An IncP-2 plasmid sublineage associated with dissemination of blaIMP-45 among carbapenem-resistant Pseudomonas aeruginosa

ABSTRACT IMP-45, a variant of IMP-9, is one of the dominant metallo-β-lactamases (MBLs) in clinical carbapenem-resistant Pseudomonas aeruginosa (CRPA) isolates in China. The aim of this study was to investigate the distribution and mechanism of dissemination of blaIMP-45. MBL genes were detected by PCR in 173 non-duplicate CRPA isolates collected from Hospital HS in Shanghai and 605 P. aeruginosa isolates from a multicenter surveillance of blaIMP-45 in China. In total, 17 IMP-45-producers (14 from Hospital HS and 3 from other hospitals) were identified. Molecular typing identified an outbreak of 11 IMP-45-producing ST508 CRPA in the ICU of Hospital HS. Conjugation assays and whole genome sequencing were conducted among IMP-45-producers. Genomic comparison revealed that 16 blaIMP-45-carrying plasmids (9 from this study and 7 from GenBank) shared a similar backbone with IncP-2 blaIMP-9-carrying plasmid pOZ176 but lacked repA-oriV-parAB region. repA2 gene was presented in pOZ176, blaIMP-45-carrying plasmids (17 from this study and 7 from GenBank) and 15 megaplasmids from GenBank. Phylogenetic analysis of repA2 showed that most blaIMP-45-carrying plasmids were clustered into a sublineage separate from the one containing pOZ176. This IncP-2 plasmid sublineage contributed to the dissemination of blaIMP-45 among genetically diverse P. aeruginosa and recruited multiple resistance genes during its evolution.


Introduction
Pseudomonas aeruginosa is prone to be resistant to βlactams, aminoglycosides and quinolones. Production of metallo-β-lactamases (MBLs) is one of the primary carbapenem resistance mechanisms in this species, among which IMP and VIM are the most prevalent [1]. IMP-1, IMP-4, IMP-6, IMP-8, IMP-9, IMP-10 and IMP-45 have been reported in China [2]. IMP-9 was initially identified in P. aeruginosa isolates from Guangzhou, China [3]. Afterwards, outbreaks of IMP-9-producing P. aeruginosa were observed in this area in 2000 and from 2005 to 2007 [3,4]. IMP-45, a single amino acid substitution variant (G214S) of IMP-9, was first reported in a canine-origin P. aeruginosa from Beijing, China in 2014, showing higher level resistance to meropenem than to imipenem [5]. Afterwards, more clinical isolates of IMP-45-producing Pseudomonas were discovered [6][7][8], including one isolated from a French patient who had been repatriated from an ICU in Shanghai, China [8], implying the risk of worldwide spread of bla IMP-45 by such dissemination.
In this study, we report an outbreak of carbapenem-resistant P. aeruginosa (CRPA) co-carrying bla IMP-45 , qnrVC1 and armA in a tertiary hospital of Shanghai and the disappearance of outbreak clones after strengthened infection control measures. Subsequently, we carried out a multicenter surveillance of bla IMP-45 in P. aeruginosa clinical isolates in China and explored the role of IncP-2 plasmids in the dissemination of bla IMP-45 among P. aeruginosa.

Clinical isolates and antimicrobial susceptibility testing
One hundred and seventy-three non-duplicate CRPA isolates were collected from a tertiary hospital (Hospital HS) in Shanghai between January 2015 and April 2018. CRPA was defined as P. aeruginosa isolate resistant to either imipenem or meropenem. Additionally, a multicenter surveillance was performed with 605 nonduplicate P. aeruginosa isolates collected consecutively from 11 hospitals in 8 provinces/municipalities across China, including 3 hospitals in Shanghai, 2 in Beijing and 1 in each of the other 6 provinces (July, 2018 to February, 2019, Supplementary Table S1).
Minimal inhibitory concentrations (MICs) were determined for 13 antimicrobial agents by broth microdilution method and interpretation was according to recommendations of the CLSI [11].

Molecular typing of P. aeruginosa isolates
Pulsed-field gel electrophoresis (PFGE) was performed using SpeI (TaKaRa Bio, Dalian, China) as the restriction enzyme and with a switch time of 2 s-40 s [14,15]. The PFGE patterns were analysed by Bio-Numerics (version 4.0; Applied Maths, Inc.) and a dendrogram was generated by the UPGMA method based on the Dice coefficient.

Conjugation assay
Transfer of plasmid carrying bla IMP-45 was performed by filter mating with P. aeruginosa PAO1 Rif (rifampin resistant) as recipient. Transconjugants were selected on LB plates supplemented with meropenem (2 mg/ L) and rifampin (500 mg/L), and further confirmed by PCR amplification and sequencing of bla IMP-45 and guaA, one of the seven housekeeping genes for MLST of P. aeruginosa. The transconjugants harbouring bla IMP-45 were tested for antimicrobial susceptibility. Conjugation was also carried out using Escherichia coli J53 (azide resistant) as recipient.
For the 8 transconjugants of outbreak isolates without WGS data, the repA2 gene and genetic context surrounding bla IMP-45 were amplified by PCR and sequenced with a series of primers designed according to the genome sequence of the transconjugant of P. aeruginosa outbreak isolate HS15-101 (Table S2).

Bioinformatics analysis
The contigs were annotated using RAST (http://rast. nmpdr.org/), screened for insertion sequences with ISfinder [21], and analysed for STs, antibiotic resistance genes and plasmid typing at the Centre for Genomic Epidemiology web site (http://www.genomicepi demiology.org/). BLASTN searches were conducted to find the complete sequenced bla IMP-45 -harbouring plasmids in GenBank database using bla IMP-45 as the query sequence. BLAST Ring Image Generator (BRIG) [22] were used in the comparative analysis of the plasmids.

Results
Outbreak of bla IMP-45 -bearing P. aeruginosa in Hospital HS Fourteen out of 173 CRPA from Hospital HS were positive for bla IMP-45 : 12 CRPA isolated from 2015, 1 from 2017 and 1 from 2018. All of them exhibited resistance to antipseudomonal β-lactams excluding aztreonam, β-lactamase inhibitor combinations and aminoglycosides. The MICs of aztreonam ranged from 4 to 16 mg/L in 7 strains whereas the remaining strains were highly resistant to aztreonam (from 64 and >128 mg/L). All the bla IMP-45 -carriers were resistant to quinolones except isolate HS17-127 (Table 1).
Eleven out of 12 bla IMP-45 -bearing CRPA from 2015 were isolated from ICU patients and shared the same sequence type, ST508 (Figure 1(A)). They presented indistinguishable or closely related PFGE patterns, different from that of HS15-106 (ST3014) from an outpatient. All the 12 bla IMP-45 -bearing CRPA from 2015 were discovered in the first nine months this year as indicated by the timeline of the isolation date of the first bla IMP-45 -carrying P. aeruginosa from each patient (Figure 1(B)). Taken together, an outbreak of bla IMP-45 -bearing ST508 CRPA occurred in the ICU of Hospital HS in 2015. The other two bla IMP-45 -bearing CRPA, HS17-127 (ST369) and HS18-41 (ST357), were clonally distinct from the previous outbreak bla IMP-45 -carriers in Hospital HS.

Transferability of bla IMP-45
To examine the transferability of bla IMP-45 , all the 17 bla IMP-45 -carrying CRPA were performed conjugation with P. aeruginosa PAO1 Rif as recipients. Fifteen transconjugants harbouring bla IMP-45 were obtained from the 11 ST508 outbreak strains and HS17-127, HS18-41 from Hospital HS as well as KM18-18 and RJ19-28 from another two hospitals. All the transconjugants contained the same guaA allele with the recipient P. aeruginosa PAO1 Rif . Transconjugants displayed similar antimicrobial susceptibility profiles with their donors except that 3 transconjugants (TcHS15-158, TcHS15-172 and TcHS15-209) were susceptible to quinolones (Supplementary Table S4). However, transfer of bla IMP-45 to E. coli failed.
General features of bla IMP-45 -harbouring plasmids P. aeruginosa clinical isolate HS17-127 was fully sequenced resulting in one chromosome and one plasmid pHS17-127. The sequencing analysis revealed that repA2 and bla IMP-45 genes were on the plasmid pHS17-127 (486,963 bp), which shared a highly similar backbone with IncP-2 plasmid pOZ176 ( Figure 2). Whole genome sequences of 3 transconjugants and 5 clinical isolates in this study (Supplementary Table  S5) were compared to the fully sequenced plasmid pHS17-127 and the 7 completely sequenced bla IMP-45 -harbouring plasmids in GenBank (up to April, 2020). The 7 plasmids, varying in size from 374 kb to nearly 514 kb, were isolated in China and from P. aeruginosa except for 1 from P. putida [6] (Table  2). Comparative genome analysis revealed that these 16 bla IMP-45 -carrying plasmids ( Table 2) shared an overall similar backbone, including genes essential for replication (repA2, 1188 bp), partition (par) and conjugal transfer (tra) (Figure 2). Moreover, they contained an operon terABCDEZ conferring tellurite resistance, which is a uniform property of IncP-2 plasmids. Virulence factors, such as pilus biogenesis gene pilD and chemotaxis gene cluster, cheABRWXZ, were also identified in the backbones.
When BLASTN with the repA2 gene of pOZ176 was performed for its homologs (100% query coverage), a total of 23 fully sequenced megaplasmids were identified in NCBI database, including pOZ176 and the above 7 bla IMP-45 -harbouring plasmids ( Figure  3). They shared a similar backbone with pOZ176 as well as the bla IMP-45 -carrying plasmids in this study, even though these megaplasmids were absent of a large fragment containing repA-oriV-parAB of pOZ176 ( Figure 2). Additionally, repA2 genes were confirmed by PCR and sequencing in the remaining 8 transconjugants of outbreak strains without WGS data in this study (Supplementary Table S2). Phylogenetic analysis of the 40 repA2 genes from 17 bla IMP-45carrying CRPA in this study and 23 megaplasmids in GenBank revealed 4 distinct subgroups (Figure 3). All the bla IMP-45 -carrying plasmids with the exception of RJ19-28 were in a sublineage separate from the one containing bla IMP-9 -harbouring pOZ176. IncP-2 plasmids and other plasmid lineages, such as IncP-7 and IncP-9, were phylogenetically analysed on the basis of replication genes, revealing that IncP-2 plasmid lineage was clearly seperated from other plasmid lineages ( Figure S2). Coexistence of bla IMP-45 , arma and qnrVC1 or qnrVC6 Apart from 1 plasmid carrying only bla IMP-45 and 1 with both bla IMP-45 and qnrVC6, 14 out of 16 plasmids co-carried bla IMP-45 and armA, including 9 together with qnrVC1 and 3 with qnrVC6 ( Table 2). The genetic structures harbouring these resistant determinants were confirmed by WGS analysis or PCR and sequencing (Supplementary Table S2). As in the previously reported IMP-45 producers [5,8], bla IMP-45 was located in the variable region of In786, adjacent to a Tn1548derivative containing armA with or without qnrVC6 ( Figure S1). qnrVC1 was in class 1 integron In1237 that coexisted with the Tn1548-derivative on the megaplasmids. However, In1237 was not transferred to the recipient strain together with bla IMP-45 -harbouring plasmids from HS15-158, HS15-172 and HS15-209.
Although at least 14 incompatible groups (IncP-1∼IncP-14) of plasmids have been identified in Pseudomonas species, there is no well-established scheme for the plasmid typing of this species as of Enterobacteriaceae [27,28]. Similar to IncP-2 plasmids reported previously [9,10], the bla IMP-45 -carrying plasmids are tellurite resistant, conjugative, and transfer between P. aeruginosa and P. putida but not to E. coli. Plasmids usually undergo continuous rearrangement and mutations, sometimes occurring in regions for plasmid typing [29], resulting in novel untypeable plasmids or new plasmid lineages evolving from currently well-studied plasmid types. The incompatibility group of bla IMP-45 -carrying plasmids has not been fully clarified since they lack the region containing IncP-2 repA-oriV-parAB [9]. A previous study reported that IncP-2 repA-oriV-parAB, an auxiliary replicon, was located in an integrative and conjugative element Tn6398a [30]. Moreover, homologs of IncP-2 repA gene were seldom present in Pseudomonas spp. but more frequently found in putative genomic islands on the chromosome of a variety of other species, such as Azotobacter, Burkholderia, Stenotrophomonas and Xanthomonas [9].
In contrast, IncP-2 repA2 gene and its close relatives (>96%) have been exclusively discovered on plasmids from Pseudomonas spp., indicating that the repA2 gene is probably the actual one encoding the IncP-2 replication initiator protein and contributing to the narrow-host-range of IncP-2 plasmids for Pseudomonas spp.
Based on the phylogenetic analysis of repA2 and the comparative genome analysis in this study, all the plasmids involved are closely related genetically, belonging to the same plasmid family as IncP-2 pOZ176. The phylogenetic tree of repA2 grouped almost all bla IMP-45 -carrying plasmids into a different subgroup from the one containing pOZ176.
In summary, clonal diversity was observed in the 17 IMP-45-producing CRPA isolates identified in this study except for outbreak clone ST508 from Hospital HS. All the bla IMP-45 -carrying plasmids were related to IncP-2 plasmid pOZ176, and contributing to the dissemination of bla IMP-45 . Moreover, this IncP-2 plasmid sublineage has undergone multiple evolutionary events, recruiting bla IMP-45 , armA and qnrVC1/ qnrVC6, thus acting as a vehicle for the dissemination of carbapenem, aminoglycoside and quinolone resistance among Pseudomonas spp., with consequent compromise of therapeutic options.