Multilocus Sequence Typing of Aggregatibacter actinomycetemcomitans Competently Depicts the Population Structure of the Species

ABSTRACT We developed a multilocus sequence typing scheme (MLST) for Aggregatibacter actinomycetemcomitans based on seven housekeeping genes, adk, atpG, frdB, mdh, pgi, recA, and zwf. A total of 188 strains of seven serotypes were separated into 57 sequence types. Whole-genome sequences were available for 140 strains, and in contrast to comparison of 16S rRNA genes, phylogenetic analysis of concatenated MLST gene fragments was in accordance with the population structure revealed by alignment of 785 core genes. MLST could not decisively identify the so-called JP2 clone associated with rapidly progressing periodontitis in adolescents, but noticeable clustering of JP2 genotype strains was revealed. The MLST scheme of A. actinomycetemcomitans can be assessed at www.pubmlst.org. IMPORTANCE Accurate diagnosis of infectious disease comprise identification, typing, and antimicrobial resistance of the infective agent. Bacteria are sometimes grouped within their species according to expression of specific toxins or particular antimicrobial resistance traits, but explicit typing for infection control and survey of pathogenesis necessitates genetic analysis such as multilocus sequence typing (MLST). Schemes for the most prevalent human pathogens have been available for more than 10 years, and time has come to extend the scrutiny to second-line infectious agents. One such pathogen is Aggregatibacter actinomycetemcomitans, which is commonly involved in periodontitis, and more rarely as the cause of infective endocarditis or spontaneous brain abscess. A MLST scheme for A. actinomycetemcomitans is now available at www.pubmlst.org. Whole-genome sequencing of a large number of isolates confirms that MLST competently depicts the population structure of the species.


Major comments
Author tried to show the MLST analysis based on seven housekeeping genes, adk, atpG, frdB mdh, pgi, recA, and zwf, and/or 785 concatenated core genes had the advantage in the study of epidemiology and population structures of Aggregatibacter actinomycetemcomitans. In Figure 2, I think that the MLST analysis could extract the clade associated with periodontal disease pathogenic population, JP2 strains. In this point, I agree with the opinion of the author and this article is meaningful in the field of clinical periodontology. On the other hand, in Figure 3, why is not the NJ phylogenetic tree based on seven housekeeping genes provided? I think that the NJ tree based on 785 concatenated core genes could well divided the populations associated with diseases, such as periodontitis and bacteremia. If the NJ tree based on seven housekeeping genes could also divided those populations, it would be more meaningful as the rapid and reliable diagnostic equipment. Thus, I request that the NJ tree based on seven housekeeping genes is provided in Figure 3.
10. Page 10, line194-198 This sentence is too long and complicated. Please check.

Reviewer #2 (Comments for the Author):
This manuscript describes the development of a seven allele MLST typing scheme for Aggregatibacter actinomycetemcomitans and its application to elucidate the population structure of this organism. The MLST scheme is based on previous research that established a six allele protocol and its novelty is based on the addition of a seventh allele and the application of the new scheme. There are two main issues with the manuscript.
1. On line 89 the authors state their zwf primers "were able to amplify fragments of the expected size from 232 isolates of serotypes a to f included in a previous investigation [23]...". The referenced study included 257 isolates. The authors need to make clear whether they amplified the gene from 232 out of 232 isolates or if their method failed to amplify the gene from any isolates. If only 232 samples were tested why were these samples selected? If the primers failed to amplify zwf from any members of the species its use in an MLST protocol is in doubt and would need to be defended. 2. The justification for developing the new MLST protocol is that the previous one only used alleles from six housekeeping genes and was not widely accepted. Whereas the use of seven alleles is common there is no absolute requirement for this number. The difference in resolving the population structure of A. actinomycetemcomitans using the different schemes, for example as shown in Figure 1, should be compared and included.

Minor points:
The exact zwf allele used for MLST analysis should be identified. Presumably it would be trimmed to not include primer sites.
The genome location of the zwf gene relative to the other genes used in the MLST scheme should be provided.

Preparing Revision Guidelines
To submit your modified manuscript, log onto the eJP submission site at https://spectrum.msubmit.net/cgi-bin/main.plex. Go to Author Tasks and click the appropriate manuscript title to begin the revision process. The information that you entered when you first submitted the paper will be displayed. Please update the information as necessary. Here are a few examples of required updates that authors must address: • Point-by-point responses to the issues raised by the reviewers in a file named "Response to Reviewers," NOT IN YOUR COVER LETTER. • Upload a compare copy of the manuscript (without figures) as a "Marked-Up Manuscript" file. • Each figure must be uploaded as a separate file, and any multipanel figures must be assembled into one file. • Manuscript: A .DOC version of the revised manuscript • Figures: Editable, high-resolution, individual figure files are required at revision, TIFF or EPS files are preferred For complete guidelines on revision requirements, please see the journal Submission and Review Process requirements at https://journals.asm.org/journal/Spectrum/submission-review-process. Submissions of a paper that does not conform to Microbiology Spectrum guidelines will delay acceptance of your manuscript. " Please return the manuscript within 60 days; if you cannot complete the modification within this time period, please contact me. If you do not wish to modify the manuscript and prefer to submit it to another journal, please notify me of your decision immediately so that the manuscript may be formally withdrawn from consideration by Microbiology Spectrum.
If you would like to submit an image for consideration as the Featured Image for an issue, please contact Spectrum staff.
If your manuscript is accepted for publication, you will be contacted separately about payment when the proofs are issued; please follow the instructions in that e-mail. Arrangements for payment must be made before your article is published. For a complete list of Publication Fees, including supplemental material costs, please visit our website.
Corresponding authors may join or renew ASM membership to obtain discounts on publication fees. Need to upgrade your membership level? Please contact Customer Service at Service@asmusa.org.
Thank you for submitting your paper to Microbiology Spectrum.
This manuscript describes the development of a seven allele MLST typing scheme for Aggregatibacter actinomycetemcomitans and its application to elucidate the population structure of this organism. The MLST scheme is based on previous research that established a six allele protocol and its novelty is based on the addition of a seventh allele and the application of the new scheme. There are two main issues with the manuscript.
1. On line 89 the authors state their zwf primers "were able to amplify fragments of the expected size from 232 isolates of serotypes a to f included in a previous investigation [23]…". The referenced study included 257 isolates. The authors need to make clear whether they amplified the gene from 232 out of 232 isolates or if their method failed to amplify the gene from any isolates. If only 232 samples were tested why were these samples selected? If the primers failed to amplify zwf from any members of the species its use in an MLST protocol is in doubt and would need to be defended. 2. The justification for developing the new MLST protocol is that the previous one only used alleles from six housekeeping genes and was not widely accepted. Whereas the use of seven alleles is common there is no absolute requirement for this number. The difference in resolving the population structure of A. actinomycetemcomitans using the different schemes, for example as shown in Figure 1, should be compared and included.
Minor points: The exact zwf allele used for MLST analysis should be identified. Presumably it would be trimmed to not include primer sites.
The genome location of the zwf gene relative to the other genes used in the MLST scheme should be provided.

Reviewer #1 (Comments for the Author):
Major comments Author tried to show the MLST analysis based on seven housekeeping genes, adk, atpG, frdB mdh, pgi, recA, and zwf, and/or 785 concatenated core genes had the advantage in the study of epidemiology and population structures of Aggregatibacter actinomycetemcomitans. In Figure 2, I think that the MLST analysis could extract the clade associated with periodontal disease pathogenic population, JP2 strains. In this point, I agree with the opinion of the author and this article is meaningful in the field of clinical periodontology. On the other hand, in Figure 3, why is not the NJ phylogenetic tree based on seven housekeeping genes provided? I think that the NJ tree based on 785 concatenated core genes could well divided the populations associated with diseases, such as periodontitis and bacteremia. If the NJ tree based on seven housekeeping genes could also divided those populations, it would be more meaningful as the rapid and reliable diagnostic equipment. Thus, I request that the NJ tree based on seven housekeeping genes is provided in Figure 3.
Reply: Figure 3 has been updated to include a NJ tree based on seven housekeeping genes. "30 cycles of 94{degree sign}C-1 min/60{degree sign}C-1 min/72{degree sign}C-2 min" is incomprehensible. "30 cycles of 94{degree sign}C for 1 min, 60{degree sign}C for 1 min and 72{degree sign}C for 2 min" or "30 cycles of 1 min at 94{degree sign}C, 1 min at 60{degree sign}C and 1 min at 72{degree sign}C" seems to be comprehensible.

Prof. Niels Nørskov-Lauritsen Odense University Hospital Clinical Microbiology Odense C Denmark
Re: Spectrum01085-21R1 (Multilocus sequence typing of Aggregatibacter actinomycetemcomitans competently depicts the population structure of the species) Dear Prof. Niels Nørskov-Lauritsen: Your manuscript has been accepted, and I am forwarding it to the ASM Journals Department for publication. You will be notified when your proofs are ready to be viewed.
The ASM Journals program strives for constant improvement in our submission and publication process. Please tell us how we can improve your experience by taking this quick Author Survey.
As an open-access publication, Spectrum receives no financial support from paid subscriptions and depends on authors' prompt payment of publication fees as soon as their articles are accepted. You will be contacted separately about payment when the proofs are issued; please follow the instructions in that e-mail. Arrangements for payment must be made before your article is published. For a complete list of Publication Fees, including supplemental material costs, please visit our website.