Emergence of Ceftazidime- and Avibactam-Resistant Klebsiella pneumoniae Carbapenemase-Producing Pseudomonas aeruginosa in China

ABSTRACT Klebsiella pneumoniae carbapenemase (KPC)-producing Pseudomonas aeruginosa (KPC-PA) has been reported sporadically. However, epidemiological and antimicrobial susceptibility data specific for KPC-PA are lacking. We collected 374 carbapenem-resistant P. aeruginosa (CRPA) isolates from seven hospitals in China from June 2016 to February 2019 and identified the blaKPC-2 gene in 40.4% (n = 151/374) of the isolates. Approximately one-half of all KPC-PA isolates (n = 76/151; 50.3%) were resistant to ceftazidime-avibactam (CAZ-AVI). Combining Kraken2 taxonomy identification and Nanopore sequencing, we identified eight plasmid types, five of which carried blaKPC-2, and 13 combination patterns of these plasmid types. In addition, we identified IS26-ΔTn6296 and Tn1403-like–ΔTn6296 as the two mobile genetic elements that mediated blaKPC-2 transmission. blaKPC-2 plasmid curing in 28 strains restored CAZ-AVI susceptibility, suggesting that blaKPC-2 was the mediator of CAZ-AVI resistance. Furthermore, the blaKPC-2 copy number was found to correlate with KPC expression and, therefore, CAZ-AVI resistance. Taken together, our results suggest that KPC-PA is becoming a clinical threat and that using CAZ-AVI to treat this specific pathogen should be done with caution. IMPORTANCE Previous research has reported several cases of KPC-PA strains and three KPC-encoding P. aeruginosa plasmid types in China. However, the prevalence and clinical significance of KPC-PA are not available. In addition, the susceptibility of the strains to CAZ-AVI remains unknown. Samples in this study were collected from seven tertiary hospitals prior to CAZ-AVI clinical approval in China. Therefore, our results represent a retrospective study establishing the baseline efficacy of the novel β-lactam/β-lactamase combination agent for treating KPC-PA infections. The observed correlation between the blaKPC copy number and CAZ-AVI resistance suggests that close monitoring of the susceptibility of the strain during treatment is required. It would also be beneficial to screen for the blaKPC gene in CRPA strains for antimicrobial surveillance purposes.


Response to Reviewer Comments
We thank reviewers for all their invaluable comments. Our responses are shown below and modifications are highlighted in the revised manuscript.
Reviewer #1: This resubmitted manuscript addressed most of my previous comments. I like the newly added plasmid curing results, although it could be more convincing if additional blaKPC gene knockout experiments can be conducted in selected isolates, which will directly prove the causal effect of increased blaKPC copy number and CAZ-AVI resistance.
I personally like the original title more. The current title in fact reduced the importance of this study. In my opinion, the study has at least two significant findings. One is the high prevalence of CAZ-AVI resistant KPC-PA in China, and the second one is the molecular mechanism of CAZ-AVI resistance is due to the increased blaKPC copy numbers in different plasmid backgrounds. The authors may consider "Emergence of ceftazidime-avibactam resistant KPC-producing Pseudomonas aeruginosa in China" or "High prevalence of ceftazidime-avibactam resistant KPC-producing Pseudomonas aeruginosa in China" for the new title.

Response:
We are pleased to learn that the reviewer likes our study. We agree that bla KPC gene knock-out would be more convincing. Therefore, we have knocked out the single-copy bla KPC-2 gene on the plasmid in strain SRRSH1408. Both of the SRRSH1408 strains, bla KPC-2 -KO and plasmid-cured, shared the same susceptibility to CAZ-AVI, 4mg/L. This indicates that the plasmid-curing approach has a comparable effect with the gene knock-out approach.
Knocking out multiple-copy bla KPC-2 genes was not successful despite several attempts. We hope the reviewer understands that we would like to publish our study at this stage. We also thank the reviewer for his recommendation of titles for our paper and gratefully chose the first one "Emergence of ceftazidime-avibactam resistant KPC-producing Pseudomonas aeruginosa in China" as the title of our revised paper.  Table S5. For plasmid contigs, we further filtered out contigs belonging to two main plasmid types (Type I and II) by mummer. The criterium is the ratio of matched length to the contig length greater than 50%. The remaining contigs potentially belong to other uncharacterized plasmids. We extracted unclassified plasmid contigs longer than 8 kb (n=74). We classified these 74 contigs into 22 clusters by CD-HIT-EST (options: -c 0.9 -A 0.9). Representative contig from each cluster were aligned in GenBank database by BLASTN.
2. What are the definitions/differences of plasmid type (e.g. line 375), pattern (line 377) and group (line 378)? It doesn't seem to be practical to assign all plasmids simply based on the short-reads assemblies. The comprehensive plasmid grouping for each isolate may not add much value to the study. I would suggest the authors simplify the analysis.

Response:
We define plasmid type characterized by the replication or transfer system. Plasmid pattern refers to the composition of plasmids in a single strain. We have now corrected the word "group" and refer to it as plasmid pattern. We have followed the reviewer's comment to simplify the analysis.
3. Based on the mapping or blast analysis, the genomes with high identities and query coverages against the reference plasmids may be assigned to the corresponding plasmid type (type I, type II and unclassified?), as the analysis confirms the presence of the reference plasmid backbones. Selected strains with type I, II and unclassified plasmid types can be further characterized by Nanopore sequencing, in order to probe the molecular mechanisms underlying the increased blaKPC copy numbers.

Response:
We agree with the reviewer. We first identified the presence of Type I and II plasmids by mapping analysis and found further plasmids by contig blast analysis. We selected representative strains from each plasmid pattern for Nanopore sequencing, which generally confirmed our plasmid cluster analysis of the NGS data.
4. The results of the study seem interesting. However, the current manuscript generally lacks focus, and is hard to follow. The method section is lengthy. The method section can be trimmed and extra details can be moved to suppl data. Numerous grammar issues were found (e.g line 64, 100-102, 324 etc) through the manuscript. All these elements should be improved in the resubmission.

Response:
We thank the reviewer's suggestion. The method section has been simplified.
We have moved details of methods to the supplemental information. Grammar issues are corrected (Line 64, 101, 224-226) in the revised manuscript.
Minor comments 1. Abstract: Line 54-55: better to say "blaKPC plasmid curing in xx strains re-sensitize the CAZ-AVI susceptibility, suggesting xxx ". Molecular mechanisms of increased blaKPC copy numbers should be added in the abstract.

Response:
We thank the reviewer for his/her suggestion and have modified this sentence in the revised manuscript (Line 53-54).
Response: Please see our response above (major comment 1).
6. Line 172-176: As I commented above, Kraken2 based method cannot assign the contigs in specific plasmids.

Response:
We agreed with the reviewer that it cannot assign the contigs into specific plasmids. The Kraken2-based contigs clustering is the first step of plasmid composition analysis. It should be further confirmed by long-read sequencing. We have corrected this statement in the revised manuscript (line 153-154).
7. Line 179-181: The statement is not accurate. I don't think this approach can determine the "exact" content, and additional details on how the plasmid "patterns" were classified should be provided.

Response:
We agreed with the reviewer this approach does have limitations as we pointed out in the discussion (line 428-438). The plasmid patterns classification should be further confirmed by long-read sequencing. We have corrected this statement as a preliminary classification in the revised manuscript (line 167-168).
Response: 22 strains were sequenced by Nanopore. We have added this information in Line 170 in the revised manuscript.
9. Line 205: Why don't use more than one plasmid gene as references (similar as the single copy chromosome genes)?
Response: We agree with the reviewer that it is more reasonable to use more than one plasmid gene as reference. Since we have removed the plasmid copy number analysis in the revised manuscript and only kept the analysis of blaKPC-2 gene copy number, its expression and CAZ-AVI MIC, it becomes meaningless to determine the plasmid copy number. Therefore, we have removed this sentence in the Methods & Materials section.  I'm please to inform that your manuscript has been accepted, and I am forwarding it to the ASM Journals Department for publication. For your reference, ASM Journals' address is given below. Before it can be scheduled for publication, your manuscript will be checked by the mSystems senior production editor, Ellie Ghatineh, to make sure that all elements meet the technical requirements for publication. She will contact you if anything needs to be revised before copyediting and production can begin. Otherwise, you will be notified when your proofs are ready to be viewed.
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