Reviewer’s report
Title:  The analysis of virulence factors and antibiotic resistance between Helicobacter pylori strains  
isolated from gastric antrum and body
Version: 0 Date: 10 Mar 2019
Reviewer: Wei-Lun Chang
Reviewer's report:
This study reported the differences of virulence factors and antibiotic resistance patterns of H. pylori  
strains isolated from different locations of the stomach in the same patient (i.e. co-infection with  
different strains). They found 10 co-infections out of 80 infected patients. Of the 10 co-infections, 7  
had different antibiotic resistance pattern, and 5 had different virulence factors.  They concluded that  
co-infection  could  undermine  the  success  of  eradication  therapy and  should  be considered  when  
interpreting the results of antimicrobial susceptibility tests. The study addressed an important question  
of H. pylori infection. However, it is better to include the single-infection group for comparison to  
know the true impact of co-infection. 
Major comments:
1. To understand the clinical impacts of co-infection and its difference from single infection, it is better  
to include the data of patients with single infection as the "control group". With both co-infection group  
and single-infection group, the author can answer the following question: 
2. Did the co-infection group have higher prevalence of virulent strain than the single-infection group?  
Such as EPIYA motif and vacA subtype, oipA on/off status…etc (Helicobacter. 2015 Oct;20(5):397-
404).
3. Did the co-infection group have higher prevalence of precancerous lesion (atrophy and intestinal  
metaplasia) or higher OLGIM (operative link on gastric intestinal metaplasia) and OLGA (operative  
link on gastric atrophy) score than the single-infection group?
4. Did the co-infection group have higher antibiotic MICs or antibiotic resistance rate than the single-
infection group? 
Minor comments:
1. In table 2, the definition of activity was not provided.
2. In table 2, only antrum was given the data of intestinal metaplasia. How about the body?
3. In table 3, not everyone is familiar with oipA on and off status. The expression of oipA on and off  
status is not clear, please modify or explain in the footnote.  
4. In table 4, although the antibiotic MIC of each isolate was provided, it is suggested to add antibiotic  
susceptibility of each isolate (either sensitive or resistant according to the breakpoint). This can help  
readers identify the co-infectors with different antibiotic susceptibility. 
5. According to the recommendations by the Clinical & Laboratory Standards Institute (CLSI) and the  
European Committee on Antimicrobial Susceptibility Testing (EUCAST), the susceptibility breakpoints  
for amoxicillin, clarithromycin and tetracycline are ≤0.125, ≤0.25 and ≤1 mg/L, respectively. The  
author should use these breakpoints to report their data (Table 4). The antibiotic resistance pattern will  
be different from their original data if the correct breakpoint is used. 
6. The discussion should be more focused on the data provided, such as the cause of different co-
infection rate in different regions (Infect Genet Evol. 2016 Jan;37:43-8) and the impact of co-infection  
in H. pylori eradication and carcinogenic effect (Clin Microbiol Infect. 2009 Mar;15(3):253-9).
7. Not only co-infection patients can have different antibiotic profile. Actually, previous study showed  
isolates from different gastric sites of the same patient can have similar PCR-RAPD profiles, but differ  
in  their  antibiotic  profiles  (Malays  J  Pathol.  2009  Jun;  31(1):29-34).  Therefore,  the  etiology  
heteroresistant  antibacterial  phenotypes  is  complex  and  should  be  discussed  (Helicobacter.  2005  
Jun;10(3):240-8). 
8. The author may want to suggest a strategy of H. pylori culture in clinical practice. Should we obtain  
specimen from different gastric sites and culture them separately?
Are the methods appropriate and well described?
If not, please specify what is required in your comments to the authors.
Yes
Does the work include the necessary controls?
If not, please specify which controls are required in your comments to the authors.
No
Are the conclusions drawn adequately supported by the data shown?
If not, please explain in your comments to the authors.
No
Are you able to assess any statistics in the manuscript or would you recommend an additional  
statistical review?
If  an  additional  statistical  review  is  recommended,  please  specify  what  aspects  require  further  
assessment in your comments to the editors.
I am able to assess the statistics
Quality of written English
Please indicate the quality of language in the manuscript:
Needs some language corrections before being published
Declaration of competing interests
Please complete a declaration of competing interests, considering the following questions:
1. Have you in the past five years received reimbursements, fees, funding, or salary from an
organisation that may in any way gain or lose financially from the publication of this
manuscript, either now or in the future?
2. Do you hold any stocks or shares in an organisation that may in any way gain or lose
financially from the publication of this manuscript, either now or in the future?
3. Do you hold or are you currently applying for any patents relating to the content of the
manuscript?
4. Have you received reimbursements, fees, funding, or salary from an organization that holds
or has applied for patents relating to the content of the manuscript?
5. Do you have any other financial competing interests?
6. Do you have any non-financial competing interests in relation to this paper?
If you can answer no to all of the above, write 'I declare that I have no competing interests' below. If  
your reply is yes to any, please give details below.
I declare that I have no competing interests.
I agree to the open peer review policy of the journal. I understand that my name will be included on my  
report to the authors and, if the manuscript is accepted for publication, my named report including any  
attachments I upload will be posted on the website along with the authors' responses. I agree for my  
report to be made available under an Open Access Creative Commons CC-BY license  
(http://creativecommons.org/licenses/by/4.0/ ). I understand that any comments which I do not wish to  
be included in my named report can be included as confidential comments to the editors, which will not  
be published.
I agree to the open peer review policy of the journal
Reviewer’s report
Title:  The analysis of virulence factors and antibiotic resistance between Helicobacter pylori strains  
isolated from gastric antrum and body
Version: 0 Date: 10 Mar 2019
Reviewer: Wei-Lun Chang
Reviewer's report:
This study reported the differences of virulence factors and antibiotic resistance patterns of H. pylori  
strains isolated from different locations of the stomach in the same patient (i.e. co-infection with  
different strains). They found 10 co-infections out of 80 infected patients. Of the 10 co-infections, 7  
had different antibiotic resistance pattern, and 5 had different virulence factors.  They concluded that  
co-infection  could  undermine  the  success  of  eradication  therapy and  should  be considered  when  
interpreting the results of antimicrobial susceptibility tests. The study addressed an important question  
of H. pylori infection. However, it is better to include the single-infection group for comparison to  
know the true impact of co-infection. 
Major comments:
1. To understand the clinical impacts of co-infection and its difference from single infection, it is better  
to include the data of patients with single infection as the "control group". With both co-infection group  
and single-infection group, the author can answer the following question: 
2. Did the co-infection group have higher prevalence of virulent strain than the single-infection group?  
Such as EPIYA motif and vacA subtype, oipA on/off status…etc (Helicobacter. 2015 Oct;20(5):397-
404).
3. Did the co-infection group have higher prevalence of precancerous lesion (atrophy and intestinal  
metaplasia) or higher OLGIM (operative link on gastric intestinal metaplasia) and OLGA (operative  
link on gastric atrophy) score than the single-infection group?
4. Did the co-infection group have higher antibiotic MICs or antibiotic resistance rate than the single-
infection group? 
Minor comments:
1. In table 2, the definition of activity was not provided.
2. In table 2, only antrum was given the data of intestinal metaplasia. How about the body?
3. In table 3, not everyone is familiar with oipA on and off status. The expression of oipA on and off  
status is not clear, please modify or explain in the footnote.  
4. In table 4, although the antibiotic MIC of each isolate was provided, it is suggested to add antibiotic  
susceptibility of each isolate (either sensitive or resistant according to the breakpoint). This can help  
readers identify the co-infectors with different antibiotic susceptibility. 
5. According to the recommendations by the Clinical & Laboratory Standards Institute (CLSI) and the  
European Committee on Antimicrobial Susceptibility Testing (EUCAST), the susceptibility breakpoints  
for amoxicillin, clarithromycin and tetracycline are ≤0.125, ≤0.25 and ≤1 mg/L, respectively. The  
author should use these breakpoints to report their data (Table 4). The antibiotic resistance pattern will  
be different from their original data if the correct breakpoint is used. 
6. The discussion should be more focused on the data provided, such as the cause of different co-
infection rate in different regions (Infect Genet Evol. 2016 Jan;37:43-8) and the impact of co-infection  
in H. pylori eradication and carcinogenic effect (Clin Microbiol Infect. 2009 Mar;15(3):253-9).
7. Not only co-infection patients can have different antibiotic profile. Actually, previous study showed  
isolates from different gastric sites of the same patient can have similar PCR-RAPD profiles, but differ  
in  their  antibiotic  profiles  (Malays  J  Pathol.  2009  Jun;  31(1):29-34).  Therefore,  the  etiology  
heteroresistant  antibacterial  phenotypes  is  complex  and  should  be  discussed  (Helicobacter.  2005  
Jun;10(3):240-8). 
8. The author may want to suggest a strategy of H. pylori culture in clinical practice. Should we obtain  
specimen from different gastric sites and culture them separately?
Are the methods appropriate and well described?
If not, please specify what is required in your comments to the authors.
Yes
Does the work include the necessary controls?
If not, please specify which controls are required in your comments to the authors.
No
Are the conclusions drawn adequately supported by the data shown?
If not, please explain in your comments to the authors.
No
Are you able to assess any statistics in the manuscript or would you recommend an additional  
statistical review?
If  an  additional  statistical  review  is  recommended,  please  specify  what  aspects  require  further  
assessment in your comments to the editors.
I am able to assess the statistics
Quality of written English
Please indicate the quality of language in the manuscript:
Needs some language corrections before being published
Declaration of competing interests
Please complete a declaration of competing interests, considering the following questions:
1. Have you in the past five years received reimbursements, fees, funding, or salary from an
organisation that may in any way gain or lose financially from the publication of this
manuscript, either now or in the future?
2. Do you hold any stocks or shares in an organisation that may in any way gain or lose
financially from the publication of this manuscript, either now or in the future?
3. Do you hold or are you currently applying for any patents relating to the content of the
manuscript?
4. Have you received reimbursements, fees, funding, or salary from an organization that holds
or has applied for patents relating to the content of the manuscript?
5. Do you have any other financial competing interests?
6. Do you have any non-financial competing interests in relation to this paper?
If you can answer no to all of the above, write 'I declare that I have no competing interests' below. If  
your reply is yes to any, please give details below.
I declare that I have no competing interests.
I agree to the open peer review policy of the journal. I understand that my name will be included on my  
report to the authors and, if the manuscript is accepted for publication, my named report including any  
attachments I upload will be posted on the website along with the authors' responses. I agree for my  
report to be made available under an Open Access Creative Commons CC-BY license  
(http://creativecommons.org/licenses/by/4.0/ ). I understand that any comments which I do not wish to  
be included in my named report can be included as confidential comments to the editors, which will not  
be published.
I agree to the open peer review policy of the journal
Please review this paper.
user
Reviewer’s report
Title:  The analysis of virulence factors and antibiotic resistance between Helicobacter pylori strains  
isolated from gastric antrum and body
Version: 0 Date: 10 Mar 2019
Reviewer: Wei-Lun Chang
Reviewer's report:
This study reported the differences of virulence factors and antibiotic resistance patterns of H. pylori  
strains isolated from different locations of the stomach in the same patient (i.e. co-infection with  
different strains). They found 10 co-infections out of 80 infected patients. Of the 10 co-infections, 7  
had different antibiotic resistance pattern, and 5 had different virulence factors.  They concluded that  
co-infection  could  undermine  the  success  of  eradication  therapy and  should  be considered  when  
interpreting the results of antimicrobial susceptibility tests. The study addressed an important question  
of H. pylori infection. However, it is better to include the single-infection group for comparison to  
know the true impact of co-infection. 
Major comments:
1. To understand the clinical impacts of co-infection and its difference from single infection, it is better  
to include the data of patients with single infection as the "control group". With both co-infection group  
and single-infection group, the author can answer the following question: 
2. Did the co-infection group have higher prevalence of virulent strain than the single-infection group?  
Such as EPIYA motif and vacA subtype, oipA on/off status…etc (Helicobacter. 2015 Oct;20(5):397-
404).
3. Did the co-infection group have higher prevalence of precancerous lesion (atrophy and intestinal  
metaplasia) or higher OLGIM (operative link on gastric intestinal metaplasia) and OLGA (operative  
link on gastric atrophy) score than the single-infection group?
4. Did the co-infection group have higher antibiotic MICs or antibiotic resistance rate than the single-
infection group? 
Minor comments:
1. In table 2, the definition of activity was not provided.
2. In table 2, only antrum was given the data of intestinal metaplasia. How about the body?
3. In table 3, not everyone is familiar with oipA on and off status. The expression of oipA on and off  
status is not clear, please modify or explain in the footnote.  
4. In table 4, although the antibiotic MIC of each isolate was provided, it is suggested to add antibiotic  
susceptibility of each isolate (either sensitive or resistant according to the breakpoint). This can help  
readers identify the co-infectors with different antibiotic susceptibility. 
5. According to the recommendations by the Clinical & Laboratory Standards Institute (CLSI) and the  
European Committee on Antimicrobial Susceptibility Testing (EUCAST), the susceptibility breakpoints  
for amoxicillin, clarithromycin and tetracycline are ≤0.125, ≤0.25 and ≤1 mg/L, respectively. The  
author should use these breakpoints to report their data (Table 4). The antibiotic resistance pattern will  
be different from their original data if the correct breakpoint is used. 
6. The discussion should be more focused on the data provided, such as the cause of different co-
infection rate in different regions (Infect Genet Evol. 2016 Jan;37:43-8) and the impact of co-infection  
in H. pylori eradication and carcinogenic effect (Clin Microbiol Infect. 2009 Mar;15(3):253-9).
7. Not only co-infection patients can have different antibiotic profile. Actually, previous study showed  
isolates from different gastric sites of the same patient can have similar PCR-RAPD profiles, but differ  
in  their  antibiotic  profiles  (Malays  J  Pathol.  2009  Jun;  31(1):29-34).  Therefore,  the  etiology  
heteroresistant  antibacterial  phenotypes  is  complex  and  should  be  discussed  (Helicobacter.  2005  
Jun;10(3):240-8). 
8. The author may want to suggest a strategy of H. pylori culture in clinical practice. Should we obtain  
specimen from different gastric sites and culture them separately?
Are the methods appropriate and well described?
If not, please specify what is required in your comments to the authors.
Yes
Does the work include the necessary controls?
If not, please specify which controls are required in your comments to the authors.
No
Are the conclusions drawn adequately supported by the data shown?
If not, please explain in your comments to the authors.
No
Are you able to assess any statistics in the manuscript or would you recommend an additional  
statistical review?
If  an  additional  statistical  review  is  recommended,  please  specify  what  aspects  require  further  
assessment in your comments to the editors.
I am able to assess the statistics
Quality of written English
Please indicate the quality of language in the manuscript:
Needs some language corrections before being published
Declaration of competing interests
Please complete a declaration of competing interests, considering the following questions:
1. Have you in the past five years received reimbursements, fees, funding, or salary from an
organisation that may in any way gain or lose financially from the publication of this
manuscript, either now or in the future?
2. Do you hold any stocks or shares in an organisation that may in any way gain or lose
financially from the publication of this manuscript, either now or in the future?
3. Do you hold or are you currently applying for any patents relating to the content of the
manuscript?
4. Have you received reimbursements, fees, funding, or salary from an organization that holds
or has applied for patents relating to the content of the manuscript?
5. Do you have any other financial competing interests?
6. Do you have any non-financial competing interests in relation to this paper?
If you can answer no to all of the above, write 'I declare that I have no competing interests' below. If  
your reply is yes to any, please give details below.
I declare that I have no competing interests.
I agree to the open peer review policy of the journal. I understand that my name will be included on my  
report to the authors and, if the manuscript is accepted for publication, my named report including any  
attachments I upload will be posted on the website along with the authors' responses. I agree for my  
report to be made available under an Open Access Creative Commons CC-BY license  
(http://creativecommons.org/licenses/by/4.0/ ). I understand that any comments which I do not wish to  
be included in my named report can be included as confidential comments to the editors, which will not  
be published.
I agree to the open peer review policy of the journal
assistant