Review for "Multifactorial Chromosomal Variants Regulate Polymyxin Resistance In Extensively Drug-Resistant Klebsiella pneumoniae"

Completed on 17 May 2017 by Willem Van Schaik . Sourced from http://biorxiv.org/content/early/2017/05/08/134684.

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This is an interesting whole-genome sequencing based study to identify mechanisms that contribute to colistin resistance in K. pneumoniae. Mutations in mgrB, phoPQ and pmrAB are identified and complemented to confirm their role in colistin resistance. The major weakness of this study is that the authors are limited in their choice of isolates: they do not have the susceptible counterpart of each resistant strains, so it is impossible to identify all SNPs and indels that have accumulated in the resistant strain. This limits the scope of the study as the authors now only study the ‘known knowns’ outlined above. It would be good if the authors include this limitation of their study in the discussion.

We thank you for your insightful response and assisting us with identifying several sections which could be written clearer. We have attempted to address your concerns in an updated preprint: https://www.biorxiv.org/content/early/2017/10/10/134684.
We understand that our selection of strains were scarce and that there were no paired isolates for example, samples collected before and after treatment or sampling a specific clinical outbreak. Although we present only the common pathways (MgrB, PhoPQ, PmrAB), further analysis was conducted to detect additional pathways altered especially those regarding LPS processing. As you mentioned, it is very difficult to call the contribution of these mutations towards resistance without adequate controls however, we found the common candidate genes were underpinning polymyxin resistance.
We acknowledge that this pathway of resistance has been abundantly established as mentioned thoroughly in our discussion, however, we have built on the current understanding of the regulation of this resistance. Within this small sample size, we have identified several interesting strains including two polymyxin resistant populations co-existing that have infected two patients, potential partial suppressor mutations which influence the MIC and variability which can arise in differencing sequence types (ST383, uncommonly reported) or species such as K. quasipneumoniae which do not contribute to resistance.

Some additional comments and suggestions are outlined below:

The abstract lacks quantitative data. l. 30 Please provide an exact number, l. 31. ‘most common’: provide number of strains.

The exact number impacted by an alteration in mgrB was 17 of the 19 polymyxin-resistant isolates and for the ISKpn26-like element, 9 of the 19 isolates harbored this change. To keep within word limits we have not updated the abstract, but this information is presented on line 205

The relevance of the ST2401 K. quasipneumoniae strain in the context of this study is unclear. It does not merit inclusion in the abstract, in my opinion.

This strain was initially characterized as colistin-susceptible K. pneumoniae but further analysis revealed this was a clinical K. quasipnemoniae colonizing in a patient with a previously unobserved sequence type. We have kept it in the abstract for now although as you say, it is not the major point of this study.

l. 49: better to write plasmid-encoded carbapenem resistance genes

This has now been modified (Line 65).

l. 54. The mortality associated with polymyxin-resistant Klebsiella infections seems awfully high. I believe the attributable mortality due to PMX-resistance is still not clear. See this interesting blog post: https://reflectionsipc.com/... for further insights on this topic.

We have added the following sentence to reflect the variability in mortality associated with polymxyin-resistance Klebsiella: "There is considerable debate regarding the mortality associated with polymyxin-resistant infections. Combining several clinical cohorts has provided an overall mortality estimate ranging from 20 to 100% which was dependent on early detection of the outbreak." which refers to a 2014 paper by Ah et al.

l. 58. I apologize for being a pedant, but the disturbance of the LPS leaflet of the outer membrane will not allow PMX to act on intracellular targets. For that to happen, the inner membrane needs to be disrupted as well.

We have reworded this line to emphasise the requirement for polymyxins to traverse the inner membrane as well on line 76: "This causes the displacement of Mg2+ and Ca2+, disrupting bacterial outer membrane integrity, allowing the polymyxins to traverse the inner membrane and act on intracellular targets."

In the discussion on mgrB it may be good to refer to Kidd et al., 2017. EMBO Mol Med who were the first to systematically study the role of this gene in K. pneumoniae.

We have now made note of this in the discussion (Line 297).

l. 67. Specify that mcr-1 confers colistin resistance. It may also be relevant to note that mcr-1 appears to be relatively rare in Klebsiella.

We understand that mcr-1 has predominantly been detected in E. coli with the rare instance of Klebsiella and have now addressed this (Line 86).

l. 97. ‘glycerol was added to 20% (v/v)’ may be a better way of phrasing this line

This is now updated, Line 101.

l. 107. I assume cation-adjusted Muller-Hinton broth was used? Please specify.

Apologies for the misunderstanding and we used cation-adjusted MHB (Line 111). We have further expanded our repertoire of antibiotics tested in this study to access the extent of resistance in these isolates (Table 1, page 29).

l. 130 – 132. I would really like to see a maximum-likelihood core genome tree here with additional reference isolates (downloadable from public databases), rather than a Neighbour-Joining tree of seven concatenated MLST alleles. It now is impossible to assess whether some of these strains (having the same ST) are truly clonally related.

A core genome MLST tree has now been constructed (Fig. 1. Page 33; Results, Line 188-203), using the Ridom SeqSphere+ v4.0.1 software.

l. 166. Incision should probably be replaced by introduction

Updated, Line 169.

l. 203. Provide exact number.

17 of the 19 polymyxin-resistant isolates has now been noted, Line 205.

l. 225. It is not immediately obvious what is meant by (65, 66% variant allele frequency)

The 65% refers to the phoQ (V446G) alteration and 66% is the pmrB (P158R) change which has now been reworded, Line 227-229.

l. 235 – 237. Is it also not a possibility that in these strains mgrB has reverted to its wild-type state by excision of the IS element?

This is an interesting point. It is possible that the excision of the IS element could be present in this sample to evoke susceptibility. To further test this, we selected 3 colonies which were reverted either by TOPO-mgrB or TOPO-pmrB. These colonies were passaged three times in non-supplemented LB broth to encourage the loss of this plasmid. After these 3 passages, consistently observed was an MIC of ≥64 mg/L for colonies reverted for by TOPO-mgrB and 16 mg/L for those via TOPO-pmrB. This identifies that the pmrB (P158R) variation was also causing polymyxin resistance in these isolates (Line 240-248).

l. 252 – 270. This section is difficult to follow. While some mutations are proposed to act as suppressors, it appears that experimental evidence cannot confirm this, so it may be better to rewrite this paragraph to reflect this key finding.

We understand that it is difficult to gain a definitive result on the suppressor mutations with complementation assays alone (Line 341). This would further require the removal of the phoPQ locus and then the introduction of phoPQ mutations (similar to assays conducted in Miller et al. 2011 (ref 51)). However, we witnessed a reduction in MIC especially for the phoP (P74L, A95S) alterations. When complemented into a background only possessing the mgrB IS element disruption, pTOPO-phoP complementation did not alter the initial MIC of ≥64 mg/L. Complementation of the mutated phoP resulted in a consistent MIC of 16 mg/L.

l. 275 – 276. I am not entirely sure that it is correct to single out Brazil and Greece here.

This sentence has been reworded, however, these locations have been pinpointed as having a relatively high prevalence of polymyxin resistance (Line 278; ref 10, Giamarellou H. 2016).

l. 292. I am not entirely sure whether this claim of primacy is relevant. Clearly, a truncation is a loss-of-function mutation and those have been complemented previously.

This has now been reworded (Line 293-300).