Pseudomonas aeruginosa High-Risk Sequence Type 463 Co-Producing KPC-2 and AFM-1 Carbapenemases, China, 2020–2022

We report the clonal spread and evolution of high-risk Pseudomonas aeruginosa sequence type 463 co-producing KPC-2 and AFM-1 carbapenemases isolated from hospital patients in China during 2020–2022. Those strains pose a substantial public health threat and surveillance and stricter infection-control measures are essential to prevent further infections.

Alignments and visualization of plasmids were generated by BLAST Ring Image Generator (BRIG) software version 0.95 (https://sourceforge.net/projects/brig).

Phylogenetic Analysis
A phylogenetic tree for all ST463 P. aeruginosa genomes from the NCBI database and our collection was constructed according to their SNPs by using Snippy version 4.4.5 (https://github.com/tseemann/snippy)and FastTree (7).The genome of strain B1122 (GenBank accession no.JAMWMN000000000) was used as a reference.As of September 2022, a total of 6,885 P. aeruginosa assembled genomes had been deposited in the NCBI Reference Sequence database (https://www.ncbi.nlm.nih.gov/refseq),among which 68 genomes were assigned to ST463 and were downloaded for phylogenetic analysis.

Plasmid Conjugation
The transferability of plasmids p94 and p1214 was evaluated by conjugation experiments; the filter mating method was used with a rifampin-resistant derivative of P. aeruginosa, PAO1, as the recipient strain.Transconjugants were selected on Mueller-Hinton agar plates supplemented with rifampin (800 μg/mL) and meropenem (4 μg/mL).Experiments were independently repeated 3 times.

Stability of blaKPC-2-Carrying Plasmid and Chromosome-Encoded blaAFM-1
To determine the stability of the blaKPC-2-carrying plasmid and chromosomal blaAFM-1 gene, a passaging experiment was completed under antibiotic-free conditions.Briefly, 3 separate bacterial cultures were prepared by inoculating 3 mL Luria-Bertani (LB) broth without antibiotics and incubating overnight at 37°C, then a serial passage of 3 μL overnight culture into 3 mL LB was prepared each day.Samples were collected and diluted on Mueller-Hinton agar plates on day 10.Then, 48 colonies were randomly selected for validation of the presence of blaKPC-2, repA, and blaAFM-1 by PCR with gene-specific primers.Retention was calculated as the percentage of cells with blaKPC-2, repA, and blaAFM-1 in the 48 selected colonies.

Biofilm Quantification and Desiccation Tolerance
Biofilm quantification was performed by using crystal violet staining as described previously (10).Biofilm formation capabilities were assessed according to a published study (11).The capacity to survive on dry surfaces over time was evaluated as described previously with some minor modifications (12).Briefly, 100 μL of an overnight bacteria culture was serially diluted and counted the next day.Another 100 μL of the same culture was transferred into a 96well microtiter plate; 3 technical replicates were prepared.The plate was air dried overnight and then incubated at 37°C for 8 days.Subsequently, 100 μL/well of fresh LB broth was added to the plate, and the plate was incubated with shaking at 37°C for 3 h.From each well, 100 μL of suspension was collected, serially diluted, and counted the next day.Experiments were conducted in triplicate.

Mouse Peritonitis Model
Male immunocompetent BALB/c mice (6-8 weeks of age, weighing 20-25 g) were injected intraperitoneally with 100 μL phosphate-buffered saline solution containing 1 × 10 7 CFU of a P. aeruginosa strain in exponential growth phase (10 mice/group).PA14 was used as a hypervirulent reference strain, and P. aeruginosa ATCC9027 was a low-virulence reference strain (13).After inoculation, mice were monitored for 5 days, and the number of dead mice was assessed each day.All animal experiments were approved by the Institutional Animal Care and Ethics Committee of the First Affiliated Hospital of Zhejiang University School of Medicine.

Statistics
Statistical analysis was performed by using GraphPad Prism software, version 9.0 (GraphPad, https://www.graphpad.com).The Mann-Whitney U-test was applied for pairwise comparison of bacterial groups by using mean values.Survival curves for the mouse infection model were analyzed by log-rank (Mantel-Cox) test.p values of <0.05 were statistically significant.

of complete blaKPC- 2 -Appendix Figure 4 .
containing plasmids from 8 carbapenem-resistant P. aeruginosa isolates obtained in this study.Plasmid p94 from strain ZY94 is the reference sequence.B) Genetic organization of archetypal type I plasmid pZPPH1-KPC (GenBank accession no.CP077990) and comparison with p94 isolated in this study.Structure of pZPPH1-KPC consists of backbone and insertion regions (shaded gray).Outermost ring is an annotation of the reference plasmids (p94 and pZPPH1-KPC) and shows the direction of the transcriptional open reading frame.GC-rich regions (GC skew) are indicated.CDS, coding sequence.Comparison of plasmid p94 from this study and homologous plasmids isolated from other Pseudomonas aeruginosa strains.BLAST Ring Image Generator (https://sourceforge.net/projects/brig) diagram shows p94 in the innermost ring and alignments with 40 related plasmids.Colored rings indicate different P. aeruginosa sequence types.The outermost black ring shows genes on p94.CDS, coding sequence; ST, sequence type.