Molecular Characterization and Comparative Genomics of IncQ-3 Plasmids Conferring Resistance to Various Antibiotics Isolated from a Wastewater Treatment Plant in Warsaw (Poland)

As small, mobilizable replicons with a broad host range, IncQ plasmids are widely distributed among clinical and environmental bacteria. They carry antibiotic resistance genes, and it has been shown that they confer resistance to β-lactams, fluoroquinolones, aminoglycosides, trimethoprim, sulphonamides, and tetracycline. The previously proposed classification system divides the plasmid group into four subgroups, i.e., IncQ-1, IncQ-2, IncQ-3, and IncQ-4. The last two subgroups have been poorly described so far. The aim of this study was to analyze five newly identified IncQ-3 plasmids isolated from a wastewater treatment plant in Poland and to compare them with all known plasmids belonging to the IncQ-3 subgroup whose sequences were retrieved from the NCBI database. The complete nucleotide sequences of the novel plasmids were annotated and bioinformatic analyses were performed, including identification of core genes and auxiliary genetic load. Furthermore, functional experiments testing plasmid mobility were carried out. Phylogenetic analysis based on three core genes (repA, mobA/repB, and mobC) revealed the presence of three main clusters of IncQ-3 replicons. Apart from having a highly conserved core, the analyzed IncQ-3 plasmids were vectors of antibiotic resistance genes, including (I) the qnrS2 gene that encodes fluoroquinolone resistance and (II) β-lactam, trimethoprim, and aminoglycoside resistance genes within integron cassettes.


Introduction
In the natural environment, as a result of the presence of subminimal inhibitory concentrations of antibiotics, resistance genes encoded by mobile genetic elements, such as plasmids, transposons, and integrons, can be disseminated through horizontal gene transfer (HGT) [1][2][3][4][5][6]. Wastewater treatment plants (WWTPs) are the entry routes for antibiotic-resistant bacteria (ARBs), including pathogenic and multidrug-resistant (MDR) strains [7]. As sites where both antibiotics and favorable conditions for bacterial growth occur, WWTPs promote horizontal gene transfer (HGT) [8,9]. HGT events may lead to the rapid emergence and dissemination of antibiotic resistance among bacteria both in clinical and natural environments [10] and also to genetic exchange between clinical and WWTP strains [11,12].

Structural and Functional Genomics of Five Novel IncQ Plasmids
In the course of our previous studies, a plethora of β-lactamase-resistant bacterial strains harboring various extrachromosomal replicons were identified [20,21]. Sequencing of their plasmidomes revealed a group of five small (7.6-9.6 kb) plasmids that were present in 16 clonally unrelated Gram-negative multireplicon strains. Based on an analysis of their replication systems, the plasmids were classified into the IncQ-3 subgroup. Interestingly, in all 16 strains various sets of plasmids accompanying IncQ-3 replicons were observed, which may exemplify HGT events among bacteria inhabiting the analyzed wastewater treatment plant, as observed previously for other environments [34][35][36]. Based on their genetic structure and gene load, the identified plasmids were divided into three groups: (i) p5.4_c4 and p115_p2, (ii) p458_p3 and p426_p3, (iii) p435_c4 ( Figure 1, Table 1).

Figure 1.
Linear maps showing the genetic structure of the circular IncQ-3 plasmids identified in the study. Genes assigned to specific modules are highlighted in different colors: Yellow-mobilization to plasmid transfer module, red-replication module, green-antibiotic resistance genes, blueintegrase. Red and yellow dots represent oriV and oriT, respectively. Comparative analyses were performed using 90% nucleotide sequence identity threshold (gray areas connecting DNA regions). Footnotes: RW-raw wastewater; AS-activated sludge; TW-treated wastewater.
Comparative nucleotide analysis of the five newly identified plasmids indicated four core genes encoding: RepA (helicase), MobA-RepB (fusion gene of relaxase-primase), MobC (protein required for DNA cleavage), and a hypothetical protein. In all these plasmids, the mobA and repB genes were fused, which was previously observed as a characteristic feature of many plasmids within all IncQ groups. Previous studies did not clearly determine if the relaxase-primase gene fusion is expressed as two separate polypeptides or as a single peptide with two domains [43]. However, there seems to exist a strong selection for these fused transcripts among the IncQ plasmids, which suggests that both processes (i.e., replication and conjugation) may be coupled. Plasmids of the IncQ group possess replication systems operating via the strand-displacement mechanism. This system consists of three genes, repBAC, which form a cluster and an intergenic region of replication initiation (oriV) containing iterons, where the replication starts [44]. In plasmids analyzed in this study, three conserved direct repeats (DR)-putative iterons-were found within the oriV regions. The consensus iteron sequence is as follows: 5 -CCCCCACGGTAACTCNNCCC-3 . The NN represents an ambiguous dinucleotide: (i) CA in plasmids p435_c4, p458_p3, p426_p3, or (ii) TC in p5.4_c4 and p115_p2. Within each oriV, the following DNA regions were identified besides iterons: (a) G+C-rich region, (b) A+T-rich region, (c) 15-bp region, highly conserved in IncQ-like plasmids (5 -CTGCGCCTAGTGGAG-3 ) and (d) two palindromic sequences (5 -CCGCGCCGAAGGGGCGCGG-3 and 5 -ACCCCCGGAGGGGGT-3 ). The repB, repA, and repC replication genes are highly similar in the identified plasmids (95-100% of nucleotide sequence identity). However, in plasmid p435_c4, no equivalent of the repC gene was identified, which suggests that the product of that gene may be delivered in trans or that the gene is unnecessary for plasmid replication. The latter hypothesis seems to be more probable, since the sequences of two other, highly similar IncQ-3 plasmids, also deprived of the repC gene, were found in the NCBI database, i.e., pJF-707 (KX946994) and pUL3AT (HE616889).
The genetic module responsible for the mobilization to conjugal transfer is also highly conserved within all identified plasmids. It consists of two genes-mobA and mobC. The oriT sites of the analyzed IncQ-3 plasmids were identified based on the homology to the previously identified oriTs [30] and all 29 plasmids contain the same, highly conserved, putative 12-bp region (5 -TTACACCTTGCT-3 ) comprising an origin of transfer.
The analyzed plasmids were tested for their ability to be transferred into two hosts, E. coli DH5a (Rif r ) and Pseudmonas aeruginosa PAO1161 (Rif r ), via bi-and triparental conjugation (Table S1 Supplementary Material). Interestingly, only two plasmids, p5.4_c4 and p115_p3, originally carried by Aeromonas spp. and Raoultella spp., were transferable, which is in agreement with previous findings concerning highly similar IncQ plasmids [28,29,38]. Transconjugants were ceftazidime-resistant, which also confirm that beta-lactamase genes carried by IncQ plasmids were truly expressed. In contrast, plasmids p426_p3, p458_p3, and p435_c4 carried by Aeromonas spp. and Kluyvera sp. were unable to be transferred via conjugation into the tested strains, although these plasmids were successfully introduced to both recipient strains by transformation (as shown in a control experiment). Therefore, we speculate that their mobilization systems may be inactive or that the plasmids need a mobilizing system different from the RK2 mating pair formation system (present in plasmid pRK2013) that was provided in the tri-parental mating.
Other identified genetic modules of the analyzed plasmids include IS4 family transposase tnp (plasmid p435_c4), ATPase subunits of an ABC transporter (p435_c4), type 3 integrases intI3 (p5.4_c4 and p115_p2), and an aerotaxis sensor receptor protein (p458_p3) (Figure 1). A summary of the identified genes, including the size of the encoded proteins, their position, and predicted function is presented in Table S2 (Supplementary Material).
The first cluster contains 16 plasmids, including p458_p3 and p426_p3. Apart from pAER-ba17, all plasmids in this cluster carry qnrS2 gene, which encodes fluoroquinolone resistance. Plasmids in this cluster have diverse oriVs, differing in iteron number and sequence.
The second cluster contains three branches formed by nine plasmids, including p5.4_c4 and p115_p2 (Figure 2a). The first and second branch consist of three plasmids: pFECR, pAHH04, and pAB5, which carry qnrS2 genes, similarly to those in cluster I. The third separate branch of cluster II consists of six replicons, which carry various antibiotic resistance genes (conferring resistances to β-lactams, trimethoprim, and aminoglycosides) that were located within class 1 or 3 integrons.
Interestingly, each plasmid of the second branch of cluster II carries a variant of the bla GES gene (β-lactamase). The first cluster contains 16 plasmids, including p458_p3 and p426_p3. Apart from pAER-ba17, all plasmids in this cluster carry qnrS2 gene, which encodes fluoroquinolone resistance. Plasmids in this cluster have diverse oriVs, differing in iteron number and sequence.
The second cluster contains three branches formed by nine plasmids, including p5.4_c4 and p115_p2 (Figure 2a). The first and second branch consist of three plasmids: pFECR, pAHH04, and pAB5, which carry qnrS2 genes, similarly to those in cluster I. The third separate branch of cluster II consists of six replicons, which carry various antibiotic resistance genes (conferring resistances to βlactams, trimethoprim, and aminoglycosides) that were located within class 1 or 3 integrons. Interestingly, each plasmid of the second branch of cluster II carries a variant of the blaGES gene (βlactamase).
The last cluster (III) consists of four plasmids divided into two branches. The former includes p435_c4, pJF-707, and pUL3AT and the latter contains one plasmid-pRGRH0378. Characteristic features of these plasmids are: (i) Differences in the structure of oriVs and (ii) the presence of variable set of ARGs, which makes them the most heterogeneous cluster. Furthermore, the lack of repC gene was observed within three plasmids in this cluster, i.e., p435_c4, pJF-707, and pUL3AT ( Figure 2). Plasmid pRGRH0378 was obtained from a rat gut metagenome from hospital sewage in Denmark (LN853034.1). The plasmid possesses repC gene and all other core genes characteristic of the rest of IncQ-3a plasmids and only one additional gene encoding a hypothetical protein. That makes pRGRH0378 not only the smallest reported plasmid of the IncQ-3 group (5.8 kbp) but also the most conserved one.

IncQ-3 Plasmids as a Reservoir of Antibiotic Resistance Genes and Biogeography of IncQ-3 Plasmids
Comparative genomics of all 29 known IncQ-3 plasmids showed that 27 of them carry at least one antibiotic resistance gene that confers resistance to quinolone, β-lactams, or trimethoprim (Tables The last cluster (III) consists of four plasmids divided into two branches. The former includes p435_c4, pJF-707, and pUL3AT and the latter contains one plasmid-pRGRH0378. Characteristic features of these plasmids are: (i) Differences in the structure of oriVs and (ii) the presence of variable set of ARGs, which makes them the most heterogeneous cluster. Furthermore, the lack of repC gene was observed within three plasmids in this cluster, i.e., p435_c4, pJF-707, and pUL3AT ( Figure 2). Plasmid pRGRH0378 was obtained from a rat gut metagenome from hospital sewage in Denmark (LN853034.1). The plasmid possesses repC gene and all other core genes characteristic of the rest of IncQ-3a plasmids and only one additional gene encoding a hypothetical protein. That makes pRGRH0378 not only the smallest reported plasmid of the IncQ-3 group (5.8 kbp) but also the most conserved one.

IncQ-3 Plasmids as a Reservoir of Antibiotic Resistance Genes and Biogeography of IncQ-3 Plasmids
Comparative genomics of all 29 known IncQ-3 plasmids showed that 27 of them carry at least one antibiotic resistance gene that confers resistance to quinolone, β-lactams, or trimethoprim (Tables 1  and 2). Two main groups of antibiotic resistance vectors were identified within investigated plasmids: (1) qnrS2-carrying plasmids that confer quinolone resistance, and (2) plasmids carrying β-lactam resistance genes-bla GES , bla OXA , and bla FOX -comprising integron gene cassettes (Figure 2c).
The qnrS2-carrying plasmid group consists of 18 replicons that have been found worldwide, mainly in wastewater [29,42] or other aqueous environments, including river water [28], hospital effluents [28], or infected fish [31,40]. These plasmids originated from diverse geographical regions, including Israel [42], Germany [29], China ( [28], CP032896), Portugal (MH133192), USA (MK191840), Canada (MF554639), South Korea [40], and Poland (p458_p3 and p426_p3 found in this study) (Figure 3). The qnrS2 genes belong to the most prevalent plasmid-mediated quinolone resistance (PMQR) gene group. Plasmid-mediated quinolone resistance was first identified in a Klebsiella pneumoniae clinical isolate from the United States in 1998 [45]. Since then, wide dissemination of qnr genes has been confirmed in both clinical and environmental strains. The dissemination is often associated with transposable elements (TEs) harbored by plasmids [46]. It was suggested that the qnrS2 gene was potentially introduced into some of the IncQ-3 plasmids by transposition [28]. In seven of the analyzed plasmids, qnrS2 is a part of a putative, nonautonomous, mobile insertion cassette, bordered by inverted repeats (IRs) and flanked by 5-bp long direct repeats (DRs). The insertion site in plasmids pAB5 [28] and pAHH04 [40] is different from that in the remaining counterparts, which suggests two independent transposition events.
Antibiotics 2020, 9, x FOR PEER REVIEW 8 of 14 plasmids: (1) qnrS2-carrying plasmids that confer quinolone resistance, and (2) plasmids carrying βlactam resistance genes-blaGES, blaOXA, and blaFOX-comprising integron gene cassettes (Figure 2c). The qnrS2-carrying plasmid group consists of 18 replicons that have been found worldwide, mainly in wastewater [29,42] or other aqueous environments, including river water [28], hospital effluents [28], or infected fish [31,40]. These plasmids originated from diverse geographical regions, including Israel [42], Germany [29], China ( [28], CP032896), Portugal (MH133192), USA (MK191840), Canada (MF554639), South Korea [40], and Poland (p458_p3 and p426_p3 found in this study) ( Figure  3). The qnrS2 genes belong to the most prevalent plasmid-mediated quinolone resistance (PMQR) gene group. Plasmid-mediated quinolone resistance was first identified in a Klebsiella pneumoniae clinical isolate from the United States in 1998 [45]. Since then, wide dissemination of qnr genes has been confirmed in both clinical and environmental strains. The dissemination is often associated with transposable elements (TEs) harbored by plasmids [46]. It was suggested that the qnrS2 gene was potentially introduced into some of the IncQ-3 plasmids by transposition [28]. In seven of the analyzed plasmids, qnrS2 is a part of a putative, nonautonomous, mobile insertion cassette, bordered by inverted repeats (IRs) and flanked by 5-bp long direct repeats (DRs). The insertion site in plasmids pAB5 [28] and pAHH04 [40] is different from that in the remaining counterparts, which suggests two independent transposition events. One of the plasmids pUR19829-KPC21 (MH133192) additionally encodes a blaKPC gene [41]. The carbapenemase gene is located on a putative complex mobile element, which is also present in pKP1194 plasmid identified in a clinical K. pneumoniae strain (KX756453) [41].
One of the plasmids pUR19829-KPC21 (MH133192) additionally encodes a bla KPC gene [41]. The carbapenemase gene is located on a putative complex mobile element, which is also present in pKP1194 plasmid identified in a clinical K. pneumoniae strain (KX756453) [41].
The second group consists of nine plasmids that carry β-lactamase genes from various groups: bla GES , bla OXA , and bla FOX . Simultaneously, most of these plasmids carry also other ARGs, i.e., dfrB3 and aac(6 )ib-cr, encoding trimethoprim and aminoglycoside resistance, respectively. The analyzed plasmids were detected in clinical and wastewater samples in diverse geographical locations, i.e., Switzerland [32], Poland (MH569711.1, this study), South Korea [37], UK (KX946994), Luxemburg [39], and France [38]. Three of the plasmids in our study p5.4_c4, p115_p2, and p435_c4 carry bla genes also within an integron. Plasmids p5.4_c4 and p115_p2 possess bla GES-7 gene that encodes GES class A β-lactamase [52]. GES β-lactamases are known to be encoded by integron gene cassettes originating from clinical and environmental strains [53][54][55][56]. A majority of studies identified bla GES genes within class 1 integrons. In contrast, within IncQ-3 plasmids, seven harbor class 3 integrons and only one contains a class 1 integron (Tables 1 and 2). Integrons are frequently associated with mobile elements, such as transposons, which fosters their dissemination among bacteria. The class 3 integrons were possibly acquired by IncQ-3 plasmids through transposition, as previously suggested by Barraud et al. (2013) [38]. The putative mobile element is bordered by terminal IRs, which closely resemble IRs of the Tn3 family, and is surrounded by 5-bp long DRs. Since no transposase gene was found, it is possible that the identified DNA region may constitute a nonautonomous transposable element, which may be mobilized by a functional transposase from another TE.
Horizontal transfer of integrons by nonautonomous TEs was previously proved experimentally in the case of integron mobilization units (IMUs), which formed a composite structure identified in the IncQ-1 family plasmid, pCHE-A [57]. The integron was surrounded by terminal IRs. Providing a Tn3-family transposase in trans resulted in transposition of the composite element into another plasmid (generating 5-bp long DRs). This indicates that nonautonomous TEs may play a role in spreading of integrons and-consequently-antibiotic resistance genes among the IncQ plasmids.

Isolation of IncQ-3 Plasmids
Five plasmids analyzed in this study were found in 16 strains originating from "Czajka" Wastewater Treatment Plant in Warsaw, Poland. The strains belong to Aeromonas spp. or Enterobcteriaceae (Table S3). A detailed description of sample collection, bacterial isolation, sewage treatment processing, and the identified strains was provided in our previous papers [20,21]. Previously, we investigated 162 bacterial isolates exhibiting resistance to β-lactams. In 110 strains, at least one extrachromosomal replicon was found; however, a majority of strains shared common plasmid profiles, as judged from electrophoretic analysis. Thirty strains with unique plasmid profiles were selected for further analyses. Complete nucleotide sequences of their plasmids were determined from plasmids isolated in 2018. Among the sequenced plasmids, the most numerous ones were small (7.6-9.6 kb) IncQ-3 plasmids (Table 1). These plasmids have been thoroughly analyzed in this study.

DNA Sequencing and Assembly
The complete nucleotide sequences of the plasmids were determined using Illumina MiSeq (Illumina Inc., San Diego, CA, USA) and MinION (Oxford Nanopore, Oxford, UK) sequencers yielding around 100× sequence coverage for each plasmid. Total plasmid DNA of the strains was sequenced in the DNA Sequencing and Oligonucleotide Synthesis Laboratory (oligo.pl) at the Institute of Biochemistry and Biophysics, Polish Academy of Sciences (Warsaw, Poland). Raw reads obtained were assembled de novo into contigs and scaffolds using the Unicycler hybrid approach (Unicycler v. 0. 4.6). Unicycler is open source (GPLv3) and available at github.com/rrwick/Unicycler. Final gap closure was performed by PCR amplification of DNA fragments, followed by Sanger sequencing with an ABI3730xl Genetic Analyzer (Applied Biosystems, Waltham, MA, USA).

Gene Annotation and Bioinformatics
The rapid annotation of the complete nucleotide sequences of plasmids was performed with on-line MAISEN tool [58]. Additional manual annotations and preparation of plasmid files for deposition were performed using Artemis software version 16.0.0 [59]. Comparative genomic analyses were carried out with BLASTn and BLASTx tools [60]. Comparative genomic data were visualized using EasyFig software version 2.2.4. [61]. Phylogenetic analysis was performed with MEGA7 [62]. Transposable elements were identified by comparative analyses (BLASTn, BLASTx) using ISfinder database [63].
As a control of the conjugal transfer experiments, plasmid DNA was introduced into recipient strains via chemical transformation. Plasmid DNA was isolated from the donor strains using a classical alkaline lysis procedure [65] and introduced into E. coli DH5α and P. aeruginosa PAO1 by chemical transformation [66,67].

Plasmid Sequences Accession Numbers
The complete nucleotide sequences of plasmids were deposited in GenBank (NCBI) database under the accession numbers MT231818-MT231822 (Table 1). A complete annotation table is also available in Supplementary Materials (Table S2).

Conclusions
As the reservoirs of antibiotic-resistant bacteria, WWTPs are ideal places for horizontal gene transfer of various ARGs. IncQ plasmids belong to a group of widely distributed replicons, occurring mostly in aquatic environments, including wastewater treatment plants, hospital effluents, and clinical samples. Previously proposed classification system divides IncQ group of plasmids into four subgroups: IncQ-1, IncQ-2, IncQ-3, and IncQ-4. The aim of this study was to analyze IncQ-3 plasmids whose sequences had been deposited in the NCBI database, plus five newly identified replicons (in five Aeromonas sp., ten Raoutella sp., and one Kluyvera sp. strain) from the "Czajka" WWTP in Poland. Hos strains for these novel plasmids were isolated from wastewater at various stages of its treatment: Raw wastewater, activated sludge, or treated wastewater.
Based on phylogenetic analysis of three plasmid core genes, the identified replicons were divided into three clusters. However, all these plasmids have highly conserved backbones. The majority of the studied plasmids (27 out of 29) were vectors of ARGs and collectively carried seven different genes that encode resistance to four groups of antibiotics (β-lactams, fluoroquinolones, trimethoprim, and aminoglycosides). IncQ-3 as vectors of antibiotic resistance genes were divided into two main groups: (I) Vectors of qnrS2 gene that encodes fluoroquinolone resistance and (II) vectors of β-lactam, trimethoprim, and aminoglycoside resistance genes located within an integron.
Supplementary Materials: The following are available online at http://www.mdpi.com/2079-6382/9/9/613/s1. Table S1: Results of bi-and triparental mating assays of the identified IncQ-3 plasmids. Table S2: Genes located within the analyzed IncQ-3 plasmids. Table S3: Bacterial strains used in this study. Table S4: Results of Minimum Inhibitory Concentration (MICs) for donor and recipient strains for bi-and triparental mating assays. Table S5: Primers used in this study.