Diversification and prevalence of the quinolone resistance crpP genes and the crpP-carrying Tn6786-related integrative and conjugative elements in Pseudomonas aeruginosa

ABSTRACT The quinolone resistance crpP genes can mediate decreased susceptibility to quinolones. However, diversification and prevalence of crpP genes and crpP-carrying integrative and conjugative elements (ICEs) still need to be elucidated. In this study, genome sequencing was conducted for 200 Chinese Pseudomonas aeruginosa isolates, 16 of which were fully sequenced. All the 37 available CrpP variants were collected for phylogenetic analysis, 10 CrpP enzymes were chosen to conduct cloning and antimicrobial susceptibility test, and 22 crpP-carrying Tn6786-related ICEs were selected for detail genetic dissection analysis. Then, typing/nomenclature schemes for crpP variants and crpP-carrying ICEs were established for the first time. The 10 representative CrpP enzymes were confirmed to mediate decreased susceptibility to one to three quinolones. Tn6786-related ICEs displayed high-level diversification in both nucleotide sequences and modular structures. Mainly, massive gene acquisition/loss occurred across the whole genomes of Tn6786-related ICEs. 53.5% (107/200) of the tested clinical P. aeruginosa isolates from China carried crpP genes, which were exclusively located within chromosome-borne Tn6786-related ICEs. The crpP-carrying ICEs were at active stages of evolution and had the high potential to be an important vector for the dissemination of resistance genes besides crpP. The present study furthered the understanding of the bioinformatics and epidemiology of crpP genes and crpP-carrying ICEs.


Introduction
There are at least three major mechanisms of resistance to quinolones in bacteria: mutations in genes encoding quinolone-targeting proteins; changes in expression of efflux pumps or porin channels; and acquisition of quinolone resistance genes such as crpP [1]. The CrpP enzyme can induce ATP-dependent phosphorylation of ciprofloxacin and mediate a decreased susceptibility to ciprofloxacin [2,3]. Abundant crpP homologues have been mainly identified in Pseudomonas aeruginosa [4][5][6][7][8][9], and less frequently in Acinetobacter baumannii [4], Escherichia coli, and Klebsiella pneumoniae [10]; however, a typing/nomenclature scheme for crpP variants is still lacking.
Integrative and conjugative elements (ICEs) [11][12][13] are important mobile genetic elements (MGEs) involved in the dissemination of resistance genes and other beneficial genes in bacteria. An ICE element can transfer between cells because of their self-encoded conjugation function, and it is typically composed of attL (attachment site at the left end), int (integrase), xis (excisionase), rlx (relaxase), oriT (origin of conjugative replication), cpl (coupling protein), a P (TivB)-or F (TivF)-type T4SS gene set (mating pair formation), and attR (attachment site at the right end). While ICEs have been identified as a major crpP reservoir in P. aeruginosa [14], there are still no reports on systematic classification and detailed genetic dissection of crpPcarrying ICEs.
This study presented the systematic typing/nomenclature schemes for crpP variants and crpP-carrying ICEs, and confirmed a limited decreased susceptibility to quinolones mediated by 10 Table S1), and further sequence data mining was performed as previously described [15,16]. Bacterial sequence types (STs) were identified according to the online P. aeruginosa MLST scheme (https:// pubmlst.org/paeruginosa). Indicated amino acid or nucleotide sequences were aligned using Clustal Omega 1.2.2 [17]. Unrooted maximum-likelihood phylogenetic trees were generated using MEGAX 10.1.8 [18] with a bootstrap iteration of 1000. GraphPad Prism 5.0 statistical software was used for statistical analysis. The χ2 was used for categorical variables. P < 0.05 was considered statistically significant. Additionally, different Tn numbers were used to name ICEs as previously described [19,20]. Conjugal transfer of each indicated ICE from its wild-type isolate into the rifampin-resistant P. aeruginosa ATCC 15,692 was conducted as previously described [16]. The crpP-1.1 coding region together with its immediately 288-bp upstream promoter-proximal region and 216-bp downstream terminator-proximal region from the wild-type isolate SE5416 was cloned into the lacZ gene of kanamycin-resistant cloning vector pUC57K. Similarly, the coding region of each of the other indicated crpP variants together with the above promoter-and terminator-proximal regions were synthesized and cloned into pUC57K. Each resulting recombinant plasmid was transformed through electroporation into Escherichia coli TOP10 to generate the relevant electroporant. Bacterial antimicrobial susceptibility was tested by the classic broth microdilution method, and was interpreted as per the 2020 Clinical and Laboratory Standards Institute (CLSI) guidelines [21]. The

Tn6786-related ICEs
A detailed genetic dissection analysis was applied to a total of 22 representative fully sequenced crpPcarrying ICEs (Supplementary material Table S3). The modular structure of each ICE was divided into the backbone, and the accessory modules, which were defined as exogenous DNA regions, were inserted at different sites of the backbone ( Figure 2). These 22 ICEs had similar backbone gene organizations, but they displayed >91% nucleotide identity with coverage from 28% to 98% of their backbone sequences (Supplementary material Table  S4), thus indicating high-level diversification in their nucleotide sequences and modular structures. These 22 ICEs shared the core backbone markers int, rlx, an F-type T4SS gene set, crpP-1, and 45-bp attL/R; totally, six crpP-1 variants were identified ( Figure 2, and supplementary material Table S5). The phylogenetic tree constructed from the aligned int sequences (Supplementary material Table S6) and that from the core single-nucleotide polymorphisms (SNPs) within the ICE backbones indicated that these 22 ICEs could be divided into four separate clustering groups that were designated as groups A to D ( Figure 3). As shown by pairwise comparison of int sequences, ICEs within each of these four groups shared >90% nucleotide identity, whereas <90% sequence identity was observed between different groups (Supplementary material Table S6).

Massive gene acquisition/loss in crpP-carrying Tn6786-related ICEs
At least 13 regions/sites across the ICE genomes were recognized to display the major modular differences among these 22 ICEs (Supplementary material Figure  S1 and Table S7). Firstly, a total of 97 events of exogenous gene acquisition occurred at these 13 regions/sites in these 22 ICEs, which carried different profiles of accessory modules (Supplementary material Figure S2-S8). Tn6786 was identified as the reference of this ICE group because it carried a sole accessory module and possessed the mostly intact backbone (83.7 kb in length) among these 22 ICEs. Additionally, it harbored the firstly discovered crpP variant crpP-1.1 (Figure 2). Each of the other 21 ICEs acquired two to seven accessory modules, which included IS elements, unit transposons, introns, and so called "inserted regions" [19]. Secondly, a total of 69 events of deletion of backbone regions, approximately a half of which resulted from exogenous gene acquisition, occurred at seven region/sites in 18 ICEs. Among each of these, seven experienced at least one event of deletions of >5-kb backbone regions. Tn6783 had the shortest backbone that was 25.5 kb in length and 58.2-kb shorter than Tn6786. Finally, there were totally 10 events substitutions of backbone regions at two region/sites in nine ICEs.   20 (1/ 117, 0.9%), were identified in these 107 crpPpositive isolates. Each detected crpP gene was carried by a Tn6786-related ICE, and the 117 ICEs detected in these 107 isolates were composed of 67 group A ones, 34 group B ones, 11 group D ones, and 5 group C ones (Figure 4(b), and Supplementary material Table S1). Additionally, all these ICEs were integrated at the end of the tRNA Lys gene as described in a previous report [14].

Conjugation experiments
To validate the intercellular mobility of crpPcarrying Tn6786-related ICEs, two ICEs harboring putative antibiotic resistance genes in addition to crpP (namely emrAB-carrying Tn6783, and ampCcarrying Tn6786) were chosen in this study for conjugal transfer experiments because the crpP genes were unsuitable as the selection markers based on antibiotic resistance. Yet, repeated conjugation attempts failed to transfer Tn6783 or Tn6786 from its wild-type isolate into ATCC 15,692.

Discussion
Since the crpP-1.1 gene was initially identified in Mexico in 2018 [2], a total of 37 non-redundant crpP variants have been reported in at least 16 countries across North America, Asia, Africa, Oceania, and Europe [4][5][6][7][8][9][10]. However, conflicting names of crpP variants were presented in different reports. To solve this problem, herein we established a systematic typing/ nomenclature scheme for all the available 37 CrpP variants based on their amino acid sequences. These 37 CrpP variants were divided into six primary groups, i.e. CrpP-1 to CrpP-6, where CrpP-1 comprised 32 variants, and each of CrpP-2 to CrpP-6 contained only a sole variant. Compared with CrpP-1.1, each of the other 31 CrpP-1 variants exhibited no more than 6 amino acid substitutions in total, indicating the variants within the primary group CrpP-1 were highly genetically conserved. Compared with CrpP-1.1, each of CrpP-2.1 to CrpP-6.1 displayed at least 39 amino acid substitutions in total, which indicated a much higher level of genetic diversity among different CrpP primary groups. Among these 37 CrpP variants, 10 have been previously reported to mediate a decreased susceptibility to one or two quinolones, including CrpP-1.1/2.1/3.1/4.1/ 5.1/6.1 to ciprofloxacin [2,10], and CrpP-1.2/1.17/1.18/ 1.19 to ciprofloxacin and levofloxacin [5]. In this study, CrpP-1.1/2.1/3.1/4.1/5.1/6.1 as the representatives of the six primary CrpP groups were verified to mediate a decreased susceptibility to only ciprofloxacin, while CrpP-1.10/1.16/1.20/1.31 were disclosed, for the first time, to mediate a decreased susceptibility to both ciprofloxacin and levofloxacin. It was speculated that the Gly7Asp or Gly7HiS substitution, as observed in the eight CrpP-1 variants CrpP-1.2/1.10/1.16/1.17/1.18/ 1.19/1.20/1.31 relative to CrpP-1.1, might be the cause of a decreased susceptibility to levofloxacin besides ciprofloxacin, regardless of the occurrence of other amino acid substitutions [5].
Although crpP was originally identified in plasmid pUM505 [2], ICEs were identified as a major reservoir of crpP genes in P. aeruginosa [14]. Our results revealed that all the fully sequenced crpP-carrying ICEs had the highly conserved 45-bp terminal attL/attR pairs and displayed similar backbone gene organizations, and thus were assigned into a newly identified Tn6786 family. A phylogenetic analysis based on their int sequences or on their core backbone SNPs could divide all the fully sequenced Tn6786-realted ICEs into four separate clustering groups (data not shown) that were designated as groups A to D. This showed that crpP-carrying Tn6786-related ICEs exhibited high-level diversity in nucleotide sequences.
A further detailed genetic dissection analysis was applied on the representative 22 crpP-carrying Tn6786related ICEs, which covered all the above A to D groups. This analysis identified at least 97 events of exogenous gene acquisition, 69 events of deletion of backbone regions, and 10 events substitutions of backbone regions, which occurred at 13 regions/sites across the whole genomes of these 22 ICEs and most likely resulted from complex transposition and homologous recombination. In particular, massive gene acquisition/loss occurred across the whole genomes of Tn6786-related ICEs. Taken together, these 22 ICEs revealed a highly mosaic modular structures.
Acquisitions of different kinds of beneficial genes in addition of crpP were identified in this work: the inserted region orf228 from Tn6786 harbored the putative β-lactam resistance gene ampC [25]; the inserted region dsbG from Tn6783 contained two putative multidrug resistance efflux pump genes emrAB [26]; Tn6488 [27] from three ICEs harbored mercuric resistance mer locus; the inserted region orf1482 from Tn6781 harbored the cup locus encoding bacterial adhesive organelles [28]; the inserted region orf549 from Tn6781 and the inserted region pilL from other three ICE harbored pyoS5 that mediated bactericidal activity against other P. aeruginosa isolates through encoding Pyocin S5 [29]. Acquisitions of exogenous genes involving drug resistance, virulence, and intraspecies competition would enhance the adaptation to different niches of host P. aeruginosa strains.
To the best of our knowledge, this is the first study that reported on the characterization of crpP genes and crpP-carrying ICEs in China. 53.5% of the clinical P. aeruginosa isolates from China tested carried the crpP genes, which were exclusively located within chromosome-borne Tn6786-related ICEs. The herein detected crpP-positive rates in Chinese P. aeruginosa isolates were comparable to European isolates (46%) [5] and Australian isolates (43%) [9]. The crpPcarrying P. aeruginosa isolates were identified from all the 18 hospitals in the 12 Chinese cities studied, indicating wide distribution of these isolates in the hospital settings in China. Notably, thirty-seven (34.6%) of the 107 crpP-positive P. aeruginosa isolates from China belonged to the high-risk clones ST244, ST235, ST274 and ST277, with ST235 being worldwide distributed [22,23].
Considering the high-level diversification in both nucleotide sequences and modular structures, the crpPcarrying Tn6786-related ICEs were at active stages of evolution and showed the high potential to act as an important vector for dissemination of antibiotic resistance genes besides crpP.
Although a circular form of crpP-carrying ICEs has been previously confirmed through PCR assays [5], repeated conjugation attempts failed to transfer Tn6783 or Tn6786 from their wild-type isolate into ATCC 15,692, whose chromosome (accession number NC_002516) did have the conserved tRNA Lys gene. This failure might result from the lesions in the conjugal transfer regions of these two tested ICEs.
The type III secretion system effector cytotoxin ExoU could markedly enhance the virulence of P. aeruginosa [24]. The exoU gene was also considered to be associated with fluoroquinolone-resistant strains of P. aeruginosa [9,30,31]. Our results further proved that the exoU gene was positively correlated with crpP genes. The mechanisms underlying the co-carriage of these two genes in P. aeruginosa strains need to be further studied.
In conclusion, systematic typing/nomenclature schemes for crpP variants and crpP-carrying Tn6786related ICEs were established. Ten major representative CrpP enzymes were confirmed to mediate a limited decreased susceptibility to quinolones. 53.5% of the tested clinical P. aeruginosa isolates from China carried the crpP genes, which were exclusively located within chromosome-borne Tn6786-related ICEs. Tn6786related ICEs displayed high-level diversification in both nucleotide sequences and modular structures. Especially, massive gene acquisition/loss occurred across the whole genomes of Tn6786-related ICEs. This work provided a deeper insight into the bioinformatics and epidemiology of crpP genes and crpPcarrying Tn6786-related ICEs.

Disclosure statement
No potential conflict of interest was reported by the authors.

Data availability statement
The data used to support the findings of this study are available from the corresponding author upon request (dong-shengzhou1977@gmail.com).