Plasmids Harboring a Tandem Duplicate of bla VIM-24 in Carbapenem-Resistant ST1816 Pseudomonas aeruginosa in Japan

Abstract

The aim of this study was to clarify the biological and clinical significance of a tandem duplicate of bla_VIM-24 in Pseudomonas aeruginosa ST1816 isolates. Thirteen ST1816 isolates carrying a plasmid harboring bla_VIMs were obtained from two medical settings in Japan between 2016 and 2019. Complete sequencing revealed that, of the 13 plasmids, four had a tandem duplicate of bla_VIM-24. These four plasmids harbored a replicon, a relaxase gene, and T4SS genes belonging to IncP-9, MOB_F, and MPF_T, respectively. All four plasmids transferred to PAO1 by filter mating. Cefepime marginally affected the growth of PAO1, carrying a pUCP19 harboring the tandem duplicate. Western blotting analysis showed that the relative intensity of VIM-24 metallo-β-lactamase produced by a PAO1 transformant containing a tandem duplicate was 2.6-fold higher than that produced by a PAO1 transformant containing a single copy. These results suggest that the tandem duplicate of bla_VIM-24 in plasmids may confer resistance against cefepime, enabling P. aeruginosa ST1816 strains to proliferate in hospitals in Japan.

Introduction

Metallo-β-lactamases (MBLs) cause resistance or reduced susceptibility to carbapenems, cephalosporins, and penicillins, except for monobactams, in various Gram-negative pathogens.¹ Verona integron-encoded MBLs (VIM-type MBLs) are among the most prevalent types of MBLs.² VIM-1 was first identified in a clinical isolate of Pseudomonas aeruginosa from northern Italy in 1997.³ Since then, VIM-type MBLs have been detected in various bacterial species worldwide;² to date, 80 VIM-type MBLs have been registered by the National Center for Biotechnology Information (NCBI; www.ncbi.nlm.nih.gov).

A strain of P. aeruginosa carrying a plasmid harboring bla_VIM-2 was first identified in France in 1996.² Since then, plasmids harboring bla_VIMs genes have been detected in Enterobacterale as well as in P. aeruginosa strains in several countries.² In Japan, a strain of Citrobacter freundii harboring a plasmid containing bla_VIM-2 was isolated in 2018.⁴ In addition, P. aeruginosa strains isolated in Japan were found to carry plasmids harboring bla_IMPs , but not bla_VIMs.⁵

Multilocus sequence typing (MLST) of P. aeruginosa strains is a useful molecular method to globally understand and identify international clones of P. aeruginosa, such as multidrug-resistant P. aeruginosa sequence type (ST) 235.⁶ Compared with ST235, ST1816 has been a minor clone.⁷ P. aeruginosa ST1816 isolates were first obtained in 2011 from blood samples of three children in Mexico, one each with Burkitt lymphoma, non-Hodgkin lymphoma, and Wilms tumor.⁸ These isolates were susceptible to meropenem and did not harbor any gene encoding a carbapenemase. P. aeruginosa ST1816 was isolated from a sputum sample in 2014 in Japan (PubMLST). A clinical isolate of P. aeruginosa ST1816, not harboring genes encoding any MBL, was reported in 2018 in Thailand.⁹

We recently reported the emergence and spread of VIM-type MBL-producing P. aeruginosa clinical isolates in Japan.⁷ Findings from this study showed that carbapenem-resistant P. aeruginosa ST235 and ST1816 strains harboring bla_VIMs were spreading clonally in hospitals in Japan. ST235 isolates were found to harbor bla_VIMs on their chromosomes, whereas ST1816 isolates harbored bla_VIMs on plasmids.

This study focused on plasmids harboring a tandem duplicate of bla_VIM-24 in four P. aeruginosa ST1816 clinical isolates. The bacteriological properties of these tandem duplicates were evaluated.

Materials and Methods

Bacterial strains

Thirteen clinical isolates of P. aeruginosa ST1816, each carrying a 65 kbp plasmid harboring bla_VIM genes, corresponding to bla_VIM-24, bla_VIM-60, or bla_VIM-66, were obtained from two hospitals in Japan between 2016 and 2019.⁷ Of these isolates, 12 were from hospital A in Ehime prefecture, and one was from hospital B in Hiroshima prefecture. The characteristics of these 13 isolates are shown in Supplementary Table S1, an update of Table 1 in our previous study.⁷ Antimicrobial susceptibility profiles were determined using the microdilution method, according to the criteria of the Clinical Laboratory Standards Institute (M100-S30).¹⁰ Genes associated with resistance to β-lactams, aminoglycosides, and quinolones were detected using ResFinder 3.0. Aminoglycoside resistance genes were detected using a future database¹¹ and in ResFinder 3.0.

Table 1.

Transferability of Plasmid Harboring Two Copies of bla_VIM-24 to Pseudomonas aeruginosa PAO1

Plasmid	Filter mating	Broth mating
pNCGM3596	2.5 × 10⁻⁷	<2.8 × 10⁻⁷
pNCGM3741	1.8 × 10⁻⁷	8.6 × 10⁻⁶
pNCGM3822	7.0 × 10⁻⁶	2.6 × 10⁻⁶
pNCGM3814_2	2.0 × 10⁻⁷	<2.9 × 10⁻⁷

Complete plasmid genome sequencing

The whole genome sequences of 13 isolates were determined using Miseq (Illumina, San Diego, CA) and MinION (Oxford Nanopore Technologies, Oxford, United Kingdom), according to the manufacturers' instructions. Genomic DNAs were extracted from these isolates using DNeasy Blood and Tissue kits (Qiagen, Tokyo, Japan) for Miseq, and Genomic-tip 100/G and Genomic DNA Buffer Set (Qiagen) for MinION. Summaries of Miseq and MinION were generated using CLC Genomic Workbench v.11.0.2 and NanoStat,¹² respectively. Phred quality scores (Q scores), defined as a property logarithmically related to the base calling error probabilities were determined as described.¹³ The raw reads generated by MiSeq and by MinION were assembled using Unicycler v.0.4.7.¹⁴

Replicon types, mobility (MOB) types (classification of relaxase amino acid sequences), and mating pair formation (MPF) types [classification of type IV secretion system (T4SS) involved in MPF] of plasmids^15,16 were determined using MOB-suite v.3.0.3 with a customized library that included replicon genes belonging to known incompatibility (Inc.) groups of Pseudomonas plasmids and customized parameters (—min_rep_ident 80—min_mob_ident 80—min_rep_cov 60—min_mob_cov 60).¹⁷ The complete DNA sequences of plasmids from all 13 isolates were compared using Easyfig. These sequences were analyzed using the Basic Local Alignment Search Tool (BLAST) (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and BLAST Ring Image Generator. The bla_VIM genes were detected by PCR using two sets of primers, F1/R1 and F2/R2 (Supplementary Table S2). These PCR products were sequenced using a DNA sequencer (3500xl Genetic Analyzer; Thermo Fisher Scientific).

Conjugation experiments

Plasmid transferability was tested using a rifampicin-resistant mutant of P. aeruginosa PAO1 (PAO1rifR) as a recipient strain and P. aeruginosa isolates carrying a plasmid harboring a tandem duplicate of bla_VIM-24 as donor strains. Conjugation was performed in broth¹⁸ and on membrane filters.¹⁹ The recipient and donor strains were used at the exponential phase of growth in the presence of rifampicin (50 mg/L) and meropenem (20 mg/L), respectively. In the conjugation experiments using broth, cells were harvested by centrifugation and washed with Mueller Hinton Broth (MHB) without antibiotics, and recipient and donor cells were mixed at a 4:1 ratio and incubated at 37°C for 2 hr. In the conjugation experiments using membrane filters, 2.0 mL of recipient cultures and 0.5 mL of donor cultures were mixed and filtered through membrane filters (pore size, 0.45 μm). The membrane was placed on a Mueller Hinton agar (MHA) plate and incubated at 37°C for 2 hr. The cells on the membrane were collected into 20 mL of MHB by washing the membrane.

Transconjugants were selected on MHA plates containing both meropenem (20 mg/L) and rifampicin (50 mg/L), and recipients were selected on plates containing rifampicin (50 mg/L) alone. The conjugation frequency was defined as the ratio of colony-forming units (CFUs) of the transconjugant to those of the recipient. Transconjugant DNA was subjected to PCR using primers F1/R1 (Supplementary Table S2) to detect bla_VIM-24; these DNA samples were subjected to PCR using primers F3/R3 to detect acsA, a housekeeping gene of P. aeruginosa. The PCR products were sequenced.

Transformation experiments

The open reading frames of bla_VIM-24 in P. aeruginosa NCGM3596 and armA, an aminoglycoside-resistant gene, in Escherichia coli MyJU780, along with their promoter regions, were PCR amplified using specific primer sets (Supplementary Table S2). The PCR products of bla_VIM and armA were digested with ShpI/BamHI and KpnI/SacI, respectively, and ligated into the plasmid shuttle vector pUCP19.²⁰ Plasmids harboring these PCR products were used to transform E. coli DH5α (TaKaRa Bio, Shiga, Japan). Transformants were selected on LB agar plates containing 50 mg/L ampicillin. These plasmids in E. coli DH5α were extracted and used to transform P. aeruginosa PAO1 by electroporation using an Eppenndorf Eporator (Eppendorf, Hamburg, Germany), according to the manufacturer's instructions. Transformants were selected on MHA plates containing amikacin (20 mg/L) and meropenem (20 mg/L). bla_VIM in the transformants was detected by PCR using the primers F1/R1 (Supplementary Table S2).

Growth curve and time-kill assay

The transformants, PAO1 carrying a plasmid harboring armA and bla_VIM-24 (PAO1::bla_VIM-24) and PAO1 carrying a plasmid harboring armA and a tandem duplicate of bla_VIM-24 (PAO1::bla_VIM-24bla_VIM-24), were cultured overnight. Aliquots containing 10⁶ CFU/mL each were inoculated into MHB containing meropenem (32 mg/L: 1/2 MIC) or cefepime (128 mg/L: 1/2 MIC) and further incubated at 37°C with shaking at 25 rpm in a TN-2612 incubator (Advantec, Osaka, Japan). A 100-μL aliquot of each culture was harvested at 0, 1, 3, 4, 6, and 24 hr, diluted in saline, and plated onto LB agar plates. After incubation for 24 hr at 37°C, the numbers of CFU were counted. Three independent time-kill assays were performed.

Western blotting

Aliquots containing 10⁶ CFU/mL PAO1::bla_VIM-24 and PAO1::bla_VIM-24bla_VIM-24 were inoculated into MHB and incubated for 3 hr to the exponential phase. Each strain was further incubated for 30 min in the presence or absence of cefepime (128 mg/L: 1/2 MIC). The bacterial cells were collected, solubilized in SDS-PAGE sample buffer, electrophoresed on SDS-PAGE gels (∼ 2 × 10⁷ CFU per 20 μL), and transferred to Immobilon-P membranes (Merck Millipore Ltd., Cork, Ireland). The Immobilon-P membranes were incubated with a mouse monoclonal anti-VIM-2 MBL antibody (RayBiotech, Peachtree Corners) and rabbit polyclonal anti-RecA antibodies (BioAcademia, Osaka, Japan).²¹

The membranes were subsequently incubated with the secondary antibodies, horseradish peroxidase (HRP)-linked goat anti-mouse IgG (Abcam, London, United Kingdom) and goat anti-rabbit IgG (Abcam), respectively, with signals detected by chemiluminescence (Western BLoT chemiluminescence HRP substrate; TaKaRa Bio, Shiga, Japan). The ratios of intensities of VIM-type MBL to RecA were calculated using NIH ImageJ software (https://imagej.nih.gov/ij/). Three independent Western blotting assays were performed.

GenBank accession numbers

The whole genome sequences of P. aeruginosa ST1816 using Miseq and Minion, and the complete plasmid sequences, were deposited in GenBank under accession numbers PRJDB8977 and PRJDB10522. Novel integrons were deposited in INTEGRALL under the numbers In2000, In2001, In2002, In2003, and In2004.

Results

DNA sequence accuracy

Supplementary Table S3 shows the accuracy of whole genome sequences of 13 P. aeruginosa ST1816 isolates harboring bla_VIMs. The MiSeq data for the 13 isolates consisted of 912,210 to 1,461,424 reads, of mean lengths ranging from 199.4 to 242.3 bases. The base call accuracy was high with a mean Q-score above 31, with 74.8% to 88.2% of reads having Q-scores ≥30. These Miseq data did not produce any whole DNA sequence of plasmids harboring bla_VIMs.

In contrast, the MinION data consisted of 19,424 to 56,907 reads, of mean lengths ranging from 9,484.6 to 14,276.9 bases. The base call accuracy was lower compared with Miseq data, with a mean Q-score of approximately 10, with 33.8% to 84.8% of reads having Q-scores ≥10. The MinION data alone yielded incomplete plasmid DNA sequences, despite their products being long reads of sequences (Supplementary Table S3). Assembly of both the Miseq and MinION data yielded complete DNA sequences of all 13 plasmids harboring bla_VIMs.

Complete DNA sequences of plasmids harboring bla_VIMs

Figure 1 shows a comparative analysis of plasmid sequences harboring bla_VIMs from the 13 isolates. The 12 isolates from hospital A had plasmids harboring bla_VIMs, ranging in size from 64,644 to 65,553 bp with sequence similarities >98.47% (Fig. 1A). These plasmids harbored a replicon, a relaxase gene, and T4SS genes belonging to IncP-9, MOB_F, and MPF_T, respectively. The isolate JUPA4295 from hospital B had a plasmid 59,422 bp in size harboring bla_VIM-60 and consisted of two parts. One part, consisting of nucleotides 9,514 to 28,871, had 95.46% sequence similarity to nucleotides 20,791 to 39,299 of pJUPA4001, and the second part, consisting of nucleotides −28,872 to 9,513, had 40.78% sequence similarity to nucleotides −39,300 to 20,790 bp of pJUPA4001 (Fig. 1A). This plasmid harbored a replicon gene belonging to IncP-11, whereas no relaxase or T4SS gene could be detected.

FIG. 1.

Comparative analysis of plasmid sequences harboring bla_VIMs. Genome rearrangement maps of plasmids in 13 Pseudomonas aeruginosa ST1816 isolates (A). These maps were drawn using Easyfig. Genomic environments surrounding bla_VIMs (B). All bla_VIM genes were located in class 1 integrons. Of the four types of structures, two had a tandem duplicate of bla_VIM-24.

Of the 13 plasmids harboring bla_VIM, four, pNCGM3596, pNCGM3741, pNCGM3822, and pNCGM3814_2, had two copies of bla_VIM in tandem (Fig. 1A). PCR and DNA sequencing of the PCR products confirmed that pNCGM3596 carried two copies of bla_VIM-24 in tandem, whereas pNCGN3661 carried a single copy of bla_VIM-60 (Supplementary Fig. S1).

Analysis of genetic environments surrounding the bla_VIM genes showed that all the bla_VIM genes were located in one of five class 1 integrons, In2000, In2001, In2002, In2003, and In2004 (Fig. 1B). The bla_VIM-24 genes were located in In2001 and In2002, followed by aadA1a (aadA1) and aacA31 (aac(6′)-31) or aadA1a. The bla_VIM-60 genes were located in In2000 and In2004, followed by aadA1a and aacA31 or aadA1a, aacA31, and aadA1a. The bla_VIM-66 gene was located in In2003 followed by aadA1a and aacA31 (Fig. 1B). Of the five integrons, three (In2000, In2003, and In2004, which had a copy of bla_VIMs) had a promoter variant PcS with strong promoter activity.²² The remaining two integrons (In2001 and In2002, both of which had a tandem duplicate of bla_VIM-24) had PcH1 with moderate promoter activity (Fig. 1B).²²

Comparative analysis of plasmids harboring bla_VIMs

The structures of plasmids of NCGM3596 obtained from Hospital A (pNCGM3596) (accession number LC586264) were similar to those of the plasmid in P. aeruginosa strain UNC_PaerCF16 (accession number CP080283), isolated in the United States in 2021 (query cover, 83%; identity, 99%); the plasmid pCCBH28525_KPC in P. aeruginosa strain CCBH28525 (accession number CP086065), isolated in Brazil in 2021 (query cover, 70%; identity, 99%); the plasmid in Pseudomonas mosselii strain DSM17497 (accession number CP081967), isolated in China in 2021 (query cover, 79%; identity, 99%); and the plasmid in Pseudomonas putida strain MX-2 (accession number CP046873), isolated in China in 2020 (query cover, 77%; identity, 99%) (Fig. 2A).

FIG. 2.

Comparative analysis of plasmids, pNCGM3596 and pJUPA4225, in P. aeruginosa NCGM3596 (obtained from Hospital A) and JUPA4225 (obtained from Hospital B), respectively. Comparative plasmid circular genome visualization of pNCGM3596, a plasmid in P. aeruginosa strain UNC_PaerCF16 isolated in the USA in 2021 (accession no. CP080283); pCCBH28525_KPC isolated in Brazil in 2021 (accession no. CP086065); a plasmid in Pseudomonas mosselii strain DSM17497 isolated in China in 2021 (accession no CP081967); and a plasmid in Pseudomonas putida strain MX-2 isolated in China in 2020 (accession no. CP046873) (A). Comparative circular genome visualization of pNCGM4295, pPA7790 isolated in Brazil in 2016 (accession no. CP015000), and pMS14403 isolated in Australia in 2016 (accession no. CP049162) (B). The circular genome maps were visualized using BLAST Ring Image Generator.

The plasmid from the isolate JUPA4295 from Hospital B (pJUPA4295) (accession number LC586269) had a structure similar to the plasmid pPA7790 in P. aeruginosa strain PA7790 (accession number CP015000), isolated in Brazil in 2016 (query cover, 64%; identity, 98%); and the plasmid pMS14403 in P. aeruginosa strain MS14403 (accession number CP049160), isolated in Australia in 2016 (query cover, 64%; identity, 98%) (Fig. 2B).

Transferability of plasmids harboring two copies of bla_VIM-24

The four plasmids harboring a tandem duplicate of bla_VIM-24, pNGCM3596, pNCGM3741, pNCGM3822, and pNCGM3814-2 (Fig. 1A), were transferred to P. aeruginosa PAO1-rifR by filter mating (Table 1). The plasmids pNCGM3741 and pNCGM3822 were also transferred by broth mating, but the other two were not (Table 1). Compared with PAO1, the transconjugants were resistant to cefepime, ceftazidime, imipenem, and meropenem, but remained susceptible to amikacin, aztreonam, ciprofloxacin, and colistin (Table 2).

Table 2.

Antimicrobial Susceptibilities of ST1816 Isolates Harboring a Tandem Duplication of bla_VIM-24, Transconjugants and Transformants

Strain	MICs against antibiotics (mg/L)
Strain	AMK	AZT	FEP	CAZ	CIP	CST	IPM	MPM	RIF
Reference strain
PAO1	1	4	1	1	<0.25	0.25	1	1	16
Rifampicin-resistant mutant of PAO1 for conjugation of plasmid
PAO1rifR	1	4	1	1	<0.25	0.25	1	1	>512
ST1816 isolates harboring two copies of bla_VIM-24
NCGM3596	32	32	128	64	32	0.25	256	512	4
NCGM3741	16	32	256	64	16	0.25	256	512	4
NCGM3814-2	8	32	256	32	64	0.125	256	512	8
NCGM3822	16	8	128	32	16	0.125	256	512	2
Transconjugant
PAO1rifR/pNCGM3596	1	8	512	256	<0.25	0.25	128	128	>512
PAO1rifR/pNCGM3741	1	8	512	256	<0.25	0.5	128	128	>512
PAO1rifR/pNCGM3814-2	1	4	256	256	<0.25	0.125	64	64	>512
PAO1rifR/pNCGM3822	1	8	512	256	<0.25	0.25	128	128	>512
Transformant
PAO1 carrying pUCP19/armA	>512	4	2	1	<0.25	2	1	0.5	N.D
PAO1 carrying pUCP19/armA and a copy of bla_VIM-24	>512	4	256	128	<0.25	1	16	64	N.D
PAO1 carrying pUCP19/armA and two copies of bla_VIM-24	>512	4	256	256	<0.25	2	32	64	N.D

AMK, amikacin; AZT, aztreonam; FEP, cefepime; CAZ, ceftazidime; CIP, ciprofloxacin; CST, colistin; IMP, imipenem; MEM, meropenem; RIF, rifampicin.

Kinetics of PAO1 carrying a plasmid harboring a tandem duplicate of bla_VIM-24

PAO1 carrying a plasmid harboring armA and bla_VIM-24 (PAO1::bla_VIM-24), and PAO1 carrying a plasmid harboring armA and a tandem duplicate of bla_VIM-24 (PAO1::bla_VIM-24bla_VIM-24) showed significantly higher MICs toward cefepime, ceftazidime, imipenem, and meropenem than PAO1 carrying a plasmid harboring armA, a vector control (Table 2). Both PAO1::bla_VIM-24 and PAO1::bla_VIM-24bla_VIM-24 showed essentially the same MIC values toward these drugs (Table 2).

To clarify the biological significance of the tandem duplicate of bla_VIM-24, growth kinetics assays were performed using these transformants (Fig. 3). PAO1::bla_VIM-24 and PAO1::bla_VIM-24bla_VIM-24 showed similar growth curves in the absence of drug, with both starting to grow exponentially 3 hr after inoculation (Fig. 3A, B). In the presence of 1/2 MIC of meropenem (32 mg/L), both PAO1::bla_VIM-24 and PAO1::bla_VIM-24bla_VIM-24 did not start to grow until 9 hr after inoculation, followed by exponential growth (Fig. 3A).

FIG. 3.

Kinetics of PAO1::bla_VIM-24 and PAO1::bla_VIM-24bla_VIM-24 growth in the presence or absence of drugs. PAO1::bla_VIM-24 (circle) and PAO1::bla_VIM-24bla_VIM-24 (triangle) were cultured in the presence (open symbols) or absence (closed symbols) of 1/2 MIC of meropenem (MPM; 32 mg/L) (A), or 1/2 MIC of cefepime (FEP; 128 mg/L) (B), and bacterial density was monitored automatically. Data in (A) and (B) are representative of three independent experiments. PAO1::bla_VIM-24 (circle) and PAO1::bla_VIM-24bla_VIM-24 (triangle) were cultured in the presence (open symbols) or absence (closed symbols) of 1/2 MIC of FEP (128 mg/L) (C), and the numbers of CFU were counted at indicated times. Data are the mean ± SD of CFUs from three independent experiments.

In the presence of 1/2 MIC of cefepime (128 mg/L), PAO1::bla_VIM-24 did not start to grow until 12 hr after inoculation, followed by an exponential growth, whereas PAO1::bla_VIM-24bla_VIM-24 started to grow exponentially 4 hr after inoculation, with the start slightly delayed compared with the same strain in the absence of drugs (Fig. 3B). Similar delay to start growth of PAO1::bla_VIM-2 was observed in the presence of 1/2 MIC of ceftazidime (64 mg/L) compared with that of PAO1::bla_VIM-24bla_VIM-24 in the presence of the same concentration of ceftazidime (1/4 MIC), although the MICs of ceftazidime were different between these two isolates (128 and 256 mg/L, respectively) (Supplementary Fig. S2).

Counting of viable cells showed essentially the same growth curves of PAO1::bla_VIM-24 and PAO1::bla_VIM-24bla_VIM-24 in the absence of drug (Fig. 3C). In the presence of 1/2 MIC of cefepime (128 mg/L), the number of CFUs of PAO1::bla_VIM-24 declined until 6 hr after inoculation, subsequently increasing to the same number of CFUs observed in the absence of cefepime at 24 hr after inoculation (Fig. 3C). In contrast, the number of CFUs of PAO1::bla_VIM-24bla_VIM-24 did not change until 4 hr after inoculation, subsequently increasing to the same number of CFUs observed in the absence of cefepime (Fig. 3C).

Production of VIM-24

Western blotting analysis showed that, in the absence of cefepime, PAO1::bla_VIM-24bla_VIM-24 produced a larger amount of VIM-24 than PAO1::bla_VIM-24, whereas the two strains produced similar amounts of RecA (Fig. 4A). Cefepime had little effect on the production of both VIM-24 and RecA by these strains (Fig. 4A). Densitometric analysis showed PAO1::bla_VIM-24bla_VIM-24 produced a 2.6-fold higher amount of VIM-24 MBL than PAO1::bla_VIM-24 (p < 0.01), with the amount of VIM-24 MBL not enhanced in the presence of cefepime (Fig. 4B).

FIG. 4.

VIM-24 metallo-β-lactamase production by transformants. PAO1::bla_VIM-24 and PAO1::bla_VIM-24 were incubated for 3 hr and then further incubated for 30 min in the presence or absence of cefepime at 1/2 MIC. The production of VIM-24 MBL and RecA was detected by Western blotting using antibodies specific to each protein (A). The intensity of each signal was quantified using NIH ImageJ software, and the ratios of the intensity of VIM-24 MBL to that of RecA were determined. The ratios represent the mean ± SD of three independent experiments. *Represents significance at p < 0.01 (B).

Discussion

Acquisition of multiple copies of genes encoding carbapenemases is a strategy adopted by drug-resistant P. aeruginosa to survive and expand clonally in medical settings. This study clearly indicated that a tandem duplicate of bla_VIM-24 conferred more resistance against 1/2 MIC of cefepime than a single copy of bla_VIM-24, although the tandem duplicate did not change the MIC values. The tandem duplicate also conferred more resistance against sub-MICs of ceftazidime than the single copy. The results may be explained by the twofold higher production of VIM-24 in PAO1 harboring a tandem duplicate of bla_VIM-24 than a single copy. A carbapenem-resistant P. aeruginosa isolate carrying a plasmid with a class 1 integron containing three copies of bla_VIM-1 was reported in Spain in 2015.²³ A PAO1 transformant carrying this plasmid showed twofold higher MICs toward carbenicillin, ceftazidime, and meropenem than a PAO1 transformant carrying a plasmid harboring a single copy of bla_VIM-1, although these plasmids did not have isogenic structures.²³ A carbapenem-resistant P. aeruginosa isolate carrying a plasmid harboring four copies of bla_GES-5 was reported in China in 2018.²⁴ These four bla_GES-5 copies were located within a class 1 integron, and a PA1280 transformant carrying the plasmid showed a 2.07-fold higher expression of bla_GES-5 and twofold higher MICs toward piperacillin-tazobactam, cefepime, meropenem, and imipenem than a PA1280 transformant with the same plasmid harboring a single copy of bla_GES-5.²⁴

The presence of a tandem duplicate of bla_VIM-24 in four plasmids, pNGCM3596, pNCGM3741, pNCGM3822, and pNCGM3814-2, may be due to the enzymatic properties of VIM-24. In this study, the growth of PAO1::bla_VIM-24bla_VIM-24 was marginally affected by cefepime, but not meropenem, at 1/2 MIC. VIM-24 was first detected in a clinical isolate of Klebsiella pneumoniae, which was highly resistant to ceftazidime and cefepime, but moderately resistant to imipenem.²⁵ VIM-24 had higher hydrolytic activities toward cefepime and ceftazidime than toward imipenem.²⁶ The four plasmids harboring a tandem duplicate of bla_VIM-24 may enhance the survival rates of P. aeruginosa clinical isolates in the presence of sub-MIC levels of cephalosporins, but not carbapenems.

Although all four plasmids could be transferred to PAO1 during filter mating, two of these plasmids, pNGCM3596 and pNCGM3814-2, did not transfer to PAO1 in broth. These differences may be due to MPF types. The four plasmids encoded MPF_T, which forms short and rigid pili.²⁷ The transferability of plasmids with MPF_T is more efficient on solid surfaces than in liquid.²⁸ The conjugation frequency of a plasmid belonging to MPF_T, pWW0, was reported to be 18-fold higher during filter than broth maiting.²⁹

In conclusion, this study found that a tandem duplicate of bla_VIM-24 on plasmids confers resistance against sub-MICs of cefepime and ceftazidime, but not meropenem, on P. aeruginosa ST1816. This tandem duplicate resulted in the spread of this strain in a hospital in Japan.

Ethical Approval

This study was approved by Biosafety Committee, 226 Juntendo University (approval number: BSL2/29-1).

Authors' Contributions

The article was seen and approved by all the authors and is not under consideration elsewhere. All the authors contributed to the work in this study. T.H. performed all experiments and wrote the initial draft of the article; T.T. designed this study and edited the article; M.T. performed genomic analyses; M.Shin. performed conjugation experiments; M.Su. conducted in silico plasmid typing; M.Shim. collected clinical isolates; and T.K. supervised this study. All authors read and approved the final article.

Footnotes

Disclosure Statement

No competing financial interests exist.

Funding Information

This study was supported by grants from Japan Society for the Promotion of Science (grant numbers: 18K07120, 20K16252, and 22K15460) and Japan Agency for Medical Research and Development (grant numbers: JP21fk0108604, JP22gm1610003, JP22fk0108139, JP22fk0108133, JP22fk0108642, JP22wm0225008, JP22wm0225004, JP22wm0225022, JP22wm0325003, JP22wm0325022, and JP22wm0325037).

Supplementary Material

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Supplementary Material

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Plasmids Harboring a Tandem Duplicate of bla VIM-24 in Carbapenem-Resistant ST1816 Pseudomonas aeruginosa in Japan

Abstract

Introduction

Materials and Methods

Bacterial strains

Complete plasmid genome sequencing

Conjugation experiments

Transformation experiments

Growth curve and time-kill assay

Western blotting

GenBank accession numbers

Results

DNA sequence accuracy

Complete DNA sequences of plasmids harboring blaVIMs

Comparative analysis of plasmids harboring blaVIMs

Transferability of plasmids harboring two copies of blaVIM-24

Kinetics of PAO1 carrying a plasmid harboring a tandem duplicate of blaVIM-24

Production of VIM-24

Discussion

Ethical Approval

Authors' Contributions

Footnotes

Disclosure Statement

Funding Information

Supplementary Material

References

Supplementary Material

Complete DNA sequences of plasmids harboring bla_VIMs

Comparative analysis of plasmids harboring bla_VIMs

Transferability of plasmids harboring two copies of bla_VIM-24

Kinetics of PAO1 carrying a plasmid harboring a tandem duplicate of bla_VIM-24