https://orcid.org/0000-0002-9437-0234
Figures
Abstract
We aimed to determine the molecular characteristics of carbapenem-resistant Pseudomonas aeruginosa strains 18081308 and 18083286, which were isolated from the urine and the sputum of two Chinese patients, respectively. Additionally, we conducted a comparative analysis between Tn6411 carrying blaIMP-1 in strain 18083286 and transposons from the same family available in GenBank. Bacterial genome sequencing was carried out on strains 18081308 and 18083286 to obtain their whole genome sequence. Average nucleotide identity (ANI) was used for their precise species identification. Serotyping and multilocus sequence typing were performed. Furthermore, the acquired drug resistance genes of these strains were identified. The carbapenem-resistant P. aeruginosa strains isolated in the present study were of sequence type ST865 and serotype O6. They all carried the same resistance genes (aacC2, tmrB, and blaIMP-1). Tn6411, a Tn7-like transposon carrying blaIMP-1, was found in strain 18083286 by single molecule real time (SMRT) sequencing. We also identified the presence of this transposon sequence in other chromosomes of P. aeruginosa and plasmids carried by Acinetobacter spp. in GenBank, indicating the necessity for heightening attention to the potential transferability of this transposon.
Citation: Zheng L, Wang Z, Guo J, Guan J, Lu G, Jing J, et al. (2024) Comparative genomics of Tn6411 transposons carrying the blaIMP-1 gene in Pseudomonas aeruginosa. PLoS ONE 19(7): e0306442. https://doi.org/10.1371/journal.pone.0306442
Editor: Mohamed O. Ahmed, University of Tripoli, LIBYA
Received: January 15, 2024; Accepted: June 15, 2024; Published: July 9, 2024
Copyright: © 2024 Zheng et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The contig sequences of strains 18081308 and 18083286 have been submitted to GenBank under accession numbers GCA_024718375.1 and GCA_024714245.1. The complete sequence of 18083286 has been submitted to GenBank under accession number CP110368.
Funding: Funding for study design, data collection, data generation, and publication costs was provided by the National Science and Natural Science Foundation of China (Grant agreement 31872486) awarded to Zhu LW. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Pseudomonas aeruginosa is a zoonotic opportunistic bacterial pathogen that is ubiquitous in diverse environments, including water, animal-related food, the surface of medical instruments, and sewage systems in hospitals [1,2]. It carries a large variety of virulence factors and can cause bacteremia, ventilator-associated pneumonia, cystic fibrosis, and chronic obstructive pulmonary disease. It has the ability to form biofilms and attach to the surface of medical instruments and food [1]. It can spread in healthcare settings from one person to another through contaminated hands or surfaces. It is easily disseminated within hospitals; it caused an estimated 32,600 infections among hospitalized patients and resulted in approximately 2,700 deaths in the United States according to the Threat Estimate 2019 report [3]. Clinically, P. aeruginosa infection is usually treated by antimicrobial therapy, and prolonged use of antibiotics to achieve bacterial cure is commonly practiced [4]. Resistance genes can be acquired through the transfer of mobile genetic elements (such as plasmids, transposons, and integrative and conjugative elements) among bacterial strains, resulting in the development of multidrug-resistant P. aeruginosa in chronically infected patients [5,6]. Carbapenems are the most important antibiotics for treating multidrug-resistant bacterial infections, but P. aeruginosa is currently also resistant to carbapenems due to the acquisition of carbapenemases, among other reasons, impeding treatment.
In the present study, two P. aeruginosa isolates (18081308 and 18083286) were obtained from two patients (urine and sputum samples, respectively) admitted to a public hospital (Changchun, China) in August 2018. Their whole genome sequences and molecular characteristics were determined. Both isolates belonged to the same multilocus sequence type (ST865) and serotype (O6). They both carried blaIMP-1. Detailed genetic dissection was applied to a Tn7-like transposon carrying blaIMP-1 to display its genetic environment. The data presented here provide a deeper understanding of drug resistance gene acquisition in P. aeruginosa from a genomic and bioinformatic point of view.
Materials and methods
Bacterial isolation and identification
In the present case, a 72-year-old man (patient A) was admitted with cardiovascular disease (CVD) in August 13, 2018. Fourteen days later, another patient (patient B), a 56-year-old man was admitted with respiratory disease. Strains 18083286 and 18081308 were isolated from the sputum (patient A) and urine specimens (patient B) of the patients. The species was determined based on the partial sequence of the 16S rRNA gene [7].
Minimum inhibitory concentrations (MICs) of amikacin, gentamicin, meropenem, imipenem, cefazolin, ceftazidime, cefotaxime, cefepime, aztreonam, ampicillin, piperacillin, amoxicillin-clavulanate, ampicillin-sulbactam, piperacillin-tazobactam, trimethoprim-sulfamethoxazole, chloramphenicol, ciprofloxacin, levofloxacin, moxifloxacin, and tetracycline against strains 18083286 and 18081308 were tested by BD Phoenix-100, using Escherichia coli ATCC25922 as a control. Drug resistance and sensitivity were judged based on the Clinical and Laboratory Standards Institute guidelines (2019).
Next-generation sequencing, sequence assembly and annotation
Bacterial genomic DNA was extracted from strains 18081308 and 18083286 using the UltraClean Microbial Kit and sequenced using an Illumina NovaSeq PE150 platform. Trimmomatic V10 was used to remove the PCR adapters and low-quality reads, and SPAdes (http://cab.spbu.ru/software/spades/) was used for sequence assembly [8]. Precise species identification was performed by pairwise average nucleotide identity (ANI) (http://www.ezbiocloud.net/tools/ani) analysis between genome sequences and the P. aeruginosa reference genome PAO1 (GenBank ID: NC_002516.2). An ≥95% ANI cut-off was used to define bacterial species [9]. PAst (https://cge.food.dtu.dk/services/PAst/) was used to perform serotyping. Multilocus sequence types (STs) were obtained by uploading their genomes, including the seven conserved housekeeping genes acsA, aroE, gtaA, mutL, nuoD, ppsA, and trpE, to pubMLST (https://pubmlst.org/). Online databases, including CARD [10] (https://card.mcmaster.ca/) and ResFinder 4.0 [11] (https://cge.cbs.dtu.dk/services/ResFinder/), were used to identify resistance genes.
Single molecule real-time sequencing, annotation and comparison
The nucleotide identity between strains 18083286 and 18081308 was evaluated using ANI. Due to the discontinuity and incompleteness of the next-generation sequencing (NGS) results, there was an interference effect on the analysis of nucleobase absences. In this study, strain 18083286 was randomly selected to undergo another DNA extraction step, and the newly obtained DNA was subsequently single molecule real-time (SMRT) sequenced using a PacBio RSII sequencer. Based on the aforementioned sequencing data, Canu software (version 2.0) was utilized for genome assembly from reads, yielding initial assembly results that reflect the genomic status of the sample. Subsequently, Rcon software (version 1.4.13) was employed for three rounds of error correction based on third-generation sequencing data, followed by three rounds of Pilon software (version 1.22) error correction using second-generation reads, resulting in the final assembly outcome. Then, the sequenced DNA was annotated to identified mobile genetic elements (MGEs), and the MGE sequences were used to generate linear alignment maps with other sequences from the same family in GenBank. RAST 2.0 [12] and BLASTP/BLASTN [13] searches were conducted to predict open reading frames (ORFs). The CRAD [10] and ResFinder 4.0 [11] databases were used to identify drug resistance genes, again. ISfinder [14] (https://www-is.biotoul.fr/; last database update 2021-9-21), TnCentral (https://tncentral.ncc.unesp.br), INTEGRALL (http://integrall.bio.ua.pt/) [15], and ICEberg 2.0 (http://db-mml.sjtu.edu.cn/ICEberg/) [16] were used to identify mobile elements. Pairwise sequence comparisons were carried out by BLASTN. Gene organization diagrams were drawn by Inkscape 1.0 (http://inkscape.org/en/).
Results and discussion
Strains 18083286 and 18081308 were identified as P. aeruginosa by the BD Phoenix-100 identification system and based on the 16S rRNA gene. Table 1 shows the drug resistance spectrum of strain 18083286, which was consistent with that of strain 18081308.After Illumina NovaSeq PE150 sequencing (basic information about the Illumina sequencing results is provided in Table 2), it was found that their ANI values were more than 95% with the reference strain P. aeruginosa PAO1 (GenBank ID: NC_002516.2), and they were confirmed to be P. aeruginosa (ANI values of P. aeruginosa 18083286 and 18081308 are provided in S1 Table).
Both isolates belonged to the same multilocus sequence type (ST865) and serotype (O6) based on MLST and PAst screening. There are 20 serotypes of P. aeruginosa, of which serotype O6 is one of the most common [17]. ST865 is not a pandemic clonal group. Until 2022, the MLST database contained a total of four strains of P. aeruginosa ST865: strains 18081308 and 18083286 isolated in this study, strain AZPAE14882 of unknown origin, and strain AUS151 isolated from soft tissue in Australia in 2008.
They were resistant to many antibiotics in addition to imipenem, including: gentamicin, meropenem, cefazolin, ceftazidime, cefotaxime, cefepime, ampicillin, amoxicillin-clavulanate, ampicillin-sulbactam, trimethoprim-sulfamethoxazole, chloramphenicol, and tetracycline. The results are summarized in Table 1. Aminoglycoside resistance genes (aac(6”)-II, aac(3)-IId, and aph(3”)-IIb), an amphenicol resistance gene (catB7), β-lactam resistance genes (blaOXA-486, blaIMP-1, and blaPAO), and a fosfomycin resistance gene (fosA) were identified by ResFinder in these strains.
Since the two strains had the same resistance profile, ST type, serotype, acquired resistance genes, and an ANI value of 99.99% (ANI values are provided in S1 Table) [18], one of the two strains was randomly selected for genetic environment analysis of the carbapenem resistance gene blaIMP-1. SMRT sequencing (basic information about SMRT sequencing results is provided in Table 2) showed that the chromosome of strain 18083286 was 6.4 Mb and its GC content was 66.3%; no plasmid was detected. blaIMP-1 was located in a Tn7-like transposon in the bacterial chromosome.
A 37.53-kb transposon was inserted at a glmS (glucosamine-fructose-6-phosphate aminotransferase) site in the chromosome of P. aeruginosa 18083286. It had a complete set of Tn7-family core transposon-encoded proteins (TnsABCDE), but with very low levels of nucleotide identity with Tn7 counterparts. This structure had the closest phylogenetic relationship with Tn6411 (a Tn7-like family transposon) in P. aeruginosa 12939 (GenBank ID: CP024477.1; coverage: 100%, identity: 100%); thus, this Tn7-like transposon was identified as Tn6411. It was first discovered in a P. aeruginosa strain from China in 2018 [19].
Until October 2022, only 10 transposons with the same TnsA as Tn6411 were indexed in GenBank (Table 3 shows strain information). Among them, strains 12939, 18083286, and HB2011305RE, and plasmid p201330 carried Tn6411, and others carried its derived structures, named Tn6411-like. They mainly included P. aeruginosa from China, but Acinetobacter spp. from Sydney, Australia, and P. aeruginosa from India were also found. Except for Tn641118083286, Tn6411-likeSE5430, and Tn6411-likePA34 transposons, the others contained 20-bp TnsB-binding sites plus 26 bp inverted repeats (IRs), which were terminal flanking regions (S2 Table). They contained complete TnsABC+TnsD/E proteins, which were encoded by genes inserted into attTn7 or plasmids capable of transfer between bacteria. Except for Tn6411-likePA34 (Indian), all others carried a truncated aacC2-tmrB region. The intact structure (IS26-aacC2-tmrB region remnant-blaTEM-1) was found in pEl15573 [20,21]. It was derived from transposon Tn2. The structure (IS26-aacC2-tmrB region remnant) was found in the IncR/IncP6 fusion plasmid pCRE3-KPC carried by Citrobacter braakii [22]. The truncated aacC2-tmrB region identified in this study was likely to be an intact structure from which first blaTEM-1 and then IS26 was deleted.
All ten Tn6411 transposons (until February 2022) listed in GenBank are shown in Fig 1. An integron carrying blaIMP-1 (a carbapenem resistance gene) and aac(6”)-II (an aminoglycoside resistance gene), named In992, was inserted between pinR (encoding a DNA site-specific recombinase) and a gene (encoding a methyltransferase domain protein) that serves the backbone of Tn641112939 (Beijing, China), Tn641118083286 (Changchun, China), Tn6411HB2011305RE (Changchun, China), and Tn6411p201330 (Changchun, China). One DR copy (ATGCCCGC) of In992 was found upstream of pinR (Tn6411-likeP8W, Tn6411-likeP9W, and Tn6411-likeSE5430), which enabled the insertion of In992, resulting in a bilateral DR sequence (ATGCCCGC). Tn6411 was carried by other strains that do not carry In992 or other integrons. The truncated Tn402 transposition module in In992 had undergone the deletion of the partial TniA sequence and the complete TniBQR sequence, resulting in the loss of its self-transfer capability.
The backbone region is shown in black, In992 is shown in light blue, the aacC2-tmrB region remnant is shown in orange, ISAba14 is shown in brownish red, and ISCfr1 is shown in pink. The shaded region represents a region with >90% nucleotide identity. All transposons encoded complete TnsABC+TnsD/E proteins. Except for Tn6411-likePA34, all others carried a truncated aacC2-tmrB region remnant. An integron named In992 carrying blaIMP-1 and aac(6”)-II was inserted between pinR and a gene encoding a methyltransferase domain protein from the backbone of Tn641112939, Tn641118083286, Tn6411HB2011305RE, Tn6411p201330, and Tn6411 carried by other strains that do not carry In992 or other integrons. The truncated Tn402 transposition module in In992 had undergone the deletion of the partial TniA sequence. Although Tn6411-likepE47 and Tn6411-likepWM98B from A. baumannii and A. nosocomialis did not carry In992, one or two copies of ISAba14 were inserted downstream of the aacC2-tmrB region remnant. Tn6411-likepE47 had a one-copy ISAba14 difference from Tn6411-likepWM98B in addition to a 1777-bp sequence difference.
One or two copies of ISAba14 were inserted upstream of the aacC2-tmrB region remnant, although Tn6411-likepE47 and Tn6411-likepWM98B from Acinetobacter baumannii (Sydney, Australia) and Acinetobacter nosocomialis (Sydney, Australia) did not carry In992. When one copy of ISAba14 and the 1777-bp neighbor base sequence were lost and the other copy of ISAba14 was retained, a Tn6411-likepWM98B (Tn6411-aacC2-tmrB region remnant-ISAba14-ISAba14) changed into Tn6411-likepE47 (Tn6411-aacC2-tmrB region remnant-ISAba14) (Fig 1).
Tn6411-likeP8W, Tn6411-likeP9W, Tn6411-likeSE5430, and Tn6411-likePA34 did not have an accessory module (the sequence of Tn6411-likePA34 was discontinuous and incomplete, and no analysis was conducted). One copy of the DR sequence (ATAT) of ISAba14 was found upstream of the aacC2-tmrB region remnant of Tn6411-likeP8W, Tn6411-likeP9W, and Tn6411-likeSE5430, which enabled double copy of the ISAba14 sequence in the same direction to insert its Tn6411-like structure (Fig 1).
Compared with Tn6411-likeSE5430, Tn6411-likeP8W and Tn6411-likeP9W lacked a 26-bp sequence downstream of the aacC2-tmrB region remnant, but the IRL of Tn6411-likeSE5430 lacked a 14-bp sequence. TnsB binding site 1 had an 8-bp deletion as Tn641118083286, but the difference was that TnsB binding sites 2 and 3 of Tn6411-likeSE5430 were also missing. Therefore, the formation of the structure of Tn6411-likeP8W and Tn6411-likeP9W might have occurred before the formation of Tn6411-likeSE5430 (S2 Table).
Genetic sequence analysis revealed the potential structure change and transfer of the Tn6411 transposons. The aacC2-tmrB region remnant-Tn6411 backbone was the earliest structure, and then in the evolutionary process, the element might form two evolutionary modes; one was localized in chromosome by TnsD for vertical transmission within species, and the other was localized in plasmid by TnsE for horizontal transmission between different species (Fig 2).
The figure shows the aacC2-tmrB region remnant-Tn6411 backbone as a local structure. ISCfr1 is shown in pink, attTn7 (glmS) is shown as a red dot, In992 is shown in light blue, and ISAba14 is shown in brownish red. (a) A Tn6411-like transposon (aacC2-tmrB region remnant-Tn6411backbone) was localized in the chromosome by TnsD; (b) In992 carrying blaIMP-1 and aac(6”)-II was inserted into the Tn6411 backbone. The truncated Tn402 transposition module in In992 had undergone the deletion of the partial TniA sequence and complete TniBQR sequence and lost its self-transfer capability, making it stable, forming Tn6411. (c) Tn6411-like (aacC2-tmrB region remnant-Tn6411) was localized in a plasmid by TnsE for horizontal transmission. (d) Two copies of ISAba14 were inserted into the Tn6411 backbone. (e) One copy of ISAba14 and a 1777-bp neighbor base sequence were lost, and the other copy of ISAba14 was retained.
In conclusion, genetic sequence analysis suggested that Tn6411 transposons could act as vectors and capture type 1 integrons containing blaIMP-1.Although the Tn6411 transposons sequences were commonly situated within the chromosome of P. aeruginosa, they had also been identified in the plasmids carried by Acinetobacter spp.. The absence of experimental verification for horizontal gene transfer also presented constraints on this research, rendering its transferability uncertain. The presence of Tn6411 sequences in various species indicated that there should be a closer monitoring of and investigation into the conditions under which this transfer occurs.
Supporting information
S1 Table. The ANI value of P. aeruginosa in this study.
https://doi.org/10.1371/journal.pone.0306442.s001
(XLSX)
S2 Table. The sequence of TnsB-binding sites and IRs.
https://doi.org/10.1371/journal.pone.0306442.s002
(XLSX)
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