Characterization of a Carbapenem-Resistant BKC-1-Producing Clinical Isolate Belonging to the Pseudomonas putida Group from Brazil

ABSTRACT

Since its first report, the class A Brazilian Klebsiella carbapenemase (BKC) has been detected only among Enterobacterales isolates from Brazilian hospitals. In this study, we characterized a multidrug-resistant Pseudomonas juntendi clinical isolate and identified a 43.3-kb plasmid carrying bla_BKC-1 and a class 1 integron (In1996) containing the arr-2, qnrVC1, dfrA21, and aac(6′)-Ib′ gene cassettes. Our results confirm the ability of Pseudomonas putida group isolates to acquire antimicrobial resistance determinants and further act as resistance reservoirs.

INTRODUCTION

The emergence of carbapenemase-producing Pseudomonas putida group (PpG) species is a concern, since multidrug-resistant (MDR) PpG strains have already been responsible for several opportunistic nosocomial infections and outbreaks (1, 2). Among clinical and environmental PpG isolates, carbapenem resistance has been linked to the production of metallo-β-lactamases (MβLs) associated with mobile genetic elements (MGEs) (3, 4). Therefore, PpG’s role as a platform for spreading antimicrobial resistance genes (ARGs) to further pathogens in nosocomial environments represents a significant epidemiological and clinical threat.

In 2015, a novel class A carbapenemase was characterized in Brazilian Klebsiella pneumoniae clinical isolates belonging to clonal complex (CC) 442 (5). BKC-1 efficiently hydrolyzes β-lactams, including amino- and ureidopenicillins and first- and second-generation cephalosporins, and confers low-level resistance against third- and fourth-generation cephalosporins, monobactam, and carbapenems (5, 6). The frequency of bla_BKC-1 in Klebsiella spp. remains low, and it was thought to be strictly related to K. pneumoniae, as this gene was initially detected in a 10-kb nonconjugative IncQ1 plasmid (p60136) (5). However, recent studies demonstrated the presence of bla_BKC-1 and a novel variant (bla_BKC-2) in similar genetic backgrounds of Citrobacter freundii and Enterobacter hormaechei subsp. xiangfangensis clinical isolates, respectively (7, 8). Nonetheless, to the best of our knowledge, this gene has not yet been observed in nonfermenting Gram-negative bacilli (NF-GNB). Here, we characterized a BKC-1-producing NF-GNB clinical isolate belonging to the PpG.

(This work was partially presented as an e-poster [P3422] at the 31st European Congress of Clinical Microbiology and Infectious Diseases [ECCMID], 2021, which was held online only.)

A carbapenem-resistant Pseudomonas isolate (P-12.273) was recovered from a urine sample of a 16-year-old female patient admitted to a tertiary-care hospital in the city of São Paulo, Brazil, in September 2012. Whole-genome sequencing (WGS) was performed with both short- and long-read strategies. For short-read sequencing, genomic DNA of P-12.273 was extracted using the DNeasy blood and tissue kit (Qiagen, Hilden, Germany) and quantified using a Qubit 2.0 fluorometer (Thermo Scientific, Waltham, MA, USA). Libraries were prepared using the Nextera XT DNA library preparation kit (Illumina Inc., CA, USA) and barcoded using IDT Nextera DNA unique dual index set C (Illumina, CA, USA). Short-read sequencing was performed using the Illumina MiSeq platform (Illumina, CA, USA) in paired-end mode to obtain 2 × 250-bp reads with the MiSeq reagent kit v2 and v3 (Illumina, San Diego, CA, USA). For long-read sequencing, genomic DNA was obtained using a ZymoBIOMICS DNA miniprep kit (Zymo, USA), and libraries were prepared using the rapid sequencing gDNA barcoding kit (SQK-RBK004; Oxford Nanopore Technologies [ONT], Oxford, UK). Sequencing was performed on a Nanopore MinION Mk1C using a FLO-MIN106D (R9.4.1) flow cell (ONT); the fast5 files were base called and demultiplexed, and adapters were trimmed with Guppy 5.1.12 (ONT).

The draft genome of P-12.273 was obtained by de novo hybrid assembly using both short and long reads using Unicycler v0.4.8 in “–mode normal.” Its estimated total length was 5,675,710 bp, present in 11 contigs, with an average G+C content of 62.38%. Additional assembly statistics are provided in Table S1 in the supplemental material. Multilocus sequence analysis (MLSA) of 16S rDNA and the gyrB, rpoB, and rpoD genes (9), followed by genome-based identification performed on the Type Strain Genome Server (https://tygs.dsmz.de), clustered isolate P-12.273 with Pseudomonas juntendi, showing digital DNA-DNA hybridization (dDDH; d₄) values of 85.6% against P. juntendi PP_2463 (GenBank accession no. CP091088.1), a clinical isolate co-carrying five β-lactam resistance genes recovered from a urinary tract infection in China (10).

MICs were determined by the cation-adjusted broth microdilution method and interpreted according to BrCAST/EUCAST guidelines (http://brcast.org.br). Susceptibility to novel β-lactams and β-lactam–β-lactamase inhibitor combinations were also determined by disk diffusion (ceftazidime-avibactam, cefiderocol, imipenem-relebactam, and meropenem-vaborbactam), following the same guidelines. P-12.273 displayed an MDR profile (11), with high MICs to β-lactams, aminoglycosides, and fluoroquinolones, but remained susceptible to polymyxin B (MIC, 1 μg/mL) (Table 1). It also remained susceptible to cefiderocol, ceftazidime-avibactam, imipenem/relebactam, and meropenem-vaborbactam. Phenotypic carbapenemase activity was detected by Blue-Carba (12), with a positive result after 90 min of incubation. MGEs and ARGs were annotated in the draft genomes using CARD (13), ISFinder (14), and INTEGRALL (15) and revealed the presence of arr-2, qnrVC1, dfrA21, aac(6′)-Ib′, aphA6, bla_BKC-1, AmpC, qacE, and sul1. Moreover, P-12.273 displayed point mutations in the quinolone resistance-determining region (QRDR) of gyrA, leading to a T₈₃I amino acid substitution. Together, these ARGs and point mutations could explain the MDR profile displayed by isolate P-12.273.

TABLE 1.

MICs and resistance determinants identified from the genome sequence of isolate P-12.273

Antibiotic class	Antimicrobial resistance gene(s)	Antimicrobial agent	MICa (μg/mL)
β-Lactams	blaBKC-1, AmpCb	Aztreonam	>128
		Piperacillin-tazobactam	128/4
		Ceftazidime	>128
		Ceftriaxone	>128
		Cefepime	128
		Imipenem	16
		Meropenem	64
		Meropenem-vaborbactamc	S
		Cefiderocolc	S
		Ceftazidime-avibactamc	S
		Imipenem-relebactamc	S
Fluoroquinolones	gyrA (T83I), qnrVC1	Ciprofloxacin	64
		Levofloxacin	>64
Aminoglycosides	aac(6′)-Ib′	Amikacin	256
	aphA6	Tobramycin	>8
Polymyxins		Polymyxin B	1
Folate synthesis inhibitors	sul1, dfrA21	Trimethoprim-sulfamethoxazole	>64
Rifamycins	arr-2	Rifampin	>256

^aMICs above BrCAST/EUCAST clinical breakpoints are in bold, when available.

^bThe role of AmpC-mediated β-lactam resistance in PpG remains to be elucidated.

^cSusceptibility profile determined by disk diffusion, following BrCAST/EUCAST guidelines.

Further resistome analysis showed that apart from aphA-6, and bla_BKC-1, the remaining six detected ARGs were located in the chromosome of P-12.273. Except for AmpC, the five remaining ARGs were found as gene cassettes of a novel class 1 integron named In1996 (Fig. 1A). This MGE was composed of four gene cassettes, namely, aac(6′)-Ib′, dfrA21, qnrVC1, and arr-2, as well as the 3′ conserved segment (3′-CS) containing the qacEΔ1 and sul1-Δorf5 genes (Fig. 1A). Sequence analysis suggests that In1996 is part of a composite transposon belonging to the Tn21 family, flanked upstream by the excepted imperfect 38-bp inverted repeats (IR) and the tnpAR module, as previously described (16, 17). Additionally, In1996 was flanked downstream by a copy of IS6100 (880 bp; accession number X53635), and the truncated urf2, followed by the mercury resistance operon merRTPAD, in accordance with the proposed genetic background. Furthermore, In1996 is part of a 14.6-kb cluster of ISs and transposons, where the Tn21-family tnpAR module was disrupted by the insertion of intact copies of IS1071 and ISPa91, as previously observed in Tn6960, a Tn6346-derived (EU696790) Tn3-family unit transposon (16, 17).

FIG 1.

Schematic representation of antimicrobial resistance-associated MGEs in P. juntendi P-12.273. Arrows indicate coding sequences (CDS) and directions of transcription. Unlabeled CDS (in gray) encode hypothetical proteins. ARGs are highlighted in red. Conjugation machinery is highlighted in green. Partitioning and replication genes are highlighted in pink, and MGEs are indicated in blue. Figures were created Proksee (https://proksee.ca/) and CAGECAT (https://cagecat.bioinformatics.nl/). (A) Gene cassette array and genetic map of In1996. The 5′-CS and 3′-CS are highlighted. The mercury resistance operon (mer) is highlighted in black. (B) Circular representation of pP12273. The positions of the 5-bp direct repeats (TTACT) and 50-bp inverted repeats flanking ISKpn23 are indicated by dark blue squares and circles, respectively. (C) Linear comparison of pP12273 with pBA7816 (accession number MN240297), pLD209 (accession number MT192131), and p60136 (accession number KP689347).

Considering the strong association between bla_BKC-1 and IncQ plasmids, the presence of extrachromosomal elements was assessed, as previously described (18, 19). Plasmid DNA extraction, followed by agarose gel electrophoresis, revealed that P-12.273 harbored a single ~45-kb plasmid. Southern blot hybridization using a bla_BKC-1-specific probe was conducted using digoxigenin (DIG) washing and blocking buffer and DIG DNA labeling and detection kits (Sigma-Aldrich, St. Louis, MO, USA) and confirmed the plasmid location of this gene (data not shown). To investigate potential plasmid transfer, filter-mating conjugation experiments were carried out (5). P-12.273 was used as the donor strain, while Escherichia coli J53 (azide resistant) and Pseudomonas aeruginosa PAO1 (rifampin resistant) were used as the recipient strains. Luria-Bertani (LB) agar supplemented with sodium azide (200 μg/mL) or with rifampin (32 μg/mL) and ceftazidime (4 μg/mL) was used to select transconjugants when E. coli J53 and P. aeruginosa PAO1 were used as recipients, respectively. Despite multiple attempts, it was not possible to retrieve any transconjugants. However, we were able to transfer the bla_BKC-1-harboring plasmid to electrocompetent E. coli DH5α cells. The transformants displayed elevated MICs of β-lactams (Table S2), including carbapenems, suggesting that the transferred BKC-1 was functional.

WGS analysis further revealed that the bla_BKC-1 gene was part of a 43,339-bp circular contig (pP12273) and was flanked up- and downstream by ISKpn23 and aphA6, respectively, as previously observed in bla_BKC-1-harboring plasmids (Fig. 1B). However, the similarity between these plasmids was restricted to this 5,457-bp fragment, including a partial sequence of repC. In fact, when the 43-kb contig was queried with the PLSDB database using mash dist (https://ccb-microbe.cs.uni-saarland.de/plsdb), it was noticed that most of the remaining 37,882 bp showed high (97.6%) nucleotide similarity with pBA7816, a fully characterized plasmid from a Pseudomonas asiatica clinical isolate (accession number MN240297) (3) (Fig. 1C). This shared backbone included replication, partitioning, and stability modules (28,039 bp), as well as repA and oriV. Interestingly, pBA7816 carried the class 1 integron In899, with bla_VIM-2 as the only gene cassette (Pseudomonas chlororaphis M11740; accession number KJ668595) (20), which was lost in pP12273. In fact, other than the intI1 gene, the remaining sequences were replaced by a putative chloride dismutase-encoding gene. Moreover, the 5,457-bp fragment shared with p60136 was inserted between repA and the tniABQC module, together with a 937-bp sequence of unknown origin, encoding only hypothetical proteins. That insertion also caused the loss of a 1,769-bp fragment from pBA7816 containing tnpC and hypothetical-protein-encoding genes. It should be noted that, although the presence of ISKpn23 upstream of bla_BKC-1 is essential for its expression (19, 21), it is not responsible for its mobilization, whose mechanism remains to be elucidated.

Previous studies have shown that the pBA7816 backbone is commonly disseminated among PpG species (3) and is likely derived from the pLD209 lineage, first detected in 2009 in a carbapenem-resistant P. putida isolate from Argentina (21, 22). Furthermore, pLD209 not only circulated among PpG species but also seems to be capable of self-mobilization to P. aeruginosa and E. coli due to the presence of a complete cluster of mating pair formation genes (namely, virB2 to virB11, virD4, traC4, and mobC). It should be noted that at least six other derived plasmids displaying high nucleotide sequence identity (85% to 99%) to pLD209 were detected in other clinical isolates, including extensively drug-resistant (XDR) Serratia marcescens and P. aeruginosa strains (23, 24).

To the best of our knowledge, this is the first description of bla_BKC-1 in a NF-GNB isolated only 4 years after the first BKC-1-producing K. pneumoniae description. Our results highlight the potential of non-P. aeruginosa Pseudomonas strains to acquire ARGs, which can be further mobilized and transferred not only to Pseudomonas spp. but also to Enterobacterales.

Ethical approval for the study was obtained from the Research Ethics Committee from Universidade Federal de São Paulo (UNIFESP)/HSP (process number CEP N3716171219).

Data availability.

The complete genome of P. juntendi P-12273 strain was submitted to GenBank under accession number SAMN15663395.