Emergence of carbapenem-resistant Pseudomonas aeruginosa ST179 producing both IMP-16 and KPC-2: a case study of introduction from Peru to Spain

ABSTRACT We describe four cases of a novel carbapenem-resistant Pseudomonas aeruginosa ST179 clone carrying the blaKPC-2 or blaKPC-35 gene together with blaIMP-16, imported from Peru to Spain and isolated from leukemia patients. All isolates were multidrug-resistant but remained susceptible to fosfomycin, cefiderocol, and colistin. Whole-genome sequencing revealed that blaKPC-2 and blaKPC-35 were located in an IncP6 plasmid, whereas blaIMP-16 was in a chromosomal type 1 integron. This study highlights the global threat of multidrug-resistant P. aeruginosa clones and underscores the importance of monitoring and early detection of emerging resistance mechanisms to guide appropriate treatment strategies. The importation and spread of such clones emphasize the urgent need to implement strict infection control measures to prevent the dissemination of carbapenem-resistant bacteria. IMPORTANCE This is the first documented case of a Pseudomonas aeruginosa ST179 strain carrying the blaKPC-35 gene, and it represents the first report of a P. aeruginosa co-harboring blaIMP-16 and either blaKPC-2 or blaKPC-35, which wre imported from Peru to Spain, highlighting a threat due to the capacity of spreading carbapenem-resistance via plasmid conjugation.

We describe the emergence of IMP-16, KPC-2, and KPC-35 carbapenemase-producing P. aeruginosa, isolated from three Peruvian patients who arrived from Peru and one Spanish patient with no recent travel history, who had shared a room with one of the Peruvian patients.

Patients and isolates
From August 2022 to February 2023, five isolates of P. aeruginosa producing both KPC and IMP carbapenemases were recovered from either surveillance or clinical samples of four leukemic patients admitted to the hematology and ICU units at the Hospital Clínic of Barcelona.

DNA extraction and Illumina and Oxford Nanopore sequencing
DNA was extracted using the ZymoBIOMICS DNA miniprep Kit (Zymo Research, Irvine, USA), according to the manufacturer's protocol.DNA quality and quantity were determined using NanoDrop 2000 spectrophotometer and Quantus Fluorometer and QuantiFluor dsDNA System (Promega, Madison, USA), respectively.
Illumina libraries were prepared with the Illumina DNA Prep kit (Illumina, San Diego, USA) and Nextera DNA CD Indexes (Illumina, San Diego, USA) using approximately 400 ng of input DNA.The libraries were normalized using a bead-based procedure and were pooled at equal volumes.The pooled library was denatured and sequenced using MiSeq reagent version 2 (Illumina, San Diego, USA) on an Illumina MiSeq platform with a 2 × 150 paired-end chemistry.

Genome assembly and quality assessment
Live basecalling was selected, and reads with a quality score lower than 8 and <200 bp were discarded.Hybrid assembly was performed as follows.A first assembly was created using ONT reads with Unicycler v0.5.0 (6).Subsequently, an ONT-based polishing step was performed using Medaka v.1.7.2 (7).Illumina FASTQ files were mapped to the polished assemblies using BWA v0.7.12 (8).A consensus Illumina-based polishing step was performed with Polypolish v0.5.0 (9), from which the final ONT-Illumina polished assemblies were obtained, except for 23-169, since no Illumina data were generated for this sample.
The completeness of the final assemblies was assessed using a double approximation of CheckM v1.2.2 (10) and BUSCO v5.3.2 (11).For a review of the assembly performance, see Supplementary Information.

Medical history
Patient 1 was an 81-year-old male born in Spain and residing in Barcelona.He was diagnosed with acute myeloid leukemia (AML) in June 2022 and started treatment at our center in July 2022.
Patient 2 was a 23-year-old male born in Peru.He was diagnosed with early T acute lymphoblastic leukemia (ALL) in December 2017 in Peru.On the 6th of July 2022, immediately after arriving in Spain from Peru, he was admitted to the emergency unit of our center because of fever and cutaneous lesions compatible with ecthyma gangreno sum.
Patient 3 was a 23-year-old male born in Peru diagnosed with ALL in August 2017 and underwent induction treatment between August 2017 and March 2020.In July 2022, the patient's symptoms worsened, and he did not respond to treatment with cytarabine and mitoxantrone.He traveled to Spain to obtain a second medical opinion and was admitted to the emergency unit of our center on the 17th of August because of fever.
Patient 4 was a 65-year-old woman who was native to Cuzco, Peru.She had been residing in Barcelona for over a month before being admitted to the emergency unit of our center.In June 2022, she was diagnosed with ALL and monitored in Peru.She was currently receiving bimonthly supportive treatment with 6-mercaptopurine.Upon admission to our center on the 2nd of February 2023, the patient suffered from a symptomatic central nervous system affection.On 20th February, she was transferred to the ICU because of respiratory-origin septic shock.
Patient 1 shared a two-bed hospital room with patient 2 when a carbapenem-resist ant P. aeruginosa isolate producing both IMP metallo-beta-lactamases and a KPC-type carbapenemase was recovered first from a urine sample and a surveillance sample in patient 1 and also from a wound sample from patient 2. Subsequent isolates with a similar phenotype and carrying carbapenemase-encoding genes were also recovered from patients 3 and 4 (see Table 1).

Antibiotic susceptibility profile
Susceptibility testing showed that all isolates were resistant to aminoglyco sides, cephalosporins, carbapenems, and novel beta-lactam/beta-lactamase inhibitor combinations, such as ceftazidime/avibactam and ceftolozane/tazobactam, whereas they were only susceptible to cefiderocol, fosfomycin, and colistin (Table 2).

Genomic analysis
All isolates belonged to sequence type ST179.ANI demonstrated that nucleotide identity between the five P. aeruginosa isolates ranged from 99.98% to 100% (Fig. 1).Interest ingly, a IMP-16 P. aeruginosa ST179 isolate (17387) collected in Lima, Peru, in 2021 and submitted at the NCBI in 2022 (27), presented identity values with the isolates from this study that ranged from 99.95% to 99.98%, while the ANI values of additional ST179 isolates retrieved from NCBI and recovered in Australia, United Kingdom, and Canada ranged from 99.62% to 99.82%.Isolates belonging to sequence types other than ST179 had ANI values ranging from 98.41% to 99.32%.
Phylogenetic analysis based on single-nucleotide polymorphisms (SNPs) of the five P. aeruginosa isolates recovered in the study and the genomes retrieved from the NCBI showed a cluster of all ST179 isolates, as expected, with isolates recovered in this study being closely related and clustered together with isolate 17387, recovered from a Peruvian patient in 2021, in good agreement with results from the ANI analysis (Fig. 2).
In silico analysis described 15 antibiotic resistance genes (ARGs) (Table S1), high lighting the presence of genes encoding metallo-beta-lactamase IMP-16 and class A carbapenemases KPC-2 and KPC-35.bla KPC genes were harbored in plasmids belonging to the IncP6 incompatibility group (see below).The bla IMP-16 gene was located within a type 1 integron structure and was flanked by additional ARGs, such as bla OXA-2 , bla OXA-4 , and aadA11 (Fig. 3).The mutational resistome of the strains was studied (Table S2) as described by López-Causapé et al. (16).

IncP6 plasmid harboring the bla KPC gene
The IncP6 plasmid of approximately 34,699 bp (ranging from 34,641 bp to 34,726 bp) and 57.7% GC carried the bla KPC-2 allele in four isolates (22-690, 22-722, 22-969, and 23-169), but the bla KPC-35 allele in isolate 22-841.All IncP6 plasmids also carried an origin of transference, the repA gene coding for a replicase, and additional genes such as the partition system parABC and the relaxosome and mobilization genes mobABCDE, but lacked the genes encoding a type IV secretion system that is essential for conjugation, thus suggesting that it was not self-transferable.Genes encoding for a type IV secretion system were present in all isolates within another contig.
The bla KPC genes were located in the same plasmid and genetic context among the five isolates, which was not the canonical Tn4401 element typically associated with bla KPC but consisted of a composite transposon of approximately 17 kbp containing an ISKpn27-bla KPC-2 -ΔISKpn6 core structure as well as Tn3-associated structures, as previously described in Klebsiella pneumoniae isolates recovered from the same hospital in 2018 (28) (Fig. 3).

DISCUSSION
We describe the potential emergence and transmission of IMP-16, KPC-2, or KPC-35 carbapenem-resistant P. aeruginosa ST179 clones imported from Peru from advanced  stage leukemia-colonized patients admitted to a Spanish hospital in Barcelona.We included five isolates from four patients with different types of leukemia.Interestingly, three of the four patients were from Peru and had just recently traveled to Spain, and two of them shared the same hematological ward together with the only patient from Spain, with whom one of them had also shared a two-bed hospital room when the first carbapenem-resistant P. aeruginosa isolate was recovered.
Halat et al. reviewed the current status and global epidemiology of carbapenemases in P. aeruginosa in 2022 (29), which included several carbapenemase genes such as bla KPC , bla GES , bla VIM , bla IMP , bla NDM , and bla OXA variants, and stated that in countries such as Brazil, Peru, and Costa Rica, the rates of carbapenem-resistant P. aeruginosa were higher than 50%.In Peru, carbapenem-resistant P. aeruginosa varies geographically, being more reported in the Lima region.According to Angles-Yanqui et al., the predomi nant carbapenemase gene is bla IMP , while bla NDM and bla KPC are more frequently found among Enterobacterales (30).In Spain, the prevalence of KPC and IMP carbapenemases is not prominent.Nevertheless, various IMP carbapenemases have been reported in the region, including IMP-11 in Acinetobacter baumannii and IMP-28 in Enterobacter kobei in A Coruña (31), IMP-13 in Enterobacter cloacae (32), IMP-16 and IMP-23 in P. aeruginosa, and IMP-22 in Enterobacter in Andalusia (33).Moreover, IMP-8, IMP-8 +OXA-48, and IMP-22 +KPC-3 K. pneumoniae strains were identified at the Instituto de Salud Carlos III  (34).In 2021, Salvador-Luján et al. reported for the first time the presence of the bla KPC gene in a P. aeruginosa strain isolated from the urine of a 56-year-old male in Peru (35).Tickler et al. (27) also investigated the epidemiology of Peruvian carbapenem-resistant P. aeruginosa isolates and found that ST179 was the third most frequently encountered sequence type (n = 12, 8.5%) after ST111 and ST357 (44% and 38.3%, respectively).Of the 12 ST179 isolates recovered in 2018 (n = 8) and 2021 (n = 4), only two of them recovered in 2018 harbored the bla IMP-16 gene, and no carriage of bla KPC was reported.Moreover, ST179 presented the virulence profile exoS+/exoT+/exoY+ (T3SS), which was also shared with all the isolates described in our study.All isolates presented the same virulence factor profile (Table S3), which included the coding regions for a type 3 and a type 6 secretion system (T3SS and T6SS, respectively), two metallophores (pyoverdine and pyochelin), pili, flagella, and exotoxin A, which are all related with the pathogenesis of P. aeruginosa.T3SS and T6SS are involved in the translocation of secreted toxins that enhance disease severity, such as ExoS/T/Y secreted effector toxins, coded in the isolates from this study.For example, ExoS causes apoptotic cell death (36)(37)(38).Pyoverdine and pyochelin scavenge metals such as iron from the host (39).
The P. aeruginosa clone in this study carried the bla IMP-16 metallo-beta-lactamase gene and bla KPC-2 or bla KPC-35 carbapenemase genes.KPC-2 is one of the predominant KPC carbapenemases that has spread worldwide, including Latin America and Spain (40).The bla KPC-35 gene is an allelic variant of bla KPC-2 that differs in only one nucleotide mutation that translates into an L169P amino acid substitution located in the omega loop of the KPC-2 protein, conferring resistance to ceftazidime/avibactam, and is usually associated with ceftazidime/avibactam treatment in patients infected with carbapenemresistant K. pneumoniae isolates producing KPC-2 variants (41)(42)(43)(44).
In this study, a P. aeruginosa isolate producing KPC-35  was recovered from a patient previously carrying a clonally related isolate that harbored the bla KPC-2 allele and received antimicrobial treatment with ceftazidime/avibactam, thus most likely promot ing the emergence of the mutation in the bla KPC-2 sequence that gave place to bla KPC-35 .However, as this P. aeruginosa isolate also carried metallo-beta-lactamase bla IMP-16 , we could not detect any changes in the MIC values against ceftazidime/avibactam.To our knowledge, this is the first report of a P. aeruginosa isolate carrying the bla KPC-35 variant, as well as the first time that a P. aeruginosa strain is accounted for simultaneously producing KPC-2 or KPC-35 together with IMP-16.
Interestingly, the genetic region containing the bla KPC gene includes different insertion sequences located within a Tn3 structure, as depicted in Fig. 3, including the ISKpn27, ISApu2, and ISApu1 insertion sequences, all flanked by incomplete ΔISKpn6 and ΔISEc33 insertion sequences.The KPC plasmid described in this study is very similar to the IncP6 plasmid described by Dai et al. (45), p10265-KPC (~38.9 kbp, accession number KU578314.1 differing only by two missing regions: (i) p10265-KPC presents a Tn5563 transposon with a tnpR-orf2-pilT-tnpA-merP-merT-merR structure that in all isolates from this study lacks a fragment of the pilT gene (only 43.34% of coverage), the full tnpA gene, and the full merP gene; and (ii) the plasmid in our study also lacks the Δbla TEM-1 gene that is located next to bla KPC in p10265-KPC.
The P. aeruginosa isolates included in this study had the bla IMP-16 gene located within a type 1 integron in the chromosome (Fig. 3).IMP-16 carbapenemase was first described in 2004 by Mendes et al. (46) in a P. aeruginosa isolate obtained from a 60-year-old man in Brazil and, to our knowledge, it has not been previously reported in Spain until now, most likely being introduced by Peruvian patients admitted to our hospital.The genetic content of the integron carrying bla IMP-16 differs from that described by Mendes et al.; the integron contained three aminoglycoside resistance genes (aac(6')−30, aac(6')-Ib', and aadA1), whereas the one in our study only contained the aadA11 gene.
In conclusion, to the best of our knowledge, this is the first documented case of a Pseudomonas aeruginosa ST179 strain carrying the bla KPC-35 gene, and it represents the first report of a P. aeruginosa strain co-harboring bla IMP-16 and either bla KPC-2 or bla KPC-35 .The importation and spread of this clone from Peru to Spain emphasizes the critical need for monitoring carbapenemase-resistant bacteria as it poses significant challenges by limiting the available antibiotic options for treating infections.Further more, the potential emergence of new resistance mechanisms against new agents used to combat carbapenem-resistant microorganisms highlights the urgent need to promptly identify and describe such resistance to guide appropriate treatment, rather than relying on empirical approaches.Data from this study suggest that KPC/IMP-16-pro ducing P. aeruginosa ST179 is either widespread in Peru or that all three patients from Peru might have been admitted at the same particular hospital where this strain is prevalent.Unfortunately, all efforts to gather additional information regarding previous admissions of such patients in hospital at their country of origin were unsuccessful, which we acknowledge as one of the main limitations of the study.

FIG 2 FIG 3
FIG 2 SNP-based phylogenetic tree of ST179 P. aeruginosa isolates.The group of isolates in this study is highlighted in blue.Image visualized with FigTree.

TABLE 1
Isolates and patients included in this study a NA: isolate not available for the study.

TABLE 2
Antimicrobial susceptibility profile of P. aeruginosa isolates from this study