Novel Chromosome-Borne Accessory Genetic Elements Carrying Multiple Antibiotic Resistance Genes in Pseudomonas aeruginosa

Pseudomonas aeruginosa is noted for its intrinsic antibiotic resistance and capacity of acquiring additional resistance genes. In this study, the genomes of nine clinical P. aeruginosa isolates were fully sequenced. An extensive genetic comparison was applied to 18 P. aeruginosa accessory genetic elements (AGEs; 13 of them were sequenced in this study and located within P. aeruginosa chromosomes) that were divided into four groups: five related integrative and conjugative elements (ICEs), four related integrative and mobilizable elements (IMEs), five related unit transposons, and two related IMEs and their two derivatives. At least 45 resistance genes, involved in resistance to 10 different categories of antibiotics and heavy metals, were identified from these 18 AGEs. A total of 10 β-lactamase genes were identified from 10 AGEs sequenced herein, and nine of them were captured within class 1 integrons, which were further integrated into ICEs and IMEs with intercellular mobility, and also unit transposons with intracellular mobility. Through this study, we identified for the first time 20 novel MGEs, including four ICEs Tn6584, Tn6585, Tn6586, and Tn6587; three IMEs Tn6853, Tn6854, and Tn6878; five unit transposons Tn6846, Tn6847, Tn6848, Tn6849, and Tn6883; and eight integrons In1795, In1778, In1820, In1784, In1775, In1774, In1789, and In1799. This was also the first report of two resistance gene variants blaCARB-53 and catB3s, and a novel ST3405 isolate of P. aeruginosa. The data presented here denoted that complex transposition and homologous recombination promoted the assembly and integration of AGEs with mosaic structures into P. aeruginosa chromosomes.


INTRODUCTION
Pseudomonas aeruginosa is a major opportunistic pathogen and a leading cause of morbidity and mortality in cystic fibrosis patients and immunocompromised individuals (Alvarez-Ortega et al., 2011). P. aeruginosa displays resistance to multiple classes of antibiotics (De Oliveira et al., 2020), being in the critical priority list of antibiotic resistant bacteria created by the World Health Organization (Tacconelli et al., 2018). One of the main mechanisms driving to the antibiotic resistance in P. aeruginosa is the production of b-lactamases, which are capable of hydrolyzing chemical compounds containing a blactam ring (Bush, 2018) and are the most common determinant for resistance to bacterial b-lactam antibiotics (Bush and Bradford, 2020). b-lactamases can be divided into four Ambler classes A to D, where classes A/C/D b-lactamases utilize a serine moiety while class B b-lactamases (also known as metallo-blactamases) need a zinc ion at its active site (Ambler, 1980). Besides the intrinsic narrow-spectrum b-lactamase (NSBL) gene bla OXA-50 (Girlich et al., 2004), P. aeruginosa has evolved to acquire various extended-spectrum b-lactamase (ESBL) and even carbapenemase genes through horizontal gene transfer mediated by different mobile genetic elements (MGEs) (Botelho et al., 2019). MGEs play critical roles in the accumulation and spread of antibiotic resistance genes in P. aeruginosa (Botelho et al., 2019).
Various ICEs, IMEs, and unit transposons have been identified and found to be important vehicles for antibiotic resistance genes in P. aeruginosa (Stokes et al., 2007;Mathee et al., 2008;Roy Chowdhury et al., 2016;Botelho et al., 2018a;Botelho et al., 2018b;van der Zee et al., 2018;Chew et al., 2019). Deep understanding of the MGEs at a genomic level will provide the theoretical basis for inhibiting the emergence of multidrugresistant P. aeruginosa and overcoming the challenge of limited antimicrobial chemotherapy measures. However, few studies have dedicated to detailedly and accurately dissect the genetic structures of these MGEs from P. aeruginosa.
Our previous studies (Zhan et al., 2018;Zeng et al., 2019) have dissected the genetic characteristics of three carbapenemaseencoding novel transposons (bla IMP-1 -containing ISPa17-based transposition unit Tn6394, bla VIM-4 -containing ICE Tn6413, and bla IMP-1 -containing unit transposon Tn6411) located within P. aeruginosa chromosomes. This follow-up study presented the complete sequences of 10 b-lactamase-encoding accessory genetic elements (AGEs; encoding four NSBLs CARB-2/-53, TEM-1B, and OXA-101; four ESBLs GES-1, PER-1, VEB-3 and OXA-10; and two carbapenemases GES-6/-15) along with three additional ones that did not encode any b-lactamase genes (but harbored dfrA12 and aphA6 and strAB) from the chromosomes of the sequenced P. aeruginosa strains. A detailed genetic dissection was applied to these 13 AGEs together with additional five reference/prototype ones from GenBank. Data presented here provided a deeper understanding of diversification of chromosomal MGEs (especially those carrying b-lactamase genes) in P. aeruginosa.

Bacterial Strains
Nine P. aeruginosa isolates (Table S1) were recovered either from sputum, airway secretions, urine, or blood of nine patients with nosocomial infections in five different Chinese public hospitals from 2011 to 2019. Bacterial species identification was performed using genome sequence-based average nucleotide identity analysis (http://www.ezbiocloud.net/tools/ ani) (Richter and Rossello-Mora, 2009).

Sequencing and Sequence Assembly
Bacterial genomic DNA was isolated using the UltraClean Microbial Kit (Qiagen, NW, Germany) and sequenced from a sheared DNA library with average size of 15 kb (ranged from 10 to 20 kb) on a PacBio RSII sequencer (Pacific Biosciences, CA, USA), as well as a paired-end library with an average insert size of 350 bp (ranged from 150 to 600 bp) on a HiSeq sequencer (Illumina, CA, USA). The paired-end short Illumina reads were used to correct the long PacBio reads utilizing proovread (Hackl et al., 2014), and then the corrected PacBio reads were assembled de novo utilizing SMARTdenovo (https://github.com/ ruanjue/smartdenovo).

Conjugal Transfer
Conjugal transfer experiments were carried out with rifampinresistant P. aeruginosa PAO1 being used as a recipient, and the indicated wild-type P. aeruginosa isolate as a donor. Three milliliters of overnight cultures of each of donor and recipient bacteria was mixed together, harvested, and resuspended in 80 mL of Brain Heart Infusion (BHI) broth (BD Biosciences). The mixture was spotted on a 1 cm 2 hydrophilic nylon membrane filter with a 0.45 µm pore size (Millipore) that was placed on BHI agar (BD Biosciences) plate and then incubated for mating at 37°C for 12 to 18 h. Bacteria were washed from filter membrane and spotted on Muller-Hinton (MH) agar (BD Biosciences) plates, for selecting a bla GES -or bla CARB -carrying PAO1 transconjugant. 1500 mg/mL rifampin (for PAO1), together with 80 mg/mL ceftazidime (for bla GES ) or 200 mg/L carbenicillin (for bla CARB ) was used for transconjugant selection.

Bacterial Antimicrobial Susceptibility Test
Bacterial antimicrobial susceptibility was tested by VITEK 2, Etest, or the classic broth microdilution method and interpreted as per the 2020 Clinical and Laboratory Standards Institute (CLSI) guidelines (CLSI, 2020).

Collection of 18 AGEs for Sequence Comparison
A detailed sequence comparison was then applied to a collection of four groups of 18 AGEs including the above 13 chromosomal AGEs together with five additional reference/prototype ones Tn6417, Tn6852, Tn6855, Tn6877, and Tn1403 from the GenBank: five related ICEs Tn6417, Tn6584, Tn6585, Tn6586, and Tn6587; four related IMEs Tn6852, Tn6853, Tn6854, and Tn6855; five related unit transposons Tn1403, Tn6846, Tn6847, Tn6848, and Tn6849; and two related IMEs Tn6877 and Tn6878 and their two derivatives dfrA12 region and bla VEB-3 region ( Table 1). At least 45 resistance genes, involved in resistance to 10 different categories of antibiotics and heavy metals, were identified in 17 of these 18 AGEs ( Figure 1 and Table S2).
Each of these five ICEs carried a single accessory module: Tn6807, Tn6808, Tn6809, Tn6532, and In1784 in Tn6584, Tn6585, Tn6586, Tn6417, and Tn6587, respectively ( Figure 2). The former four were integrated at a site upstream of the ICE backbone gene orf582 and identified as derivatives of Tn6346 (Ng et al., 2009), while the last one into the ICE backbone gene ftsk (cell division protein).
Tn6346, a prototype Tn3-family unit transposon originally identified in Achromobacter spp. AO22 (Ng et al., 2009), was a hybrid of the core transposition module tnpAR-res from Tn5051 and the mer region from Tn501 ( Figure 3). Tn6532, Tn6807, Tn6808, and Tn6809 differed from Tn6346 because of the interruption of tnpA due to the insertion of IS1071 at the same position, and that of urf2 due to the insertion of four different concise class 1 integrons In159, In1795, In1778, and In1820, respectively, at the same site; the four transposons Tn6532, Tn6807, Tn6808, and Tn6809 were bracketed by the same 5-bp direct repeats (DRs; target site duplication signals for transposition).
FIGURE 1 | Heatmap of prevalence of resistance genes. The original data are shown in Table S2.  Comparison of Four Related IMEs Tn6852, Tn6853, Tn6854, and Tn6855 Tn6852 (14.0 kb in length) was used as the prototype IME and initially found in P. aeruginosa PA38182 (Witney et al., 2014). Tn6852 carried the backbone markers attL/R, int, and the chrA region ( Figure 4). Conversely, Tn6853, Tn6854, and Tn6855 (Lv et al., 2020) carried a multidrug efflux locus nfxB-mexCD-oprJ instead of the chrA region. Both the chrA region and the nfxB-mexCD-oprJ region were considered as the IME backbone components, since none of the associated MGEs were identified for them. Tn6852 thereby had no accessory modules, but two Tn3-family unit transposons Tn6848 and Tn6849 as accessory modules were integrated at the same site within the backbone gene mexC of Tn6853 and Tn6854, respectively.  Tn6852, Tn6853, and Tn6854 were integrated into the chromosomal gene umuC (DNA polymerase V subunit) in P. aeruginosa, while Tn6855 into Tn5393c of plasmid pHNAH8I-1 in K. pneumoniae. Although plasmid-borne, Tn6855 was included in this study because it was a prototype MGE carrying an intact nfxB-mexCD-oprJ locus that was often found in Pseudomonas species.

Comparison of Two Related IMEs Tn6877 and Tn6878 and Their Derivatives dfrA12
Region and bla VEB-3 Region Tn6877 (55.9 kb in length) was used as the prototype IME and initially found in P. aeruginosa Pa58 (Espinosa-Camacho et al., 2017). Tn6877, Tn6878, and a presumed primordial Tn6877related element were all integrated at the same site between the two chromosomal genes orf2892 (a subunit of ribonucleotide reductase) and orf1248 (ß subunit), and they had essentially identical backbones, which was 21.6 kb in length and contained attL/R and two different int genes ( Figure 6). Three Tn21-related accessory modules, namely Tn6882, Tn6883, and a presumed primordial Tn21-related element were inserted at the same site within the above three AGEs, respectively. In addition, Tn6877 acquired the second accessory module ISPa1635. A huge DNA region composed of the dfrA12 region plus a 2,462.2-kb chromosomal region underwent an inversion, which was likely mediated by two copies of IS26: one located within the primordial Tn6877-related element and another inserted into the chromosomal gene orf1113 (chain length determinant protein) ( Figure 6). This inversion split the primordial Tn6877-related element into two separate parts: the dfrA12 region and the bla VEB-3 region; correspondingly, the primordial Tn21-related element in it was disrupted into the 5′-fragment and 3′-fragment.

Characterization of Two Novel Variants of Antibiotic Resistance Genes
This was the first report of a novel bla CARB-2 variant bla CARB-53 and a novel catB3 variant catB3s. The deduced CARB-53 protein differs from CARB-2 by a single amino acid substitution Glu26Lys. catB3s differed from the catB3 reference gene by one amino acid substitution Val210Ile.The bla CARB-53 or catB3s gene fragments were cloned into the cloning vector pUC57K and then transformed into E. coli TOP10 to obtain the electroporant TOP10/pUC57K-CARB or TOP10/pUC57K-catB.
As expected, TOP10/pUC57K-CARB was highly resistant to ampicillin, piperacillin, and carbenicillin but remained susceptible to cephalosporins and carbapenems ( Table 2). TOP10/pUC57K-catB showed a very high level of resistance to chloramphenicol with a minimum inhibitory concentration (MIC) value ≥256, while the two negative control strains TOP10/pUC57K and A B FIGURE 7 | Comparison of Tn6882, Tn6883, and related regions. Shown are five Tn21-related regions (A) and VR3 In1799 /VR2 In27 (B). Genes are denoted by arrows. Genes, MGEs, and other features are colored based on their functional classification. Shading denotes regions of homology (nucleotide identity ≥95%). Numbers in brackets indicate nucleotide positions within the chromosomes of strains Pa58, SE5429, and SE5458, respectively. The accession numbers of Tn21 (Partridge et al., 2001) and IS26-rmtB-qepA-IS26 unit (Yamane et al., 2007)  TOP10 were susceptible to chloramphenicol with a MIC value of 8.
Since the objective of this work was not to characterize these genes in terms of conferred phenotype, the cloning of the original genes was not done in parallel. Nevertheless, bla CARB-53 or catB3s had the drug resistance profile similar to its original variant bla CARB-2 (Korfhagen and Loper, 1975) or catB3 (Bunny et al., 1995), respectively.

ICE Transferability and Antimicrobial Susceptibility
A total of four ICEs Tn6584, Tn6585, Tn6586, and Tn6587 were identified from the nine P. aeruginosa isolates, and all these four ICEs had the essential conjugal transfer genes. After repeated conjugation experiment attempts, only Tn6587 was transferred from its rifampin-susceptible wild-type isolate into the rifampinresistant P. aeruginosa PAO1, generating the transconjugant PAO1/Tn6587. PAO1/Tn6587 was highly resistant to carbenicillin with a MIC value >512 owing to the presence of bla CARB-53 , whereas PAO1 was susceptible to chloramphenicol with a MIC value of 32.

Concluding Remarks
This work presented the complete sequences of 13 P. aeruginosa chromosomal AGEs, which could be divided into four groups: four Tn6417-related ICEs Tn6584, Tn6585, Tn6586 and Tn6587; two Tn6852-related IMEs Tn6853 and Tn6854; four Tn1403related unit transposons Tn6846, Tn6847, Tn6848 and Tn6849; and one Tn6877-related IME Tn6878 and its two derivatives dfrA12 region and bla VEB-3 region.
Besides the above 10 b-lactamase genes, there were additional 35 resistance genes identified in various subregions (including integrons, unit transposons, and putative resistance units) of these 13 chromosomal AGEs; thereby, most of these chromosomal AGEs had mosaic modular structures and encoded multiple drug resistance (Table S2).
Data presented here denoted that complex events of transposition and homologous recombination promoted the assembly and further integration of these chromosomal AGEs, carrying a large amount of resistance genes, into P. aeruginosa chromosomes.

ETHICS STATEMENT
This study uses the clinical bacterial isolates obtained from the Chinese public hospitals as listed in Table S1. The local legislation did not require the study to be reviewed or approved by an ethics committee because the bacterial isolates involved in this study were part of the routine hospital laboratory procedures. The research involving biohazards and all related procedures were approved by the Biosafety Committee of the Beijing Institute of Microbiology and Epidemiology.

AUTHOR CONTRIBUTIONS
DZ and MZ conceived the study and designed experimental procedures. TY, YJ, and FC performed the experiments. TY, LH, and XL analyzed the data. HY, JL, and ZY contributed to