Characterization of Emerging Pathogens Carrying bla KPC-2 Gene in IncP-6 Plasmids Isolated From Urban Sewage in Argentina

Untreated wastewater is a reservoir for multidrug-resistant bacteria, but its role in the spread of antibiotic resistance in the human population remains poorly investigated. In this study, we isolated a KPC-2-producing ST2787 Klebsiella quasipneumoniae subsp. quasipneumoniae (WW14A), recovered from raw sewage at a wastewater treatment plant in Argentina in 2018 and determined its complete genome sequence. Strain WW14A was resistant to all β-lactams, ciprofloxacin and amikacin. A core genome phylogenetic analysis indicated that WW14A was closely related to a GES-5-producing Taiwanese strain isolated from hospital wastewater in 2015 and it was clearly distinct from strains isolated recently in Argentina and Brazil. Interestingly, bla KPC-2 was harbored by a recently described IncP-6 broad-spectrum plasmid which was sporadically reported worldwide and had never been reported before in Argentina. We investigated the presence of the IncP-6 replicon in isolates obtained from the same sampling and found a novel non-typable/IncP-6 hybrid plasmid in a newly assigned ST1407 Enterobacter asburiae (WW19C) also harboring bla KPC-2. Nanopore sequencing and hybrid assembly of strains WW14A and WW19C revealed that both IncP-6 plasmids shared 72% of coverage (~20 kb), with 99.99% of sequence similarity and each one also presented uniquely combined regions that were derived from other plasmids recently reported in different countries of South America, Asia, and Europe. The region harboring the carbapenem resistance gene (~11 kb) in both plasmids contained a Tn3 transposon disrupted by a Tn3-ISApu-flanked element and the core sequence was composed by ΔISKpn6/bla KPC-2/Δbla TEM-1/ISKpn27. Both strains also carried genes conferring resistance to heavy metals (e.g., arsenic, mercury, lead, cadmium, copper), pesticides (e.g., glyphosate), disinfectants, and several virulence-related genes, posing a potential pathogenic risk in the case of infections. This is the first study documenting bla KPC-2 associated with IncP-6 plasmids in K. quasipneumoniae and Enterobacter cloacae complex from wastewater in Argentina and highlights the circulation of IncP-6 plasmids as potential reservoirs of bla KPC-2 in the environment.

Untreated wastewater is a reservoir for multidrug-resistant bacteria, but its role in the spread of antibiotic resistance in the human population remains poorly investigated. In this study, we isolated a KPC-2-producing ST2787 Klebsiella quasipneumoniae subsp. quasipneumoniae (WW14A), recovered from raw sewage at a wastewater treatment plant in Argentina in 2018 and determined its complete genome sequence. Strain WW14A was resistant to all b-lactams, ciprofloxacin and amikacin. A core genome phylogenetic analysis indicated that WW14A was closely related to a GES-5-producing Taiwanese strain isolated from hospital wastewater in 2015 and it was clearly distinct from strains isolated recently in Argentina and Brazil. Interestingly, bla KPC-2 was harbored by a recently described IncP-6 broad-spectrum plasmid which was sporadically reported worldwide and had never been reported before in Argentina. We investigated the presence of the IncP-6 replicon in isolates obtained from the same sampling and found a novel nontypable/IncP-6 hybrid plasmid in a newly assigned ST1407 Enterobacter asburiae (WW19C) also harboring bla KPC-2 . Nanopore sequencing and hybrid assembly of strains WW14A and WW19C revealed that both IncP-6 plasmids shared 72% of coverage (~20 kb), with 99.99% of sequence similarity and each one also presented uniquely combined regions that were derived from other plasmids recently reported in different countries of South America, Asia, and Europe. The region harboring the carbapenem resistance gene (~11 kb) in both plasmids contained a Tn3 transposon disrupted by a Tn3-ISApu-flanked element and the core sequence was composed by DISKpn6/bla KPC-2 /Dbla TEM-1 /ISKpn27. Both strains also carried genes conferring

INTRODUCTION
The spread of carbapenem-resistant Gram-negative bacteria is an urgent and critical public health priority according to the World Health Organization (Tacconelli et al., 2018). Several classes of carbapenemases have emerged in members of the Enterobacteriaceae family, including class B metallo-b-lactamases (e.g., New Delhi Metallo-b-lactamase -NDM), class D (e.g.,  and class A carbapenemases, especially Klebsiella pneumoniae carbapenemase (KPC) (Rojas et al., 2017). Antimicrobial resistance genes (ARGs) and their bacterial hosts are widely distributed in clinical settings but also through the environment, especially in surface water, sewage treatment plant effluents, soil, and animal waste (Sekizuka et al., 2018). The appearance of bla KPC gene in the environment represents an emerging environmental issue with potentially serious public health implications (Hu et al., 2019). Nevertheless, environmental contamination with carbapenemaseproducing Enterobacteriaceae (CPE) has not been fully investigated. Compared to clinical isolates, available data for CPE in wastewater are limited, with few reports on genetic characteristics such as fine-scale within-species phylogeny and virulence gene profiles. Detailed characterization of environmental CPE is important to better understand the molecular epidemiology and reservoirs of these clinically important microbes (Gomi et al., 2018). Wastewater Treatment Plants (WWTPs) act as interfaces between the human population and the aquatic environment. Several previous studies have proposed WWTPs and wastewater to be hotspots for horizontal gene transfer, facilitating the exchange of ARGs among different bacterial species. While urban population keeps growing, an increased proportion of sewage effluents get into surface waters, being the role of WWTPs essential in reducing the spread of ARGs. WWTPs and wastewater act as anthropogenic sources, reservoirs, and environmental suppliers of ARGs, making it necessary to monitor the impact on the spread of resistance to antimicrobials (Gomi et al., 2018;Suzuki et al., 2019;Furlan et al., 2020).
Multidrug-resistant hypervirulent lineages of K. quasipneumoniae subsp. quasipenumoniae, recently defined as a new species, are an emerging issue for public health worldwide. Misidentification using standard laboratory methods is common and consequently, the clinical significance of K. quasipneumoniae is imprecisely defined (Rodrigues et al., 2018). While it was originally associated with environmental niches, there is actual evidence that it is able to sustain in hospitalized patients and disseminate among patients (Mathers et al., 2019). Moreover, this species has been shown to take up plasmids from other enterobacteria and harbor several resistance plasmids belonging to different incompatibility groups such as IncU/IncX5 (bla KPC ), IncHI2 (mcr-9) and IncFII/IncFIB (mcr-8.2) (Mathers et al., 2019;Yang et al., 2019;Faccone et al., 2020).
Members of the Enterobacter cloacae complex (ECC) are part of the human gut microbiota and are considered opportunistic pathogens responsible for a wide range of health-care-associated infections and hospital outbreaks, especially in intensive care units. Due to the presence of intrinsic chromosomal AmpC cephalosporinases, along with the acquisition of plasmidmediated extended-spectrum b-lactamases, carbapenems are among the "antibiotics of choice" to treat infections caused by isolates displaying high-level cephalosporin resistance. Therefore, the increasing carriage of bla KPC by members of the ECC over the last years is worrisome (De La Cadena et al., 2018).
Interestingly, KPC-producing Enterobacteriaceae and Aeromonas isolates from hospital wastewater in Taiwan, river sediments in China or coastal waters in the United States have been recently reported, all of them associated with IncP-6 plasmids (Botts et al., 2017;Gomi et al., 2018;Hu et al., 2019). IncP types are broad-host-range plasmids that have demonstrated the potential to mediate the dissemination of ARGs among Gram-negative bacteria, especially Enterobacterales and Pseudomonas aeruginosa (Cuzon et al., 2011;Naas et al., 2013;Dai et al., 2016;Gomi et al., 2018).
The KPC-2-encoding gene has been found in several plasmids from different incompatibility (Inc) groups, usually IncFII, IncL/ M, IncN, or IncA/C, but only rarely in other plasmids like IncP-6 and IncX (Yao et al., 2017). For example, in a continent-wide study performed in Europe with 1717 K. pneumoniae isolates, using a combination of long-and short-read sequence data, bla KPC -carrying IncP-6 plasmids were found only rarely (David et al., 2020). The emergence of bla KPC gene on IncP-6 broadhost-range plasmids, capable of replicating in both E. coli (where they are assigned into the IncG group) and Pseudomonas, has facilitated its rapid dissemination to Enterobacteriaceae and other Gram-negative families (Dai et al., 2016).
The aim of this study was to demonstrate the most relevant genomic features of two KPC-2-producing strains, a Klebsiella quasipneumoniae subsp. quasipneumoniae and an Enterobacter asburiae, isolated from a WWTP in Buenos Aires, Argentina, in 2018. Both strains harbor IncP-6 plasmids, for which we describe the genetic structure, in particular the backbone surrounding bla KPC-2 , and also compare to previously described plasmids, giving an overview of IncP-6 diversity.
This work contributes to a better understanding of acquired resistance and microbial adapting features in K. quasipneumoniae and ECC from a One Health perspective and pinpoints the important role of wastewater in global dissemination of resistance markers associated with IncP-6 backbone plasmids across multiple species around the world. To our knowledge, this is also the first report of IncP-6 plasmids circulating in Argentina and supports the hypothesis that IncP-6-bla KPC-2 promiscuous plasmids may have had a geographic origin in South America prior to their introduction in other countries of Europe and Asia (Yao et al., 2017).

Sampling and Bacterial Identification
A 1000-mL sample of raw wastewater collected at a municipal sewage treatment plant in Argentina was filtered through 0.45µm membranes, which were cultivated for 18 h at 37°C in MacConkey broth supplied with 10 µg/mL meropenem, to select for bacteria with reduced carbapenem susceptibility. The resulting suspension was then streaked on MacConkey agar and colonies showing different morphologies were selected and assessed by PCR, using primers targeting bla IMP , bla VIM , bla NDM , bla OXA-48 and bla KPC (Poirel et al., 2011). Two bla KPC positive strains were isolated and bla KPC-2 genes were identified in both strains by PCR amplification with primers KPC-F (5´-ATGTCACTGTATCGCCGTCT -3´) and KPC-R (5´-TTTTCAGAGCCTTACTGCCC -3´) (Saba Villarroel et al., 2017) followed by direct amplicon DNA sequencing. Carbapenemase production was checked by a modified Hodge test and a positive result for synergy between imipenem (30 mg) and phenyl boronic acid (300 mg) containing disks. Both isolates were identified to species level by MALDI-TOF/ MS (matrix-assisted laser desorption/ionizationtime of flight mass spectrometry) (Bruker Daltonics GmbH, Bremen, Germany), and designated WW14A (Klebsiella pneumoniae) and WW19C (E. asburiae).

Mobilization Experiments
The transmissibility of plasmids was tested with the filter mating protocol using E. coli J53 (sodium azide resistant) as recipient.
Mating was initiated by mixing 0.5 mL donor bacteria suspension with 0.5 mL recipient bacteria (16 h culture in lysogeny broth; LB) followed by immobilization onto a 0.22-µm nitrocellulose membrane filter. Control cultures were prepared identically with either donor or recipient bacteria alone. Filters were incubated overnight at 35°C on LB agar plates (no antibiotics). Biomass was removed with a sterile cotton swab and resuspended in 1 mL sterile saline. Fifty µL of suspended bacteria, either donor, recipient, or the mating combination, were spread onto LB agar plates supplemented with 50 µg/mL sodium azide and 10 µg/mL imipenem and incubated at 37°C for 18 h.

Pangenome Analysis
Pangenome analysis of WW14A and WW19C was performed for constructing whole genome single nucleotide polymorphism (SNP)-based within-genus phylogenetic trees, selecting isolates analyzed in other studies (Supplementary Table S1), i.e., isolates analyzed by Chavda et al. (2016), Gomi et al. (2018) andJousset et al. (2019) for WW19C; and isolates analyzed by Gomi et al. (2018) and all Klebsiella quasipneumoniae genomes present in NCBI Assembly database (June 5 th , 2020) for WW14A, aiming to place WW19C and WW14A in broader phylogenetic contexts. Briefly, annotated genomes were used in a pangenome analysis with Roary 3.13.0 to generate a core-gene alignment (using a blastp percentage identity of 95% and a core definition of 99%) (Page et al., 2015). This core-genome alignment was used to generate a SNP alignment with SNP-sites (v 2.5.1) (Page et al., 2016) which was later used to construct a maximum likelihood (ML) phylogenetic tree with RAxML (v 8.2.12) (Stamatakis, 2014) under the generalized time reversible model (GTR) and 100 bootstrap replicates.

RESULTS AND DISCUSSION
Membrane filtration and cultivation using meropenem selection of the wastewater sample followed by PCR screening allowed the detection of CPE, from which two KPC-producing strains, WW14A and WW19C, were selected for further analysis and whole genome sequencing.
Tables 1 and 2 summarize the genomic features observed in each strain, based on annotation outputs. The most relevant results of this analysis will be detailed and discussed in the following sections.
Sequence analysis revealed that WW14A isolate includes a genome of 6.01 Mb with 57.3% GC content. It was confirmed as Klebsiella quasipneumoniae subsp. quasipneumoniae and assigned as ST2787. Moreover, a core genome phylogenetic analysis indicated that strain WW14A is closely related to a GES-5 producing Klebsiella quasipneumoniae subsp quasipenumoniae isolate from a Taiwanese hospital wastewater (Gomi et al., 2018) and is clearly distinct from strains isolated recently in Argentina (Faccone et al., 2020) and Brazil (Fuga et al., 2020;Furlan et al., 2020) (Figure 1).
According to BIGSdb and KAPTIVE databases, the closest capsular type assigned for this strain is wzi88-KL70, considering there are 2 mismatches with the reference wzi cluster 88 and 78.7% identity with KL70 locus (9 from 20 genes present). The strain displayed a negative string test, so it may not be classified as hypermucoviscous. On the other hand, virulome analysis showed a hypervirulent profile, carrying genes encoding for aerobactins, enterobactins, hemolysins, pullulanase secretion, hyperadherence and biofilm formation. One intact phagerelated sequence (Haemophilus phage SuMu [39.8 kb]) was identified, besides other ten incomplete (7.2 to 34.7 kb) or questionable phages from Pseudomonas, Salmonella, E. coli and Cronobacter. Also, two confirmed (178 and 459 bp) and four questionable (94 to 121 bp) CRISPRs signatures were identified. The presence of phages and CRISPRs in WW14A genome shows its adaptative signatures, i.e., a memory of past genetic interactions with bacteriophages and plasmids ( Table 1).
WW14A is the first non-clinical KPC-2-producing isolate from Argentina which has been fully sequenced. Klebsiella quasipneumoniae was recently defined as a new species and was originally thought to be associated exclusively with environmental niches (Venkitapathi et al., 2021). However, despite there being relatively few reports to date, the true prevalence of this organism in clinical settings is likely underestimated as it is not generally distinguished from K. pneumoniae in routine testing of clinical laboratories (Mathers et al., 2019).

Enterobacter asburiae WW19C
WW19C was identified as E. asburiae with a 5.2 Mb genome and 55.2% GC content. The phylogenomic analysis confirmed that WW19C was related to E. asburiae EN3600, a clinical isolate coproducing IMP-8, CTX-M-14, CTX-M-3, and QnrS1 from China ( Figure 2)  Regarding its virulome, when compared to WW14A, WW19C shows the same categories of virulence, but with a broader variety of genes. Four intact phage-related sequences were identified: Enterobacter phage Tyrion (46.4 kb), Escherichia phages HK75 (34 kb) and 186 (26 kb), and Salmonella phage SEN34 (42.5 kb), the last being also found as an incomplete phage sequence in WW14A. In addition, WW19C also carried two incomplete (6.8 kb and 28.1kb) and two questionable phage sequences. Three questionable CRISPRs (96, 134 and 148 bp) were also identified ( Table 1).
Members of the ECC are opportunistic pathogens responsible for a wide range of healthcare-associated infections and hospital outbreaks, especially in intensive care units (De La Cadena et al., 2018). The emergence of carbapenem-resistant ECC like WW19C is of great concern as diverse plasmids of incompatibility group IncP-6 (20 -60 kb) were the most common carriers of bla KPC among ECC clinical isolates from Colombia, suggesting that KPC dissemination is not just due to horizontal transmission of a single plasmid-borne bla KPC , but that multiple rearrangements and transposition events can occur (Rojas Coy et al., 2019).

Overview of IncP-6 Plasmids Carrying bla KPC-2
IncP-6 plasmids identified in this study, designated pWW14A-KPC2 and pWW19C-KPC2, could be completely closed, analyzed, and compared with other closed plasmids belonging to the same incompatibility group. They both share 72% of coverage, with 99.99% of sequence similarity. Figure 3 shows a comparison of IncP-6 plasmids described herein with other complete IncP-6 described in the literature (Naas et al., 2013;Dai et al., 2016;Wang et al., 2017;Yao et al., 2017).
The bla KPC-2 -containing plasmid pWW14A-KPC2 was 40,407 bp in size with an average GC content of 57.9%. It comprised 55 open reading frames (ORF) according to RAST annotation, of which 25 encoded proteins with known functions and 30 were hypothetical proteins. Further analysis showed that the structure was highly similar to those of several bla KPC-2 -containing plasmids of both environmental and clinical origin deposited in GenBank. Plasmids pKOX3-P5-KPC, from a clinical K. oxytoca in China (GenBank accession no. KY913901) (Wang et al., 2017), p121SC21-KPC2 from Spanish wastewater Citrobacter freundii 121SC21 (Genbank accession no. NZ_LT992437.1) (Yao et al., 2017), and p10265-KPC, first reported in China from a clinical P. aeruginosa 10265 (GenBank accession no. KU578314) (Dai et al., 2016) were highly similar over the entire region (Figure 3). The bla KPC-2 gene is the only determinant of antimicrobial resistance located in plasmid pWW14A-KPC2. This is the first report of a typical IncP-6 plasmid carrying bla KPC-2 in a K. quasipneumoniae isolate.
On the other hand, pWW19C-KPC2 was 34,721 bp with an average GC content of 52.9%. It comprised 46 open reading frames (ORF) according to RAST annotation, of which 25 encoded proteins with known functions and 21 were hypothetical proteins. It showed the highest similarity with pECL189-1 from Enterobacter hormaechei ECL189 (GenBank accession no. CP047966.1, 72% coverage and 100% identity), a strain isolated from a Chinese hospital co-producing KPC-2, NDM-1, TEM-1 and SHV-66 ( Figure 3).

Replication, Maintenance and Dissemination of pWW14A-KPC2 and pWW19C-KPC2
Both plasmids belong to the IncP-6 incompatibility group according to replicon-based schemes because they carry the replicase gene repA, which constitutes an IncP-6-type consecutive par-rep gene cluster, together with the partition genes parABC. The repA gene and the parABC locus of pWW14A-KPC2 and pWW19C-KPC2 are identical and show 100%, >96%, >98%, and >98% of nucleotide sequence identity with the IncP-6 plasmids p10265-KPC, Rms149, pRIO-5, and pCOL-1, respectively. RepA in Rms149 has shown to confer the plasmid's replication ability in E. coli, P. aeruginosa, and P. putida and its parABC locus is known to promote plasmid mobilization in E. coli (Haines et al., 2005), while RepA in pRIO-5 allows replication in Serratia marcescens and Acinetobacter baumannii but not in P. aeruginosa (Bonnin et al., 2012). pWW19C-KPC2 also contains and extra plasmid replication initiation protein with 100% nucleotide identity with the replicon of pVPS18EC0801-5, a short nontypeable plasmid of 4910 bp present in foodborne E. coli strain (B) Core genome phylogenetic analysis of strain WW14A. The analysis showed that strain WW14A is closely related to a Taiwanese GES-5-producing Klebsiella quasipneumoniae subsp. quasipenumoniae isolate from hospital wastewater (TTHS016) and is clearly distinct from strains isolated recently from Argentina (M17277; Faccone et al., 2020) and America. TTHS021, TTWP002 and WW14A strains are marked with a pink dot, indicating they carry an incP-6 plasmid. (Klebsiella quasipneumoniae genomes present in NCBI Assembly database were used for comparison. Data was graphically displayed using Microreact at microreact.org). 18GA07VL07-EC isolated from retail veal in the United States in 2018 (GenBank accession no. CP063722.1, unpublished). The presence of two replication initiation genes, IncP-6 replicon repA and the replication initiation protein found in pVPS18EC0801-5 suggests pWW19C-KPC2 may be a novel hybrid plasmid (Supplementary Figure S1). The presence of extra replicon genes may expand pWW19C-KPC2 ability of maintenance and dissemination. The IncP-6 backbone of pWW14A-KPC2 was compared with p10265-KPC and it contained the same plasmid maintenance genes, as follows: kfrA; a 5.6 kb MOBP family mobilization module composed of genes mobA (relaxase/primase fusion protein), mobB (oriT recognition-like protein), mobC (relaxosome protein), mobD and mobE (auxiliary proteins); the anti-oxidative system msrB-msrA-yfcG-corA-orfX gene cluster; and endonuclease paeR7IR. Within the backbone, pWW14A-KPC2 harbored two accessory modules: on one hand, a truncated Tn5045-associated mercury resistance operon disrupted by IS4321 (the latter being absent in p10265-KPC), and on the other, ISPa19. Linear comparison of the abovementioned plasmids with pWW19C-KPC2 showed notable differences (Figure 4)  i. Absence of a~10 kb region that includes kfrA, ISPa19 and the MOBP family mobilization module; ii. Absence of a~4.9 kb region that includes the antioxidative system and paeR7IR; iii. The number of copies of the 17 bp oriV iteron sequence (GCGCCTGCCTTTGAGTA) was 6 in p10265-KPC and 14 in pWW14A-KPC2, in contrast to pWW19C-KPC2 where it appeared only 8 times; iv. The presence of a~5.2 kb extra region composed by the cluster ISSba14-Tn552 invertase bin3-transmembrane sulfite exporter tauE/safE -mopA. Blast search indicated that the extra region is present in other four plasmids of diverse origins with more than 99.9% identity (Supplementary Table S2), and it is also completely shared by the IncFI2 plasmid also present in E. asburiae WW19C.
None of the plasmids could be transferred to E. coli J53 via conjugation, despite repeated attempts. The examination of plasmid sequences confirmed the absence of conjugative transfer genes involved in plasmid transfer, such as the tra or trb operons, in accordance with the fact that typical IncP-6 plasmids are mobilizable rather than self-transmissible if a conjugative plasmid is also present (Botts et al., 2017). The mob gene cluster in pWW14A-KPC2 is functional for plasmid mobilization in E. coli (Yuan et al., 2019), being mobA, mobB, and mobC essential for functionality while mobD and mobE are non-essential but greatly enhance mobilization frequency (Dai et al., 2016). In the case of pWW19C-KPC2, which lacks this module, we inferred that this function is assumed by the relaxosome proteins coded in the unique accessory region related to the short non-typeable plasmid pVPS18EC0801-5 ( Figure 4).
Genomic Comparison of the bla KPC-2 Genetic Environment From pWW14A-KPC2 and pWW19C-KPC2 With Those From Related Plasmids The region harboring the bla KPC-2 gene in pWW14A-KPC2 is 11 kb long and contains a Tn3-based transposon disrupted by an ISApu-flanked element, where the core sequence is composed by DISKpn6/bla KPC-2 -Dbla TEM-1 -ISKpn27. This core module is connected with the gene cluster korC-orfX-klcA-orfX-repB. The resulting structure matched 99% to clinical isolates C. freundii M9169 and E. cloacae M11180 from Argentina (Gomez et al., 2011) and is associated with a truncated ISEc33 element. The resulting DISEc33-associated element is not bracketed by IRs and DRs, suggesting that its mobilization could be attributed to homologous recombination-based insertion of a foreign element Tn3-ISKpn27-Dbla TEM-1 -bla KPC-2 -ISKpn6-korC-orf-klcA-repB into a pre-existent intact ISEc33 element (making it truncated at 3´end), rather than resulting from a transposition event of the whole ISEc33-associated element followed by the deletion of its adjacent extremities removing IR and DR sequences (Dai et al., 2016). This core platform was initially discovered in p10265-KPC. In the bla KPC-2 gene cluster of p10265-KPC, the primary genetic structure, Tn3-ISKpn27bla KPC-2 -DISKpn6-korC-orf-klcA-DrepB, may have undergone two evolutionary events: (i) insertion of a bla TEM-1 gene between ISKpn27 and the Tn3 IRR (right inverted repeat) and (ii) disruption of the tnpA gene (transposase) from Tn3 by insertion of a composite transposon, ISApu1-orfX-ISApu2 (Dong et al., 2020). Interestingly, pWW19C-KPC lacked DISEc33 insertion sequence, indicating that the insertion of the bla KPC-2 cluster occurred at a different position in an IncP-6 backbone and seems to have a different evolutionary history of genetic assembly and transposition ( Figure 4). The acquisition of the KPC-2 encoding region by IncP-6 replicons is in agreement with the results of previous studies, in which similar plasmids show remnants of multiple events, with intact or partial mobile elements dispersed throughout their sequences (Botts et al., 2017). Its dissemination could be an important contribution to the establishment of emerging clones as major nosocomial pathogens (Cejas et al., 2019).

CONCLUSION
This is the first report on the genomic features of two nonclinical KPC-2-producing Enterobacteriaceae isolates from Argentina in terms of resistance determinants, genetic contexts of carbapenemase encoding genes, phylogeny, and virulence potential. To our knowledge, this is also the first report of IncP-6 plasmids circulating in Argentina and provides insights into the relevance of these plasmids in the maintenance and spread of KPC through the environment.
WW14A and WW19C had IncP-6 plasmids carrying bla KPC-2 in a Tn3-derived genetic element bearing a non-Tn4401 structure and its full sequence was determined and compared with other IncP-6 plasmids, from diverse origins. The presence of a highly similar plasmid in different isolates from distant countries raises questions about mechanisms of persistence and dissemination and indicates it might play an important role in the horizontal dissemination of KPC-2 carbapenem resistance through wastewater and the spread from wastewater to humans and vice versa. Our findings underline the increasing importance of IncP-6 plasmids as environmental reservoirs and the spread potential of the resistance segments they carry through reshuffling with other plasmids.
None of the plasmids could be transferred by conjugation, due to the absence of the transfer system genes, but we hypothesize they could be mobilized by coresident plasmids present in both strains.
Given that K. quasipenumoniae and Enterobacter spp. are ubiquitous organisms isolated from a wide range of environmental niches and given the fact that K. quasipneumoniae may have been misidentified in clinical sources, being its clinical relevance underestimated, they might act as important vectors for the dissemination of plasmid-mediated carbapenem-resistance genes. Therefore, effective detection of such plasmids in carbapenem resistant isolates from wastewater may be used as a potential epidemiological indicator. Treatment methods in most WWTPs are usually not enough to mitigate resistance genetic determinants, therefore surveillance in sewage and urban effluents could provide monitoring data to understand the evolution of antimicrobial resistance in the environment. New strategies should also be developed to limit plasmid spread into bacterial populations.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi. nlm.nih.gov/, PRJNA715927.

AUTHOR CONTRIBUTIONS
BG and MD, with the cooperation of GG and PPo conceived and designed the study. BG, MD, FB, PPe, GD'A, RF-E and LN performed experiments. BF, FE and NL performed WGS. BG, MD and MSH analyzed data. JC and RF-E provided assistance with MALDI-TOF/MS analysis and interpretation. MVH granted accession and supplied the water sample. MR provided laboratory infrastructure in Brazil. GG provided laboratory infrastructure in Argentina. BG, MD and MSH wrote this manuscript. All authors reviewed the manuscript. All authors contributed to the article and approved the submitted version.

FUNDING
This work was supported by Agencia Nacional de Promocioń Cientıfca y Tecnoloǵica PICT 2018-03413 to BG, and UBACyT 2018 -20020170100473BA to GG.