Comparison of the virulence of community- and hospital- isolated Acinetobacter baumannii in HeLa cell line and insect model, Galleria mellonella
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Acinetobacter baumannii is an important nosocomial pathogen causing high infections and morbidity among affected individuals, and most studies focus on nosocomial strains. However, A. baumannii can also be isolated from healthy community individuals. This study compared the pathogenicity of hospital and community A. baumannii isolates using Galleria mellonella and human cell cultures. The insect model, G. mellonella , and in vitro HeLa cell line were used with ten A. baumannii isolates (six community and four hospital isolates from Segamat, Malaysia). G. mellonella killing assays and HeLa cell adherence, invasion and cytotoxicity assays were performed to investigate the virulence and invasion potential of the isolates. Out of the ten isolates investigated, three community and two hospital isolates were found to be highly virulent in the G. mellonella infection model, killing 100% of larvae within 96 h. These strains were also found to be invasive and have significant cytotoxicity in HeLa cells. Our study revealed that community- and hospital-isolated A. baumannii could be equally virulent judged by both model systems. Undoubtedly, besides hospital settings, the presence of highly virulent A. baumannii in community reservoirs poses a significant public health risk and requires additional investigation.
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I am pleased to tell you that your article has now been accepted for publication in Access Microbiology.
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Dear Dr. Nazmul Hasan Muza Thank you for your patience while we assessed your manuscript “Comparison of the virulence of community and hospital isolated Acinetobacter baumannii in HeLa cell line and insect model, Galleria mellon”. Your work was reviewed by members of the editorial board as well as two independent reviewers. In light of the reviews, we are pleased to inform you that the manuscript is provisionally accepted with minor revisions required. We believe that your work would be a valuable contribution to the existing literature. The second referee has highlighted concerns many of which we agree with. However, some of these points can be seen as the limitation of the study. Please ensure that you respond to each of the reviewer’s comments. In particular we would like to draw your attention to the comments 1, 5, 6,10, 11 and …
Dear Dr. Nazmul Hasan Muza Thank you for your patience while we assessed your manuscript “Comparison of the virulence of community and hospital isolated Acinetobacter baumannii in HeLa cell line and insect model, Galleria mellon”. Your work was reviewed by members of the editorial board as well as two independent reviewers. In light of the reviews, we are pleased to inform you that the manuscript is provisionally accepted with minor revisions required. We believe that your work would be a valuable contribution to the existing literature. The second referee has highlighted concerns many of which we agree with. However, some of these points can be seen as the limitation of the study. Please ensure that you respond to each of the reviewer’s comments. In particular we would like to draw your attention to the comments 1, 5, 6,10, 11 and 13 by the second reviewer. For the comment 13, we suggest you provide a relevant reference to support your stand. When you are ready to resubmit, please upload a detailed list of your responses to the reviewer comments and a description of the changes you have made in the manuscript. We hope that you find the peer review process constructive, and we look forward to your revised manuscript. Thank you again for your submission.
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Comments to Author
In this manuscript by Muzahid et al they describe the phenotype of 10 isolates of Acinetobacter baumanii based on their ability to adhere to and invade Hela cells and their virulence in an in vivo Galleria melonella infection model. Four of the isolates were acquired from patients in hospital while 6 were isolated from members of the community. The key finding of the paper is that some isolates from members of the community were more virulent than some of the isolates from hospital patients. This is a limited study due to the small number of isolates used and the limited assays used to assess virulence. In the Galleria infection model two hospital isolates and 3 isolates from the community killed the moths at a dose of 106 FU. If the dose was increased to 107 all the isolates could kill the moths, …
Comments to Author
In this manuscript by Muzahid et al they describe the phenotype of 10 isolates of Acinetobacter baumanii based on their ability to adhere to and invade Hela cells and their virulence in an in vivo Galleria melonella infection model. Four of the isolates were acquired from patients in hospital while 6 were isolated from members of the community. The key finding of the paper is that some isolates from members of the community were more virulent than some of the isolates from hospital patients. This is a limited study due to the small number of isolates used and the limited assays used to assess virulence. In the Galleria infection model two hospital isolates and 3 isolates from the community killed the moths at a dose of 106 FU. If the dose was increased to 107 all the isolates could kill the moths, indicating that virulence was dose dependent. The authors then looked at the interaction of the isolates with HeLa cells. They assessed adherence, invasion and cytotoxicity. Adherence to cells was assessed by microscopy while invasion was assessed by determination of the number of CFUs per well. How many fields of view were counted to assess bacterial adherence? It would be better if total bacterial association was assessed by measuring the number of CFU per well without genatmicin treatment. This would allow for more reliable quantitative data to be generated for direct comparison with the invasion data. Fig2 shows images demonstrating some bacteria adherent to cells and some cells are damaged by some isolates. This figure adds little to the paper. Cytotoxicity is demonstrated in Figure 4 and it is a more reliable quantitative assay. I would remove figure 2. Fig 3 shows invasion of HeLa cells for each isolate. This figure needs to be amended and a log scale should be used on the Y axis. This allows for a more reasonable comparison between isolates to be made. Fig 4 shows cytotoxicity of the isolates based on the Trypan blue exclusion assay. Three community isolates that were also virulent in the Galleria infection model are significantly more cytotoxic compared to the negative control. Lines 276-278 state "Statistically significant negative correlations were found 277 between the survival time of Galleria larvae infected with indicated strains and their HeLa cell invasion 278 (Table S6, S7, S8)." I have read the supplementary information and I am not sure what this means. Do the authors mean that there was no correlation between survival time for Galleria larvae infected and Hela cell invasion? Lines 322-325 state "Our study revealed that the in vivo Galleria infection model and in vitro cell culture could be 323 used as a routine way to detect the pathogenicity of A. baumannii. The significant statistical correlations 324 between the two tests suggest that either or both models could be used as a preliminary screen before 325 selecting strains for detailed in vivo assessment in a mammalian model system". How does this statement fit with Lines 276-278. I find the data presented in supplemental tables S6 and S7 highly confusing. Could the authors clarify in simple terms what the data is showing. Is there a correlation between virulence in the Galleria model and ability to invade HeLa cells. Other comments ul should be changed throughout the manuscript. Lines 281-183 "we 282 find that about 3% of the general community produced feces that allowed the isolation of A. baumannii 283 isolates." There is no reference supplied for this statement. Is this person observation if so what is it based on ie how many individuals were screened etc. How were the isolates identified as Acinetobacter baumanii. How were the individuals from the community selected for faecal screening for carriage of Acinetobacter baumanii.? Did these individuals have any health problems or were they all healthy. Why were HeLa cells chosen for adherence and invasion assays? Is any antimicrobial resistance data available for these isolates? This information would add greatly to the discussion. The real concern would be that MDR isolates are circulating in the community and that they are virulent. It is not surprising that isolates from the community have the potential to be virulent. This study highlight s that underlying health problems likely contribute to Acinetobacter baumanii virulence in humans and hence the propensity to find isolates in hospital patients.
Please rate the manuscript for methodological rigour
Good
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?
Yes
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Comments to Author
To: Access Microbiology Dear EIC, Dear AE; Prof. Nihal Bandara This is my review report for the manuscript ID: ACMI-D-24-00112 In this study, the authors compared several characteristics of A.baumannii between isolates from healthy people (community) and infected patients (hospital). They used an in-vivo model for comparison of virulence power and in-vitro human cell line for several pathophysiological factors such as adherence, invasion, and cytotoxicity power between these two groups. I think the results of this study, especially about the same potential virulence power of community and hospital isolates of A.baumannii, are interesting for the readers. This is one of the rare manuscripts that I can't recommend to revise the writing. I think the manuscript can add some novel things to the field. I …
Comments to Author
To: Access Microbiology Dear EIC, Dear AE; Prof. Nihal Bandara This is my review report for the manuscript ID: ACMI-D-24-00112 In this study, the authors compared several characteristics of A.baumannii between isolates from healthy people (community) and infected patients (hospital). They used an in-vivo model for comparison of virulence power and in-vitro human cell line for several pathophysiological factors such as adherence, invasion, and cytotoxicity power between these two groups. I think the results of this study, especially about the same potential virulence power of community and hospital isolates of A.baumannii, are interesting for the readers. This is one of the rare manuscripts that I can't recommend to revise the writing. I think the manuscript can add some novel things to the field. I prepared some minor comments to further level up the manuscript content. Comments * The introduction section is too long. Kindly truncate it to one or less. * Kindly add the limitations and strengths of the study to the end of the discussion section. Good luck
Please rate the manuscript for methodological rigour
Very good
Please rate the quality of the presentation and structure of the manuscript
Very 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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