Co-occurrence of ST412 Klebsiella pneumoniae isolates with hypermucoviscous and non-mucoviscous phenotypes in a short-term hospitalized patient

ABSTRACT Hypermucoviscosity (HMV) is a phenotype that is commonly associated with hypervirulence in Klebsiella pneumoniae. The factors that contribute to the emergence of HMV subpopulations remain unclear. In this study, eight K. pneumoniae strains were recovered from an inpatient who had been hospitalized for 20 days. Three of the isolates exhibited a non-HMV phenotype, which was concomitant with higher biofilm formation than the other five HMV isolates. All eight isolates were highly susceptible to serum killing, albeit HMV strains were remarkably more infective than non-HMV counterparts in a mouse model of infection. Whole genome sequencing (WGS) showed that the eight isolates belonged to the K57-ST412 lineage. Average nucleotide identity (FastANIb) analysis indicated that eight isolates share 99.96% to 99.99% similarity and were confirmed to be the same clone. Through comparative genomics analysis, 12 non-synonymous mutations were found among these isolates, eight of which in the non-HMV variants, including rmpA (c.285delG) and wbaP (c.1305T > A), which are assumed to be associated with the non-HMV phenotype. Mutations in manB (c.1318G > A), dmsB (c.577C > T) and tkt (c.1928C > A) occurred in HMV isolates only. RNA-Seq revealed transcripts of genes involved in energy metabolism, carbohydrate metabolism and membrane transport, including cysP, cydA, narK, tktA, pduQ, aceB, metN, and lsrA, to be significantly dysregulated in the non-HMV strains, suggesting a contribution to HMV phenotype development. This study suggests that co-occurrence of HMV and non-HMV phenotypes in the same clonal population may be mediated by mutational mechanisms as well as by certain genes involved in membrane transport and central metabolism. IMPORTANCE K. pneumoniae with a hypermucoviscosity (HMV) phenotype is a community-acquired pathogen that is associated with increased invasiveness and pathogenicity, and underlying diseases are the most common comorbid risk factors inducing metastatic complications. HMV was earlier attributed to the overproduction of capsular polysaccharide, and more data point to the possibility of several causes contributing to this bacterial phenotype. Here, we describe a unique event in which the same clonal population showed both HMV and non-HMV characteristics. Studies have demonstrated that this process is influenced by mutational processes and genes related to transport and central metabolism. These findings provide fresh insight into the mechanisms behind co-occurrence of HMV and non-HMV phenotypes in monoclonal populations as well as potentially being critical in developing strategies to control the further spread of HMV K. pneumoniae.

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Sincerely, Karoline Faust Editor mSystems
Reviewer #1 (Comments for the Author): 1.The qRT-PCR data may be easier to interpret if the y-axis is on a log2 scale with the x-axis crossing at 0 (i.e.decreased transcripts will drop below the x-axis).
2. Line 126 -"non-HMV isolates did not exceed 0.25" is not true as one value is 0.4.May need to revise to state that the mean was 0.25 (range = 0.1-0.4).
4. Lines 283-287 -The authors state that "rmpA is associated with higher biofilm formation ... and wbaP can lead to increased biofilm formation.Therefore, we speculate that this might contribute to the distinct phenotypes exhibited by K201054 as opposed to K201055 and K201060."The referenced phenotypes of rmpA and wbaP on biofilm formation do not logically lead to the authors conclustion.Can the authors please clarify?
In my opinion, I think that what is observed is that K201055 and K201060 are acapsular, which means they are non-mucoid too.However, K201054 is not acapsular, but is non-mucoid (due to lack of RmpD expression), so the phenotypes attributable to capsule abundance persist, but phenotypes (string test) attributable to mucoidy (ie capsule chain length) are absent.
5. Line 300 -Klebsiella uses a Wzy-dependent capsule biosynthesis pathway.It is not the ABC transporter pathway for capsule biosynthesis, which is distinct from the Wzy pathway.PMID: 32680453 6. Please add lines 35-37 of response to reviewers to methods and/or results.
7. Lines 69-70 of response to reviewers -Thank you for including your GO and KEGG analyses.It appears these are located in Table S2, which is not referenced anywhere in the manuscript text.Please reference Table S2 in the text.
Reviewer #2 (Comments for the Author): This manuscript (mSystems 00262-24) is in its third revision and is continuing to improve.I have a few additional comments for consideration.
• Several references noted were mis-formatted (e.g. 10, 5, 6).I'm seeing this frequently and believe reference programs are to blame.Careful inspection is warranted to make sure all are correct.• Line 158, the higher levels of colonization by HMV strains at 24 hours is suggestive of increased virulence, but not indicative.Sustained colonization over time or evidence of illness/disease would be needed to for use of the more conclusive term "indicative".
• Gene and protein nomenclature is consistently inaccurate regarding the mutations the authors identified.Genes do not have amino acid substitutions, but instead encode them.Some examples are lines 183,198,272,, a mutation in rmpA is described as deletion of a single nucleotide, and an easy assumption is that this led to a premature stop codon.But in Lines 272-273, this mutation is described as R96G (again mixing up gene and protein nomenclature).At the very least, the description of this mutation should be consistent in both locations to avoid confusion.
• Line 216-218, the description of ManB as a "polymerizing signal transduction for envelop biosynthesis" does not make sense with what I understand the function of this enzyme to be, which is the conversion between mannose-1-phosphate and mannose-6-phosphate.This might warrant clarification, particularly as the phrase quoted above does not make sense.
• Line 282, shouldn't these mutations lead to better sedimentation?
• Lines 300-307, the link to ABC-type CPS export is illogical here.Klebsiella capsules are exported by Wzy type complexes.The differences in transporter activity between HMV and non-HMV isolates likely has nothing to do with CPS.
• The HMV strains appear to contain genes encoding additional siderophores (Line 182).It would be appropriate to explain why these strains showed smaller zones of orange in the CAS assay.Are there caveats to this assay that could explain this presumed contradiction?
• In the response to reviewers, there is frequent mention of a modified CPS quantification method.I noted one step that deviated from established protocols (resuspension of the polysaccharide pellet in 100 mM HCl).The authors should explain why this was done, how it improves the protocol, and reference/acknowledge the source if applicable.

Dear Editors and Reviewers,
Thank you for your letter and for the reviewers' comments concerning our manuscript entitled "mSystems00262-24".The constructive criticism of Editor and Reviewers has been valuable and very helpful for revising and improving our paper, as well as for improving the significance of our research.We have studied comments carefully and have made corrections which we hope meet with approval.

Responds to the editor:
The reviewers have now assessed the revised work.Below you will find their comments as well as instructions from the mSystems editorial office.In general, please carefully check English spelling and grammar as reviewers still pointed out problems with English.Please return the manuscript within 60 days.If you do not wish to modify the manuscript and prefer to submit it to another journal, notify me immediately so that the manuscript may be formally withdrawn from consideration by mSystems.
Response: Thanks to the editor for the comments.We are very sorry for the mistakes in this manuscript and the inconvenience caused to the reviewers.We have invited native speakers to check our English spelling and grammar, and have revised the manuscript accordingly, so we expect it to meet the journal's standards.

Reviewer #1 (Comments for the Author):
1.The qRT-PCR data may be easier to interpret if the y-axis is on a log2 scale with the x-axis crossing at 0 (i.e.decreased transcripts will drop below the x-axis).
Response: Thanks for the reviewer's suggestion, which will greatly improve the quality of our manuscripts.Based on the reviewer's advice, we have placed the decreased transcripts below the X-axis in Figure 5E.
2. Line 126 -"non-HMV isolates did not exceed 0.25" is not true as one value is 0.4.May need to revise to state that the mean was 0.25 (range = 0.1-0.4).
Response: Thanks to the reviewer for observation, we are sorry for the lack of rigor.We have corrected the statement from "non-HMV isolates did not exceed 0.25" to "the mean value of non-HMV isolates did not exceed 0.25" (line 128).S4 is compelling and moving it to the main figures should be considered.

Figure
Response: We think this is an excellent suggestion.We have moved Figure S4  Response: Thanks for the reviewer's valuable advice, we apologise for the error.We looked at the references and found that both rmpA and wbaP are associated with biofilm formation.Absence of rmpA has been associated with higher biofilm formation capacity; similarly, mutations in wbaP can lead to increased biofilm formation ability.Therefore, we hypothesize that the mutations in rmpA and wbaP could be factors influencing the difference in biofilm formation between K201054 and K201055 and K201060 (line287-289).
As stated in lines 274-284, we speculate that rmpA mutations are responsible for the non-HMV phenotype of strain K201054 and also result in better natural sedimentation, which is consistent with evidence that rmpA is associated with the HMV phenotype of Klebsiella pneumoniae.The mutation in wbaP resulted in better natural sedimentation and reduced the overproduction of capsule polysaccharide in the non-HMV strains K201055 and K201060.The reduction in CPS corresponding to a non-HMV appearance on the plates.This helps explain the differences in natural sedimentation and capsular polysaccharide of strain K201054 compared to K201055 and K201060 (line274-284).Response: Thank you for pointing out this problem in the manuscript.We read this reference carefully and learned that Klebsiella synthesizes capsules via the Wzydependent pathway.We have now made the necessary corrections.In addition, in our transcriptomic study comparing HMV with non-HMV isolates, certain dysregulated genes such as cysU and cysP had been annotated to functions associated to both membrane transport and energy metabolism.And we have added that to the manuscript (line299-302).
6. Please add lines 35-37 of response to reviewers to methods and/or results.
Response: We gratefully appreciate your valuable suggestion.As suggested by the reviewers, we have added sample volume and heat-inactivated serum as negative controls to the serum resistance assay (line376-377).S2, which is not referenced anywhere in the manuscript text.Please reference Table S2 in the text.
Response: Thank you for your observation.In our revised manuscript, we have added

•
Fig 1 & 2, color scheme for HMV and Non-HMV isolates is reversed.Please be consistent.• Fig 2, why CFU per 0.1g tissue?As with the sedimentation assay, it is unclear why these authors are choosing to perform and present their data in unconventional ways.Will make it difficult to compare with other studies.[this doesn't really need to be addressed, but something for the authors to consider going forward].• Fig 3C (serum survival), as with the other panels, please indicate which strains are HMV vs Non-HMV.
to the main manuscript and renamed it Figure 4.In addition, we have revised the sentence "Introduction of the native rmpA and wbaP into the corresponding non-HMV strains restored the HMV phenotype" to "Introduction of native rmpA into non-HMV strain K201054 and wbaP into non-HMV strains K201055 and K201060 restored their HMV phenotypes" (line204-206).4. Lines 283-287 -The authors state that "rmpA is associated with higher biofilm formation ... and wbaP can lead to increased biofilm formation.Therefore, we speculate that this might contribute to the distinct phenotypes exhibited by K201054 as opposed to K201055 and K201060."The referenced phenotypes of rmpA and wbaP on biofilm formation do not logically lead to the authors conclustion.Can the authors please clarify?In my opinion, I think that what is observed is that K201055 and K201060 are acapsular, which means they are non-mucoid too.However, K201054 is not acapsular, but is non-mucoid (due to lack of RmpD expression), so the phenotypes attributable to capsule abundance persist, but phenotypes (string test) attributable to mucoidy (ie capsule chain length) are absent.

5.
Line 300 -Klebsiella uses a Wzy-dependent capsule biosynthesis pathway.It is not the ABC transporter pathway for capsule biosynthesis, which is distinct from the Wzy pathway.PMID: 32680453IF: 10.5 Q1

7.
Lines 69-70 of response to reviewers -Thank you for including your GO and KEGG analyses.It appears these are located in Table