Roles of Whole-Genome Sequencing in Determining Transmission and Risk Factors Associated with Carbapenem-Resistant Organisms at a Vietnam HealthCare Center of Orphan Children

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This article needs to be corrected for English language grammar and composition, many sentences were found to be too long and lacks clarity.Scientific names should be written following the guidelines. The presentation of results need to be improved, in some instances it seems that raw data has been presented without formal analysis. Presentation of results needs to be improved while removing repetitions. results section needs to be concised. The findings of the results needs to be compared with the already available data in the discussion chapter.

 

Comments on the Quality of English Language

Needs to be improved

Author Response

1. This article needs to be corrected for English language grammar and composition, many sentences were found to be too long and lacks clarity.

Response: Kim_comment-a

I corrected English grammar and composition in my whole manuscript and sentences to become easy to read and understand for readers. All parts corrected in my manuscript are shown in the Open Review_ Rounds 2 and 3. 

I sent my manuscript to English editing services to increase the clarity of my manuscript.

1. Scientific names should be written following the guidelines.

Response: Kim_comment-b

The scientific names are corrected, following the microbiological guidelines, and highlighted in yellow color in my manuscript.

 

1. The presentation of results need to be improved, in some instances it seems that raw data has been presented without formal analysis.

Response: Kim_comment-c

We relied on the raw data and arranged them in the order of the steps in the study, inclusive

1. to screen CROs with chromocarba plates,
2. to confirm CROs with the BD Phoenix™ CPO detection panel,
3. to perform the WGS for 12 CRO samples selected among the 36 CRO samples discovered in the study.

Thus, the results shown in our manuscript were not only original raw data.

 

1. Presentation of results needs to be improved while removing repetitions.

Response: Kim_comment-d

I presented the results as your advice in my manuscript to easily read for readers.

 

1. results section needs to be concised. The findings of the results needs to be compared with the already available data in the discussion chapter.

 

Response: Kim_comment-e

The results are improved in the result section and highlighted in yellow color.

Comments on the Quality of English Language

Needs to be improved

Response: Kim_comment

I will improve the quality of English in my manuscript and you can see it.

Submission Date

08 December 2024

Date of this review

16 Dec 2024 06:47:39

Kim responded: I edited my manuscript as advised by reviewer_1 on 22 Dec 2024

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have submitted a detailed article, which mainly using whole genome sequencing to determine transmission, risk factors associated with Carbapenem-resistant organisms (CRO), a typical kind of anti-biotic-resistant Gram-negative bacteria. The authors performed a cross-sectional study and collected fecal samples from healthcare workers, residents and environmental samples to screen, isolate, and identify bacterial species and phenotypic tests for antimicrobial susceptibility of carbapenem-resistant bacteria, and the whole genome sequencing for 12 CROs chosen to confirm the CRO transmission between HCWs and residents.

The work seems meaningful, as antimicrobial resistance is one of the most concerned public health problems. However, several mistakes should be carefully corrected.

1. All the names of bacteria need to be italic. The authors should check carefully as nearly all the names format appeared in the text are wrong.

2. Abstract, Line 24, the study was from September 2022 to December 2022, while in Section 2.2, Line 117, from September 2022 to 117 May 2023. Please confirm.

Moreover, if the samples were collected from September 2022 to December 2022, I wonder if the results are representative and if they are affected by the seasons or temperature.

3. Figure 1, the resolution is low, please provide with a clearer one.

Author Response

1. All the names of bacteria need to be italic. The authors should check carefully as nearly all the names format appeared in the text are wrong.

Response: Kim_comment 1

Firstly, I thank you so much for your kind support and recommendations

I corrected all the names of bacteria as you asked and guided. Please help me correct them if you want. I highlighted these names in yellow color in my manuscript.

2. Abstract, Line 24, the study was from September 2022 to December 2022, while in Section 2.2, Line 117, from September 2022 to 117 May 2023. Please confirm.

Response: Kim_comment 2-1

The study was from September 2022 to December 2022, and I corrected this information in Section 2.2, Line 117, by from September 2022 to December 2022

Moreover, if the samples were collected from September 2022 to December 2022, I wonder if the results are representative and if they are affected by the seasons or temperature.

Response: Kim_comment 2-2

The results collected from fecal and environmental samples were representative of study data because, in Vietnam, there are only two seasons (rainy and hot season). From September to October in the South of Vietnam, it is the middle of the rainy season. From November to December, it is the hot season. However, it is still rainy in this period, and the average temperature for the total year is from 29 to 32^o C. Thus, in South Vietnam (For instance, Cu Chi District in Ho Chi Minh City), the season and temperature don’t influence the kinds of bacteria detected in the environment, such as A.baumannii, Pseudomonas aeruginosa, P. putida, etc, can be observed and detected in the environmen.

In my studies, CROs detected in the human fecal samples (HCWs and children), for example, were often E.coli, Ent cloacae, and K pneumonia. These bacteria were resistant to many antibiotics, including carbapenems due to antibiotic overuse or contamination. The kind of bacteria shown above are not dependent on the average temperature of from 29 to 32 Celcius degree.

 

2. aeruginosais a gram-negative bacterium, and it has a rod shape and monoflagella. The optimum temperature for growth ranges from 25 to 37°C though P. aeruginosa can survive at higher temperature (∼42°C), which differs from other Pseudomonas species (Wu et al., 2015) (Chapter 41 - Pseudomonas aeruginosa , Weihui Wu ¹, Yongxin Jin ¹, Fang Bai ¹, Shouguang Jin ^1 2. Molecular Medical Microbiology (Second Edition) Volume 2, 2015, Pages 753-767 (¹ Nankai University, Tianjin, China, ² University of Florida, Gainesville, FL, USA.
3. calcoaceticus–baumannii complex grows at temperatures between 35°C and 37°C; however, specific environmental isolates thrive at temperatures between 20°C and 30°C. The only bacterium in the family able to survive at 44°C is A. baumannii (Biocide resistance in Acinetobacter baumannii: appraising the mechanisms, E.S. Milani, A. Hasani, M. Varschochi, J. Sadeghi, M.Y. Memar, A. Hasani )
4. Figure 1, the resolution is low, please provide with a clearer one.

Response: Kim_comment 3

I repaired the Figure1 with another cleared than old one.

 

Submission Date

08 December 2024

Date of this review

11 Dec 2024 02:22:43

Kim responded: I edited my manuscript as advised by reviewer_2 on 24 Dec 2024

 

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript entitled „Roles of Whole Genome Sequencing in Determining Transmission, Risk Factors Associated with Carbapenem-Resistant Organisms at a Vietnam HealthCare Center of Orphaned Children, Vietnam” describes investigations of antibiotic resistant bacteria from different sources including, human and environmental samples. This study indicates importance of WGS in detection of antibiotic resistant bacteria. The topic of this submission is an important issue, however, some parts of this manuscript should be clarified.

 

Comments

1) In the abstract this is written „fecal samples from human subjects” proper form: fecal samples from humans

 

2) Do not start a sentence with, „So”:

 „So, at this center, we…” proper form: At this center we…

„So, whole genome sequencing (WGS) plays…” proper from: Whole-genome sequencing (WGS) plays…

 

3) All bacterial names must be in italic form in all over the manuscript: E. coli, Klebsiella pneumoniae, Acinetobacter baumannii

 

4) At the definition at CRE „ESBL” and „AmpC” do not confer carbapenem resistance. Please, revise that definition.

 

5) What do you mean by „E. coli class B, E. coli class D, Ent. cloacae class B”? These are present  many times in the text and in tables. What are class B, class D, please, clarify!

 

6) In Table 6 (on page 12) in the third column „Occu” is written. What do you mean by that?

 

7) Tables are not in numerical order. Please, revise table numbering. There is no Table 1 in this manuscript, but Table 6 has a, b, c, d parts.  

 

8) In this study whole-genome sequencing was performed but no carbapenemase genes are reported. I suggest to authors to add a table and list all detected carbapenemase genes.

 

9) Clonal analysis of detected carbapenem resistant strains should be demonstrated. Based on WGS data sequence types (STs) of strains of this study should be shown in a table.

 

10) At materials and methods whole genome sequencing should be in one part, including description of work in Charles River laboratory. After this part should be „Statistical analysis” given.

 

Author Response

Comments

1) In the abstract this is written „fecal samples from human subjects” proper form: fecal samples from humans

Response: Kim: Comment 1

Firstly, I thank you so much for correcting my wrong notes. I corrected the notes you asked for. I highlighted all notes corrected in Yellow color in my manuscript.

I corrected them as follows.

At this center, we performed a cross-sectional study and collected fecal samples from humans (20 healthcare workers (HCWs).

 

2) Do not start a sentence with, „So”:

 „So, at this center, we…” proper form: At this center we…

„So, whole genome sequencing (WGS) plays…” proper from: Whole-genome sequencing (WGS) plays…

Response: Kim: Comment 2

In the comment 2, I corrected as you requested in the abstract part. Please help me to check them again. I thank you so much.

 

3) All bacterial names must be in italic form in all over the manuscript: E. coli, Klebsiella pneumoniae, Acinetobacter baumannii.

Response: Kim: Comment 3

Thank you so much for your kindness. All bacterial names must be in italic form all over the manuscript, as requested and highlighted in yellow color. Please help me to check them again. Thank you again.

 

4) At the definition at CRE „ESBL” and „AmpC” do not confer carbapenem resistance. Please, revise that definition.

Response: Kim: Comment 4

I thank you for pointing out this crucial note. I corrected the definition of CRE in my manuscript, highlighted it in yellow color, and nored it as follows.

 

CRE: Carbapenem-resistant Enterobacteriaceae (i.e., Escherichia coli, Klebsiella pneumoniae) are resistant to carbapenems and include all carbapenem resistance mechanisms (i.e., production of an AmpC/ESBL plus porin mutations or carbapenemase production)

 

I quoted the information from TABLE1: Summary of terms used to describe carbapenem resistance among Gram-negative bacilli (Gniadek TJ, Carroll KC, Simner PJ. Carbapenem-Resistant Non-Glucose-Fermenting Gram-Negative Bacilli: the Missing Piece to the Puzzle. J Clin Microbiol. 2016 Jul;54(7):1700-1710. doi: 10.1128/JCM.03264-15. Epub 2016 Feb 24. PMID: 26912753; PMCID: PMC4922101).

 

5) What do you mean by „E. coli class B, E. coli class D, Ent. cloacae class B”? These are present many times in the text and in tables. What are class B, class D, please, clarify!

 Response: Kim: Comment 5

In my study I used The BD Phoenix™ CPO detect panel to detect the rapid and correct detection of carbapenemase producing organisms (CPOs) can help inform clinician decision making on antibiotic therapy. The BD Phoenix™ CPO detect panel, as part of antimicrobial susceptibility testing (AST), detects carbapenemase activity (Positive/Negative) and categorizes CPOs according to Ambler classes. (Evaluation of the BD Phoenix CPO Detect Panel for Detection and Classification of Carbapenemase Producing Enterobacterales. Harshad Lade 1, Seri Jeong 2, Kibum Jeon 3, Han-Sung Kim 4, Hyun Soo Kim 5, Wonkeun Song 2, Jae-Seok Kim 1,)

In clinical practice, based on the results of CROs detected by the BD Phoenix™ CPO detect panel, physicians will decide which antibiotics clinical physicians can choose to treat patients infected by CROs, as shown in Table 1: Activity and Indications of New Agents Against Carbapenem-resistant Gram-negative Pathogens.

Treatment Options for Carbapenem-resistant Gramnegative Bacterial Infections. Yohei Doi ^1,2 (1.Center for Innovative Antimicrobial Therapy, Division of Infectious Diseases, University of Pittsburgh School of Medicine, Pennsylvania; and 2.Departments of Microbiology and Infectious Diseases, Fujita Health University School of Medicine, Aichi, Japan

 

Table 1. Activity and Indications of New Agents Against Carbapenem-resistant Gram-negative Pathogens
	Activity
	Enterobacteriaceae
Agent	Class A Carbapenemase (eg, KPC)	Class B Carbapenemase (eg, NDM	Class D Carbapenemase (eg, OXA-48)	P.  aeruginosae	A.  baumannii	S.  maltophili	Indications (Including Expected)	Pathogendirected Trial (Including Expected)
Ceftazidime - avibactam	Yes	No	Yes	Yes	No	No	cUTI/AP, cIAI, HABP/VABP	No
Ceftolozane -tazobactam	No	No	No	Yes	No	No	cUTI/AP, cIAI, NP	No
Meropenem-vaborbactam	Yes	No	No	No	No	No	cUTI/AP	Yes
Imipenem cilastatin- relebactam	Yes	No	No	Yes	No	No	cUTI/AP, cIAI, HABP/VABP	Yes
Cefiderocol	Yes	Yes	Yes	Yes	Yes	Yes	cUTI/AP, HABP/ VABP	Yes
Plazomicin	Yes	Variableb	Yes	Variable	No	No	cUTI/AP	Yes
Eravacycline	Yes	Yes	Yes	No	Yes	Yes	cIAI	No
Fosfomycin	Yes	Yes	Yes	Variable	No	No	cUTI/AP	No
Abbreviations: A. baumannii, Acinetobacter baumannii; AP, acute pyelonephritis; cIAI, complicated intra-abdominal infection; cUTI, complicated urinary tract infection; HABP, hospital-acquired bacterial pneumonia; KPC, Klebsiella pneumoniae carbapenemase; NDM, New Delhi metallo-β-lactamase; NP, nosocomial pneumonia; OXA, oxacillinase; P. aeruginosa, Pseudomonas aeruginosa; S. maltophilia, Stenotrophomonas maltophilia; VABP, ventilator-associated bacterial pneumonia. a Not active beyond the activity of meropenem alone. b Frequently inactive against strains that produce NDM-type metallo-β-lactamases.

 

In our study, all results of BD Phoenix™ CPO detection panel, including antimicrobial susceptibility testing (AST), carbapenemase activity detection (Positive/Negative), and category of CPOs according to Ambler classes, are data critical in choosing antibiotics appropriate to treat CRO infection in clinical practice because they are data used for research and guide for antibiotic use appropriate to treat patients clinically infected with CREs (CRO). Thus, we kept all results of CREs categorized with the BD Phoenix CPO Detect Panel for Detection and Classification of Carbapenemase Producing Enterobacterales as evidence of detecting CREs (CRO) of the rapid and automated microbiology system of BD Phoenix based on NMIC 500-CPO Detect Panel of this system.

If you don't want us to show the results of CRE associated with the category system according to Ambler classes, we will remove these parts.

 

6) In Table 6 (on page 12) in the third column „Occu” is written. What do you mean by that?

 Response: Kim: Comment 6

In Table 6, we replace the word "Occupation" with its abbreviation "Occu" because there is not enough space for the total word of Occupation if it is written.

The abbreviation words are shown below Table 6

H : HCWs, Rsd : Resident, Cc: Child carer, Cln: Cleaner, FP: Food provider, Gd: Guard, Mgr of Ct: Manager of Center, Pharm: Pharmacist, RN: Registered nurse, No_OCCU: No occupation, Drt of PW (HCW)/ Rsd (Yrs): Duration of professional work (HCW)/ residents (Orphans) (years), 7 days For PW per W: 7 days for professional work/ work per week.

 

7) Tables are not in numerical order. Please, revise table numbering. There is no Table 1 in this manuscript, but Table 6 has a, b, c, d parts.  

 Response: Kim: Comment 7

Part 1 of comment 7:

We place Table 1 in the Supplementary Tables, and you can find this information in the section quoted as shown below

“All 20 healthcare workers (HCWs) and 67 residents, (including children) participated in the study. We observed and determined 303 environmental samples in the Center of Care and Protection of Orphaned Children. But we randomly chose some samples in the environment, based on the Yamane formula [29], was n = 175, , Where n = sample size required (n =175), N = the total number of environmental samples was 307 (N) observed and determined in the center of care and protection of orphan children, e = 0.05, allowable error (%).

            The number of randomly chosen environmental samples is present in Table 1 (Supplementary Tables)”

 

Part 2 of comment 7: Table 6 has 3 parts, including a,b,c,d and e parts 

 

In Table 6, we showed all characteristics related to demography, antibiotic use, treatment, and medical procedures performed on HCWs and residents to easily read, compare, and check for readers when they contact this information between two groups, including HCWs (N=20) and residents (N=67). In Table 6, we showed this table in 5 parts, including a,b,c,d, and e parts, as described above.

 

8) In this study whole-genome sequencing was performed but no carbapenemase genes are reported. I suggest to authors to add a table and list all detected carbapenemase genes.

 

Response: Kim: Comment 8

Firstly, thank you so much for pointing out the interesting notes.

Second, I don’t present the carbapenemase genes in this paper: 1) this paper is too long. So, it will be difficult for readers because they have to focus on many subjects in one paper, and 2) I want to present the results related to carbapenemase genes of 12 CRO samples in another paper to have “space“ to show, analyze, and discuss the results of carbapenemase genes of 12 CRO isolates. Especially the transmission of carbapenemase genes between CROs with the same or different species if CRO transmission was happening.

 

9) Clonal analysis of detected carbapenem resistant strains should be demonstrated. Based on WGS data sequence types (STs) of strains of this study should be shown in a table.

 

Response: Kim: Comment 9

 

As the reason described in the comment 8, I will perform the clonal analysis of detected carbapenem resistant strains in another paper. 

 

10) At materials and methods whole genome sequencing should be in one part, including description of work in Charles River laboratory. After this part should be „Statistical analysis” given.

 

Response: Kim: Comment 10

 

Firstly, thank you for pointing out these detailed notes. I followed your advice and changed the location of materials, methods, and whole genome sequencing of 12 CROs in part 2.5 in my manuscript and highlighted these pieces of information in yellow color.

 

Submission Date

08 December 2024

Date of this review

21 Dec 2024 20:46:18

 

I edited my manuscript as advised by reviewer_3 on 29 Dec 2024.

I improved English grammar and sentences in my manuscript on 31 Dec 2024

Today, I sent my manuscript to English Editing Services to help me improve my English manuscript.

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

changes are satisfactory, article can be accepted in current format

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript has been revised. All necessay modifications have been done in thet text.