Role of megaplasmids and chromosomal integration in acquisition of CTX-M-encoding genes by Pseudomonas aeruginosa

The genes that encode CTX-M-type ESBLs have diffused extensively among Enterobacterales over the past decades.¹ Despite this epidemic success at the global level, few of these have been detected (bla_{CTX-M-1,-2,-3,-14,-15and-43}) in the distantly related opportunistic pathogen Pseudomonas aeruginosa.^1–4 The narrow host range of plasmids carrying the bla_CTX-M determinants in Enterobacterales (mainly of incompatibility group IncF) plausibly accounts for the low prevalence of these resistance genes in P. aeruginosa.⁵ On the other hand, little is known about the mobile elements that enable the acquisition of CTX-M enzymes by clinical P. aeruginosa strains. One plasmid close to broad-host-range incompatibility group IncP2 has been found to carry the gene bla_CTX-M-2, while another one belonging to IncQ harboured bla_CTX-M-3.^6,7 The present report describes the structure of a large conjugative plasmid determining a novel CTX-M variant, named CTX-M-206, and the genetic environment of a chromosomally located bla_CTX-M-15 gene in a P. aeruginosa isolate present in a second strain.

P. aeruginosa 152962 (serotype O:4) and 174411 (O:6) were isolated, respectively, from the blood and urine samples of two patients with no travel history, admitted to geographically distant French hospitals. In contrast to 174411, which belongs to sporadic ST2464 reported once in Estonia in 2013 (https://pubmlst.org/), 152962 is related to the high-risk clone ST654, known to produce various carbapenemases, such as VIM-2 in France, Poland and Tunisia, IMP-26 in Indonesia and NDM-1 in North America.^8–12

When tested for their drug susceptibility by the broth microdilution method with customized antibiotic microplates (Sensititre, ThermoFisher, Illkirch-Graffenstaden, France), both strains appeared resistant to β-lactams (MICs >32 mg/L), except for ceftazidime/avibactam (4/4 mg/L) and meropenem (2 mg/L) for 174411, according to the current EUCAST breakpoints (Table 1).¹³ The isolates were also resistant to ciprofloxacin (>16 mg/L) and amikacin (≥32 mg/L), but were still susceptible to colistin (1 mg/L). Interestingly, double-disc synergy tests using the β-lactamase inhibitor clavulanic acid (20/10 μg amoxicillin/clavulanate disc) with ceftazidime (10 μg disc) and cefepime (30 μg disc) as test molecules, respectively, were positive, strongly suggesting that the resistance profiles of bacteria were at least partly due to an Ambler class A β-lactamase. PCR-sequencing experiments (Sanger Applied Biosystems 3500 platform of Besançon University Hospital) confirmed the presence of gene bla_CTX-M-15 in 152962 and of a novel variant of gene bla_CTX-M-3 in 174411. The encoded product, dubbed CTX-M-206, differs from CTX-M-3 by a single Asn-92-Ser substitution. Accounting for the high resistance level of 152962 to carbapenems (MICs >64 mg/L; Table 1), the gene coding for MBL VIM-2 was detected in this strain, along with the determinant of penicillinase TEM-1. Finally, the gene of narrow-spectrum oxacillinase OXA-2 was identified in strain 174411.

Table 1.

Drug susceptibilities (MICs) for P. aeruginosa 152962, P. aeruginosa 174411, the P. aeruginosa PU21 transconjugant harbouring the plasmid pPWIS1 and the P. aeruginosa PU21 reference strain

Antibiotic	MIC (mg/L)
	P. aeruginosa 152962	P. aeruginosa 174411	P. aeruginosa PU21	P. aeruginosa PU21(pPWIS1)
Ticarcillin	>128	>128	16	>128
Piperacillin/tazobactam	128/4	128/4	≤4/4	128/4
Ceftazidime	>64	32	2	32
Cefepime	>64	>64	2	>64
Ceftolozane/tazobactam	>32/4	16/4	≤0.5/4	8/4
Ceftazidime/avibactam	>32/4	4/4	2/4	4/4
Aztreonam	>128	>128	4	128
Imipenem	>64	8	1	1
Meropenem	>64	2	1	1
Amikacin	>128	32	2	2
Tobramycin	>32	2	0.5	0.5
Ciprofloxacin	>16	>16	≤0.12	≤0.12
Colistin	1	1	1	1

Conjugation assays with 152962 and 174411 as donors, and a rifampicin-resistant mutant of P. aeruginosa reference strain PU21 as recipient, resulted in isolation of PU21 transconjugants harbouring the bla_CTX-M-206 gene on a large plasmid, dubbed pPWIS1. These transconjugants, while resistant to ticarcillin (>128 mg/L), piperacillin/tazobactam (128/4 mg/L), cefepime (>64 mg/L), aztreonam (128 mg/L) and ceftolozane/tazobactam (8/4 mg/L), remained susceptible to ceftazidime/avibactam (4/4 mg/L) and carbapenems (Table 1).

The pPWIS1 plasmid was extracted from a selected PU21 transconjugant and then fully sequenced using Oxford Nanopore Technology (MinION platform, Oxford Science Park, UK). After filtering out the 5% reads with the worst quality, the filtered long reads (110 685 reads for 944 172 143 bp) were de novo assembled with Flye (two contigs with 136× average read depth).¹⁴ The polishing of the plasmid sequence was then performed with medaka, Rebaler and Pilon using paired-end short Illumina reads (platform NextSeq, 2 × 150 paired-end, Microsynth AG Company).¹⁵ Its very large size (419 683 bp) was found to encompass 494 predicted coding regions showing a mean G + C content of 57% (Figure 1). pPWIS1 shares extensive sequence homologies with various megaplasmids, including p12939-PER, pPUV-17, pJB37, pOZ176, pNK546-KPC and pRBL16 (from 83% to 92% query cover and from 98% to 99% nucleotide similarity) (Figure S1, available as Supplementary data at JAC Online). With the exception of pRBL16, initially detected in a Pseudomonas citronellolis strain (SJT3-3), these megaplasmids were recovered from P. aeruginosa clinical strains isolated in Poland, Portugal and China, and were found to carry diverse β-lactamase genes, such as bla_KPC-2, bla_PER-1, bla_OXA-246,bla_VIM-2 and/or bla_IMP-9.^16–19

Figure 1.

Comparison of Inc_pRBL16 plasmids, pPWIS1, p12939-PER, pPUV17, pJB37, pOZ176, pNK546-KPC and pRBL16. (a) The two most inner circles correspond to the scale and the G+C percent, respectively. The next circles from the inside correspond to plasmid pRBL16 (83% query cover and 99.94% nucleotide similarity; accession number CP015879.1), pNK546-KPC (92% query cover and 99.97% nucleotide similarity, accession number MN433457), pOZ176 (86% query cover and 99.96% nucleotide similarity, accession number KC543497), pJB37 (464 804 bp, 87% query cover and 99.92% nucleotide similarity; accession number KY494864), pPUV-17 (87% query cover, 99.98% nucleotide similarity, accession number MT732195.1), p12939-PER (90% query cover and 98.52% nucleotide similarity, accession number MF344569), and pPWIS1 with the positions of ORFs in minus and plus strands. Only the genes encoding resistance clusters (mercury and telluric resistance), repA gene, IS222 and accessory modules (I, II, and III) are indicated. Softwares BRIG 0.95 and Inskcape (version 1.0.1) were used to construct and draw the plasmidic maps. (b) Only resistance genes, transposon-related genes (tnpA, tnpR), and insertion sequences are shown in modules I and II. Hypothetical proteins in module III are indicated as hp. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

Highlighting the close similarity of pPWIS1 to pRBL16 (accession number CP015879), the reference plasmid of the Inc_pRBL16 group, both replicons exhibited a repA_IncpRBL16 gene (replication initiation) that was 100% identical and shared more than 99.9% sequence identity regarding backbone genes, such as repA-parB2-parAB (partition region), traF/traG/virD2 and trbBCDEJLGGI (conjugative transfer region), che (chemotaxis locus) and pil (pilus assembly locus), as well as for the genes of tellurite resistance (ter), and three copies of iterons (TCGTGCTATCAGGAGTA) (Figure S1).¹⁶ Consistent with the notion of evolvability of Inc_pRBL16 plasmids, we found that pPWIS1 differed from the closest plasmid pNK546-KPC by the presence of three accessory modules (Figure 1). The first of these (5470 bp) contains a copy of Tn5393, a transposon common in environmental bacteria that confers streptomycin resistance. In the second module (7402 bp), the bla_CTX-M-206 gene is borne by a truncated transposition unit Δorf477-bla_CTX-M-206-ΔISEcp1, itself flanked by a truncated Tn2, a genetic environment that has already been reported for various bla_CTX-M genes carried by IncF plasmids in Enterobacterales.²⁰ The large number of transposition elements, including ΔIS26, ΔIS1326 and ΔTn2, found upstream of this CTX-M gene suggests that multiple rearrangements occurred in this region, which thus appears to be a hotspot for gene integration. Indeed, various resistance genes, such as bla_KPC-2, bla_VIM-2, bla_IMP-9 and bla_PER-1, have previously been localized in this region on related megaplasmids. Finally, the third module of 7510 bp contains six ORFs whose functions are unknown.

As compared with the situation described above, WGS of strain 152962 revealed that the bla_CTX-M-15 gene was part of a large region of 72 134 bp, which inserted into a gene, PA0069, of hypothetical function according to the annotation of the reference strain PAO1 genome (Figure 2) (https://pseudomonas.com).²¹ A search in databases showed that a sequence of 14 066 bp surrounding bla_CTX-M-15 in 152962 had 100% nucleotide identity, though with some gene rearrangement, with a region of a plasmid (227 989 bp) named pKPN3-307 (accession number KY271404) detected in four Italian strains of Klebsiella pneumoniae ST307 producing carbapenemase KPC-2.²² This region is flanked by IS26 and a copy of gene tnpA, and contains the ISEcp1-bla_CTX-M-15-orf477Δ transposition unit, itself inserted in a truncated Tn2 element. While strB, strA, sul2, IS5075 and tnpA are located downstream of this region in plasmid pKPN3-307, they are situated upstream in strain 152962. A recombination between the copies of strA and strB genes present on each side of the bla_CTX-M-15 region might account for this inversion (Figure 2). Reminiscent of the large DNA fragment acquired by 152962, a 65 363 bp sequence was reported to be inserted at the same location (gene PA0069) in a P. aeruginosa strain named PA99, isolated in Thailand in 2016 (accession number CP042967). Apart from the 14 066 bp bla_CTX-M-15 region described above, and two gene cassettes (bla_VIM-2 and aacA4) accounting for the high resistance of 152962 to carbapenems and amikacin, all the genes identified in 152962 are present in strain PA99. On the other hand, strain PA99 contains the mercury resistance locus merRTPADE.

Figure 2.

Schematic representation of the genetic environment of gene bla_CTX-M-15 in P. aeruginosa 152962. Linear representation of the large region (72134 bp) inserted into the chromosomal gene PA0069 containing the genes bla_TEM-1, bla_CTX-M-15 and bla_VIM-2 in the strain 152962 strain are mentioned. Resistance genes, transposon-related genes (tnpA, tnpR), insertion sequences, class 1 integrase and the genes strA and strB are indicated. The chromosomal insertion into the gene PA0069 is reported in black. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

In conclusion, this study highlights how P. aeruginosa is able to collect resistance genes (i.e. bla_CTX-M determinants) from distantly related microorganisms through genetic transfers that involve either self-transmissible broad-host-range megaplasmids of the Inc_pRBL16 incompatibility group or insertion of large DNA fragments in spots of high genomic plasticity (i.e. gene PA0069).

Nucleotide sequence accession numbers

The nucleotide sequences reported in this study and corresponding to the entire sequence of the plasmid pPWIS1 and the chromosome of the strain 152962 have been deposited in the GenBank nucleotide database under accession numbers VFEZ01000004.1 and CP069198.1, respectively.

Funding

This work was supported by the French Ministry of Health though the Santé Publique France agency.

Transparency declarations

None to declare.

Author contributions

K.J. planned the study. P.T., M.B., Racha Beyrouthy, Richard Bonnet and K.J. performed the sequencing, molecular analysis and antimicrobial susceptibility testing. K.J. and P.P. wrote the manuscript.

Supplementary data

Figure S1 is available as Supplementary data at JAC Online.