convert figure 3 into a table, or add a Y axis and remove labels above the bars. Figure 4 is completely inappropriate. A line graph denotes connectivity between the data points along the X axis, and shouldn't be used for discrete data categories. The values on the Y axis are also incorrect. Please remake this graph. Figure 5- same comments as for figure 3. Plus figure legend needs rewording "Column graph shows the percentage (%) susceptibility of different antibiotics among Staphylococcus aureus wound isolates"- the bacteria show susceptibility to the antibiotics. Figure 6- need a Y axis Table 1- please add a P value column comparing variables between sepsis and non-sepsis patients Figure 7- same comment as figures 3 and 5. I can't find reference to figure 3 in the text. Please ensue all figures and tables are referred to in the text, and are numbered in the order they appear in the text. Lines 382-383- "According to our research, 52.38% of the patients had grown a pathogen among wound samples, which seems like a quite significant percentage"- please reword to remove significant which should be reserved for cases which are statistically significant. Optional edits: The discussion is still very long winded and contains much repetition of the results. The style would also benefit from some adjustment to switch from "according to xxx" to a more streamlined style of "Our results are in line with other studies which show X result (reference)." Making these edits will improve your manuscript, but choosing not to make them will not block publication.

  2. In addition to responding to the detailed reviewer reports, please thoroughly check and amend all graphs; - Check all axis for appropriate labels. - Remove gridlines. - Graphs should not have titles, but only figure legends which make the graphs fully interpretable. - The use of 3D plots is inappropriate. Please change all plots to 2D only. - The red bars on figure 6 have no axis. - If bar plots require numbers on top to make them interpretable, consider presenting the data as tables instead.

  3. Comments to Author

    Dear Authors, Well done for the manuscript, it is informative and well written. Please allow me to give a few humble feedback, for the purpose of improving the work. As below. 1. Methodological rigour, reproducibility and availability of underlying data: - Line 278-279, "Carbapenems were resistant among (36/69, 52.17%) isolates.": Can it be more specific? please specify that carbapenems were resistant among 36/69, 52.17% of which classification of isolates (does it means, overall Gram-negative bacilli, or specific to Enterobacterales, Pseudomonas spp, or Acinetobacter spp). - Line 281, "MDRO among the isolates were 61 (62.88%)": same as the above, can it be more specific, whether the "MDRO" here means "MDRO Gram-negative bacteria"? - For the antimicrobial susceptibility testing of Gram-negative bacilli, as presented in Figure 5, I wish to suggest a major revision to clarify the testing methodology of colistin and cefoperazone-sulbactam, because this has the potential to affect the validity of the antimicrobial susceptibility tests reported here. As the authors stated that the references for antimicrobial susceptibility test is the Clinical Laboratory Standards Institute (CLSI) M-100 Ed 33, according to my understanding on reading the CLSI M-100 Guidelines: (i) Colistin susceptibility test - as the authors mentioned, the test method for susceptibility in this study is Kirby-Bauer (KB) disc method, however, CLSI M-100 Ed 33 (and the latest Ed34) stated that KB disc method is unreliable for colistin testing for all Gram-negatives, includes Enterobacterales, Pseudomonas aeruginosa/ spp and Acinetobacter spp., in which these are the common species found in this study. In CLSI, there is no interpretation breakpoints to decide whether colistin is sensitive or resistant, if using KB disc method. In the CLSI guidelines M-100 (Ed 33 and the latest Ed34), the recommended colistin testing method is either broth microdilution or CBDE (colistin broth disc elution), which the results are measured in minimum inhibitory concentration (MIC). Since the authors had reported sensitivity for colistin in Figure 5, can the authors please clarify the references for colistin susceptibility test method and the susceptibility interpretation breakpoints? If it is not the CLSI M-100 Ed 33, can the authors please state the exact references use? (ii) Same for cefoperazone-sulbactam, the test method and susceptibility interpretation breakpoints for common Gram-negatives bacteria (Enterobacterales, Pseudomonas and Acinetobacter) has not been stated in the CLSI M-100 guidelines. If the authors have references other than CLSI, please state in the references list. I humbly suggest further advise from the microbiologist in the study team. 2. Presentation of results - I think what stated in Line 120-121 under section "Objectives" hasn't being reflected in the results and discussion, or just vague. It stated "To study the changes in the pattern of bacterial etiology with follow up of patient samples", while the results presented in Figures 2-7, as well as discussion section, the species of microorganisms and antimicrobial susceptibility are listed in collective way (means the overall), and didn't reflects the "changes of bacterial etiology" according to timeline, eg: "what are the species and their susceptibility isolated from the first sample?", "then what are the species and susceptibility at 96 hour, 1 week and 2 weeks respectively", "are there increasing in resistance? or any changes in the microorganism population with the shift of the time?", "are there concerns of hospital/ nosocomial infection which intervene, especially based on the results of samples collected more than 48 hours", etc. - For Gram-positive bacteria, the authors had specifically presented the sensitivity of Staphylococcus aureus in Figure 6. I suggest the authors can do the same for Gram-negative bacteria which is more diverse, instead of collectively presented the overall susceptibility results only in Figure 5, suggest the sensitivity for a few important species can be separated and reported in different tables, such as that for Enterobacterales, Pseudomonas aeruginosa and Acinetobacter baumanii. The justification for this suggestion is, the first-line antimicrobial agents (empirically and target therapy) can be different for these; the rate of MDRO also varies among different genus/ species. - Line 297-299 which describe the other blood investigations, seem like suddenly chip in between the discussion of microbiology results. Can this be stated after the microbiology results, eg. after line 303? - Section "Figures and Tables" (line 318-366) were put in between "Results" and "Discussion", which may not be common in other papers. Suggest to check if this is the required format of this journal. 3. How the style and organization of the paper communicates and represents key findings - most of my suggestions were mentioned above 4. Literature analysis or discussion: Overall satisfactory 5. Any other relevant comments - tying error: Line 191-192, it stated that the CLSI M100-edition is 23 Ed, I believed this is a typing error, because in the reference no. 10, it stated CLSI Ed 33 published in year 2023. - "Limitation: the authors stated the limitation is only two anaerobic microorganisms being isolated. Suggest to elaborate more on this limitation, eg. was it due to the lack of optimum anaerobic facility, optimum culture media, incubation period, etc? - Also, for the two anaerobic organisms isolated, there wasn't antimicrobial susceptibility reported here, despite there are recommended testing method and susceptibility breakpoints in CLSI guidelines. If this wasn't been performed, it could be stated as a limitation, with elaboration. - The "conclusion" is too general, suggest it can be improved by stating what are the most common Gram-negative and Gram-positive pathogens in wound infection, and the antimicrobial susceptibility pattern which is worth to alert the clinicians to consider when starts empirical therapy for RTA patients. - Ethical concern: For the collection of deep wound swabs and aspirate at different interval up to 2 weeks, it doesn't define in the inclusion/ exclusion criteria whether this is for in-patient only or also involved outpatient. If this also involved outpatient, then the ethical concern would be: other than the researchers and its publication purpose, are the clinicians who treat the patients also review the results of these specimens collected on subsequent follow up (especially after discharging patients) and continuous treatment given if microorganism growth detected from the subsequent follow up samples? Above are my humble two cents and suggestions for improvement. Thank you very much and best wishes.

    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: My answer is "not sure", as the details provided on manuscript on this part is insufficient. Please see my message to the editorial, second paragraph on the concern of who will follow up the subsequent samples and will continuous treatment be provided to the patients if the subsequent samples grow microorganisms from culture, suggest to clarify with the authors.

  4. Comments to Author

    Many thanks for the authors for their piece of work. They aim to describe the bacterial profile of wound site infections among road traffic accident victims as well as evaluate potential risk factors for the progression for sepsis among this population to inform local practice. Overall, the content of the manuscript is sound but the vast quantity of grammatical errors takes away from the appreciation of the data being reported. Furthermore, there are significant queries regarding the Results being reported. Some specific comments; - Throughout the manuscript there are countless errors in the tenses being used, incorrect capitalisation, incorrect conjunctions and sentences which are disjointed. The manuscript will require a thorough proof-read and rewrite. - Throughout the manuscript there is also the use of antibiotic, then antimicrobial. Please stay consistent with terminology. - The Abstract for the most part reflects the study well but can be condensed and restructured to say the same thing but more effectively. - Line 83: Please reference why it is an urgent need to save the RTA patients, especially the young ones for economic productivity. - Line 93-96: These lines add nothing to your manuscript. Please consider omitting. - Line 102: Reference needed. - Line 105-106 - I am not sure how I feel about the repetition of the need to save RTA patients because they are young. It comes across insensitive to older individuals, and as doctors we have an onus to try and save all persons, I would omit it here as it was already stated and hopefully referenced as above. - Line 110: Reference needed. - Line 130-131: You investigated the risk factors for the development of sepsis. It would be important that you define what you used for "sepsis". Do you simply mean those who developed a bloodstream infection? Or those with organ dysfunction? - Line 139: I fail to see how Triage Care is relevant in this study. It is neither analysed further nor part of your Objectives. - Line 143: "ethical approval" - Your Methods are very well described but will benefit from referencing. - The anaerobic culture processing however, is very confusing to read. You mention that the third wound swab was kept in RCM and growth was observed by turbidity of the broth. What then happens to this broth? Is it subcultured? Then, you mention that the sample was also cultured on Wilkins Chalgren. Is this the same swab that is supposedly in RCM? Or are both methods employed, in which case I presume the swab can only be used for one method which would be a limitation if all samples are not processed in the same way. - Your bias should be better described and elaborated in the Discussion. - Quantitative variables can be significantly shortened, please restructure. Same with the Statistical Methods. - I do have some particular concerns regarding your Results. 189 patients were recruited, but only 99 wound site infections were evident. You state that these were "culture confirmed". What do you mean by this? Was it assumed that once a swab cultured an organism it was classified as a "wound infection" or was clinical acumen applied to those cases? Growth of an organism does not mean infection but can represent colonisation. I do believe you need to be more distinct in what you define as "infection" versus "culture positive" swab. - Line 239-240: 50 patients developed sepsis. Did all these patients have a culture positive wound swab? - Line 243: Males were more commonly affected, n=158. This needs to be clarified. Males suffered a greater number of RTAs. Using "affected" may imply that they were more likely to suffer a wound infection which is not what is being reported. Please be explicit in what you are saying. - Line 246: "severe trauma" - who are these patients? What is used to define "severe trauma"? - Lines 259-262: I am not sure how the site of injury is relevant to this particular piece of work. It is not what was being investigated. Similarly, consider omitting Lines 395-398. - Line 282: Save for Discussion where empiric regimens can be reported. - Figure 5: I would consider changing these number to percentages and report similarly to that in Figure 6. However, I do not know what is the small adjacent bar in Figure 6. - The way you report things are very difficult to follow. For Figure 4 it is clear that those reported microorganisms are from wound swabs, but for Figure 7 you simply state that those were the microorganisms isolated from patients with sepsis. Where were these isolated from? Blood cultures? Urine? Wound Swabs? Rectal Swabs? Where? It cannot be wound swabs only as in Figure 4 you report 4 Acinetobacter baumannii isolates and in Figure 7 there are 14. Please be clearer. - Line 309-317: Major rewrite needed. Perhaps consider a Table to display these results, similar to that in Table 1. - Line 380: Monomicrobial infections. Where is the data to support this? - Lines 414-416: Different risk factors were reported, but did you collect data on those variables? If you did not then you cannot say that it was different as you do not have the data to support that statement. - Line 432: "violating the first prophylactic principle" - What is this? Reference needed. - Line 435: Reference needed. - Lines 443-444: I am not sure how identifying risk factors for sepsis reduces mortality and length of stay. This is not a direct causal relationship. It would be more accurate to say that identifying risk factors for sepsis will allow clinicians to be more vigilant in identifying patients who may deteriorate and allow more prompt intervention. While you report a lot of interesting data it is presented poorly and requires a significant re-write.

    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