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Article

Emergence and Transfer of Plasmid-Harbored rmtB in a Clinical Multidrug-Resistant Pseudomonas aeruginosa Strain

by
Jiacong Gao
,
Xiaoya Wei
,
Liwen Yin
,
Yongxin Jin
,
Fang Bai
,
Zhihui Cheng
and
Weihui Wu
*
State Key Laboratory of Medicinal Chemical Biology, Key Laboratory of Molecular Microbiology and Technology of the Ministry of Education, Department of Microbiology, College of Life Sciences, Nankai University, Tianjin 300071, China
*
Author to whom correspondence should be addressed.
Microorganisms 2022, 10(9), 1818; https://doi.org/10.3390/microorganisms10091818
Submission received: 11 August 2022 / Revised: 3 September 2022 / Accepted: 8 September 2022 / Published: 11 September 2022
(This article belongs to the Special Issue Antimicrobial Resistance Mechanisms in Bacteria)
Browse Figures
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Abstract

:
Multidrug-resistant (MDR) Pseudomonas aeruginosa poses a great challenge to clinical treatment. In this study, we characterized a ST768 MDR P. aeruginosa strain, Pa150, that was isolated from a diabetic foot patient. The minimum inhibitory concentration (MIC) assay showed that Pa150 was resistant to almost all kinds of antibiotics, especially aminoglycosides. Whole genome sequencing revealed multiple antibiotic resistant genes on the chromosome and a 437-Kb plasmid (named pTJPa150) that harbors conjugation-related genes. A conjugation assay verified its self-transmissibility. On the pTJPa150 plasmid, we identified a 16S rRNA methylase gene, rmtB, that is flanked by mobile genetic elements (MGEs). The transfer of the pTJPa150 plasmid or the cloning of the rmtB gene into the reference strain, PAO1, significantly increased the bacterial resistance to aminoglycoside antibiotics. To the best of our knowledge, this is the first report of an rmtB-carrying conjugative plasmid isolated from P. aeruginosa, revealing a novel possible transmission mechanism of the rmtB gene.

1. Introduction

Pseudomonas aeruginosa is a common opportunistic pathogen that widely exists in water, air, human and animal skin, respiratory tracts, and intestinal environments [1,2]. Studies have shown that P. aeruginosa usually carries multiple antibiotic resistance genes (ARGs), increasing the difficulties in clinical treatment [3].
Aminoglycoside antibiotics were one of the earliest antibiotics discovered and used clinically. These antibiotics interfere with protein synthesis and ultimately cause bacterial death by binding to the highly conserved A-site of the 16S rRNA of the bacterial 30S ribosomal subunit [4,5]. They have been widely used in the treatment of lung infection caused by P. aeruginosa [6,7]. However, resistance to aminoglycosides, including gentamicin, tobramycin, and amikacin, has been increasingly reported worldwide in P. aeruginosa [8]. The resistance typically results from low membrane permeability, multidrug efflux systems, mutation of the target gene, and/or chromosome- or plasmid-encoded aminoglycoside modification enzymes (AME) and 16S rRNA methylases (16S-RMTase) [9,10,11].
AMEs inactivate aminoglycosides by modifying their amino or hydroxyl groups, including acetylation (aminoglycoside acetyltransferase [AAC]), phosphorylation (aminoglycoside phosphoryltransferase [APH]), and adenylation (aminoglycoside nucleotidyltransferase [ANT]) [12,13]. 16S-RMTases add a methyl (CH3) group to specific residues of the 16S rRNA, which significantly reduces its binding affinity to aminoglycosides, leading to high-level and extensive aminoglycoside resistance [14]. In recent years, 16S-RMTase has become more and more prevalent in clinical strains [14]. In 2003, a plasmid-mediated 16S rRNA methylase (rmtA) was first reported in Japan in a P. aeruginosa clinical strain [15], which led to high level resistance to aminoglycoside antibiotics. In recent years, aminoglycoside resistance mediated by an rmtB gene carried by plasmids has become a serious threat to almost all aminoglycoside antibiotics [14,16].
Large plasmids in bacteria are mostly conjugative [17,18], and usually carry multiple ARGs that are surrounded by mobile genetic elements (MGEs). Conjugative plasmids play important roles in facilitating the horizontal transfer of ARGs via conjugation [19,20,21,22,23,24]. Many studies have shown that large plasmids carried by P. aeruginosa contribute to the accumulation and transmission of ARGs, leading to multidrug-resistance [25,26].
In this study, we characterized an ST768 MDR P. aeruginosa strain, Pa150, and identified a 437-Kb plasmid (pTJPa150) containing a 16S rRNA methylase rmtB. We identified MGEs flanking rmtB and demonstrated the transmission of rmtB by the pTJPa150 plasmid through conjugation.

2. Materials and Methods

2.1. Strains, Plasmids, Primers

The bacterial strains, plasmids, and primers used in this study are listed in Tables S1 and S2, respectively.

2.2. Antibiotic Susceptibility Test

Overnight bacterial cultures were diluted 1:100 into fresh LB broth to an OD600 of 1. Then, the antibiotics were serially diluted in MHB broth (100 μL) in a 96-well plate. One hundred μL MHB broth containing 106 CFU/mL cells were added to each well with the diluted antibiotics. The plates were incubated at 37 °C for 24 h. The MICs were determined by the lowest antibiotic concentration with no visible bacterial growth.

2.3. Whole Genome Sequencing (WGS) and Bioinformatics Analysis

Total DNA from Pa150 was extracted using the TIANamp Bacteria DNA Kit (TIANGEN, Beijing, China) according to the manufacturer’s instructions. DNA library construction and sequencing were performed by Grandomics Biosciences Co., Ltd., Beijing, China. Single-molecule sequencing was performed with a PromethION sequencer (Oxford Nanopore Technology, Bejing, China). Genome assembly, correction, and optimization were performed on the obtained data to obtain the final genome. CGView server software was used for plasmid circle mapping and MEGA software (version 7.0, Auckland, New Zealand) was used for evolutionary analysis.

2.4. Gene Cloning and Conjugation Assays

A DNA fragment containing the rmtB gene with its native transcription terminator region was amplified by PCR. The PCR product was digested with BamHI and EcoRI, and then cloned into the pUCP20 vector to yield the recombination plasmid pUCP20-rmtB. The resultant plasmid was transferred into P. aeruginosa reference strain PAO1 via electroporation.
The conjugation assay was performed by using a tetracycline-resistant mutant of PAO1 carrying a lacZ gene (PAO1-lacZ) as the recipient strain [27,28]. The donor strain Pa150 and the recipient strain PAO1-lacZ were grown in LB broth to an OD600 of 0.8. The donor bacteria and the recipient bacteria were mixed at a ratio of 7:1. The bacteria were centrifuged at 6000 rpm for 5 min and then washed three times with 1 mL fresh LB broth. Then, 100 μL of the bacterial suspension were added onto a filter membrane, which was placed on a Nutrient-Agar (BD, Difco) plate at 37 °C for 12 h. The bacteria were washed out from the membrane by LB and plated on LB agar plates containing X-Gal (40 mg/mL), tetracycline (150 μg/mL), chloramphenicol (200 μg/mL), and isopropyl β-D-1-thiogalactopyranoside (IPTG, 20 μg/mL). Blue colonies were further streaked on the selection plates and the transconjugants were verified by PCR with the primes listed in Table S2.

2.5. Statistical Analysis of All Strains Containing the rmtB Gene

A basic local alignment search tool (BALST) was used for searching sequences sharing high similarity (>90% identity) with the rmtB gene. Information about all strains (n = 620) containing the rmtB gene was collected, including species, isolation years, sources, and countries (Table S5). The analytic results were presented by line, pie, and bar charts using the Prism software (Graphpad).

2.6. Data Availability

The genome and plasmid sequences were deposited in the NCBI Genbank database (CP094677, CP094678)

3. Results

3.1. Characterization of MDR P. aeruginosa Strain Pa150 and Conjugative Plasmid pTJPa150

A P. aeruginosa strain (named Pa150) was isolated from a diabetic foot patient in Baoding, Hebei Province, China. This isolate is resistant to almost all classes of antibiotics (Table 1), including meropenem (MEM, MIC = 64 μg/mL), aztreonam avibactam (AZT, MIC = 64 μg/mL), azithromycin (AZM, MIC = 128 μg/mL), ciprofloxacin (CIP, MIC = 32 μg/mL), amikacin (AMK, MIC > 128 μg/mL), and tobramycin (TBR, MIC > 128 μg/mL), but remains susceptible to colistin (COL, MIC = 0.25 μg/mL). Whole genome sequencing (WGS) of Pa150 was performed (Figure 1). The genome of Pa150 was around 6.6 Mb, with GC content of 65.62% (Table S3), encoding 6497 genes. Multilocus sequence typing (MLST) revealed that Pa150 belongs to ST768. ST175, ST235, ST357, and ST664 are high risk clones of P. aeruginosa, with the highest clinical isolation rates in the world [29,30,31]. However, the MDR ST768 clone is rarely reported.
Notably, Pa150 harbors a large conjugative plasmid, pTJPa150. The pTJPa150 plasmid is 436,716 bp in size with a GC content of 56.85%; it contains 464 predicted open reading frames (ORFs) (Figure 2). The pTJPa150 plasmid has the highest coverage and sequence similarity (93% + 99.14%) with the plasmid pBT2436 of the P. aeruginosa strain T2436 (GenBank accession number CP039989), which was isolated from adult male sputum in Thailand in 2013. Further sequencing analysis revealed that the pTJPa150 plasmid contains multiple ARGs, including two β-lactamase genes (blaTEM and blaOXA-10), five aminoglycoside resistance genes (aac(6′)-IIa, aac(3′)-IId, strA, strB, and rmtB), a single-component efflux pump gene cmlA and a resistance-nodulation-cell-division (RND) pump gene cluster tnfxB-tmexCD-toprJ (Table S3), indicating a potential role in the multidrug-resistance of Pa150.

3.2. Contribution of the Conjugative Plasmid pTJPa150 in Antibiotic Resistance

The conjugative transfer of bacterial plasmids is the most effective mode of horizontal gene transmission and is, therefore, considered to be one of the main causes for the increase in multidrug-resistant bacteria [18,32]. In Gram-negative bacteria, conjugation is usually mediated by the conjugative type IV secretion system (T4SS), a large transport apparatus produced by donor cells [33,34]. The pTJPa150 plasmid contains genes involved in conjugation (Figure 2). To verify the self-transmissibility of the pTJPa150 plasmid and its role in the MDR of Pa150, we performed a conjugation assay by using Pa150 and a lacZ expressing P. aeruginosa reference stain PAO1 (PAO1-lacZ) as the donor and recipient strains, respectively. The presence of the pTJPa150 plasmid in the transconjugants was verified by PCR amplification of the blaTEM and parB genes. Compared with the recipient strain, the MICs of β-lactam, macrolide, and quinolone antibiotics for the transconjugant were two- to eight-fold higher (Table 1). Particularly, the MICs of aminoglycoside antibiotics were increased by more than 128-fold, reaching the similar levels of Pa150 (Table 1), indicating a significant role of the pTJPa150 plasmid in aminoglycosides resistance. The treatment of Pa150 and the transconjugant (PAO1-lacZ-pTJPa150) with the RND pump inhibitor PAβN reduced the MICs of the β-lactam, macrolide, and quinolone antibiotics, but had no effect on the MICs of aminoglycosides (Table 1). These results demonstrate a role of TMexCD-TOprJ in the resistance to β-lactam, macrolide, and quinolone antibiotics, and additional mechanisms in the resistance to aminoglycosides.

3.3. Characterization of the Role of RmtB in Aminoglycoside Resistance

As shown in Figure 2 and Table S4, the pTJPa150 plasmid contains four aminoglycoside modification enzyme genes (aac(6′)-IIa, strA, strB, aac(3′)-IId) and a 16S rRNA methylase gene rmtB. Previous reports have shown that AAC(6′) enzymes are the most common AMEs that are widely distributed in Gram-negative and Gram-positive bacteria. However, AAC(6′)-II enzymes cannot modify amikacin. AAC(3)-II enzymes are almost exclusively found in Gram-negative bacteria and confer resistance to many antibiotics, including gentamicin, neomycin, tobramycin, and sisomicin [13]. strA and strB are currently the most widely distributed streptomycin resistance determinants. However, these four AMEs are rarely reported to contribute to high amikacin resistance [13,35,36]. RmtB was first identified in Serratia marcescens. It shares an 82% amino acid sequence identity with RmtA and has been shown to lead to high-level resistance to various aminoglycosides [37]. To verify the role of rmtB in the aminoglycoside resistance, we cloned the gene into E. coli DH5α and PAO1. The expression of rmtB significantly increased the MICs of the aminoglycosides in both strains (Table 1), indicating an important role of rmtB in the aminoglycoside resistance.

3.4. Possible Origin and Mobilization of rmtB

On the pTJPa150 plasmid, rmtB is in close proximity to blaTEM, flanked by multiple MGEs (Figure 2). Phylogenetic analysis revealed that RmtB has high amino acid homology in 53 phylogenetic groups (90%) (Figure 3A), mainly including five P. aeruginosa isolates (green), 15 E. coli isolates (blue), and 28 Klebsiella pneumonia isolates (orange). These rmtB-carrying strains were isolated from various countries since 2005, including the USA, China, Thailand, Vietnam, and Canada. In addition, the amino acid sequence of RmtB in the pTJPa150 plasmid is more closely related to that of the K. pneumoniae HS11286 plasmid pKPHS3.
Like the reported rmtB gene in S. Marcescens-95, the rmtB gene in the pTJPa150 plasmid is flanked by blaTEM and Tn3. Additionally, IS91, IS30, and IS26 transposons and the rmtB-blaTEM-Tn3 cluster form an IS91-groEL-nahP-rmtB-blaTEM-Tn3-IS30-IS26 module (Figure 3B). Sequence alignment using the rmtB-blaTEM-Tn3 cluster sequence revealed a total of 520 sequences with homology greater than 99% and query cover greater than 90% in the NCBI database. We then selected the top 11 sequences with the highest homology to the IS91-groEL-nahP-rmtB-blaTEM-Tn3-IS30-IS26 module in Pa150 as the representatives to show the alignments (Figure 3B). K. pneumoniae HS11286, the closest strain to Pa150 in the phylogenetic tree, was included in the 11 strains. Through linear sequence alignment, we found that there were insertion sequence (IS) elements on both sides of the rmtB-blaTEM-Tn3 cluster in all of the 11 strains. Of the 11 strains, the MGEs on both sides of the rmtB-blaTEM-Tn3 cluster were IS26 elements (Figure 3B), except for the plasmid pKPHS3 in K. pneumoniae strain HS11286 and the plasmid pHN21SC92-1 in Citrobater Portucalensis strain GD21SC92T (Figure 3B). Wang et al. found that IS26 elements play a vital role in the dissemination of the resistance genes (16). However, in the pTJPa150 plasmid, the rmtB-blaTEM-Tn3 cluster is flanked by IS26 and IS91, which is the same in the C. portucalensis plasmid pHN21SC92-1. Further analysis revealed that part of the IS91 sequence showed 100% identity with the ISCR15b in the K. pneumoniae plasmid pKPHS3 (Figure 3B), suggesting the transfer of the gene cluster in this plasmid may also be mediated by IS26 and ISCR15b.

3.5. Epidemic Analysis of rmtB-Carrying Strains

To date, 620 rmtB-carrying strains have been reported worldwide. Among all of the strains, only 1.77% (n = 11) are P. aeruginosa; the majority are K. pneumonia (54.03%) and E. coli (34.19%). In 90.64% of these strains, rmtB is present on plasmids (Figure 4A, Table S5), 90% of which are larger than 10 Kb. Given the important roles of plasmids in the transmission of resistance genes [38], the presence of rmtB on plasmids may account for its worldwide pread. In all of the analyzed samples, the most frequent isolation source was the clinical environment (n = 355), followed by animal sources (n = 60) and natural environments (n = 25) (Figure 4B, Table S5). It is worth mentioning that these rmtB-carrying strains are almost all pathogenic bacteria.
From another perspective, these rmtB-carrying strains were isolated from 32 different countries, with the largest total number of strains isolated from China (Figure 4D, Table S5). From 2003 to 2022, rmtB-carrying strains were isolated from 24 provinces or regions in China; they were highly prevalent in Zhejiang (17.9%), Guangdong (15.2%), Sichuan (10.22%), and Henan (11.97%) provinces (Figure 4C, Table S5). According to the dataset, rmtB was first reported in 2003, and the reported number of rmtB-carrying strains kept increasing until 2017, then decreased to 47 in 2021 (Figure 4E, Table S5).

4. Discussion

P. aeruginosa is a common clinical opportunistic pathogen and one of the main sources of nosocomial infection [39]. With the increase in clinical MDR strains, the World Health Organization (WHO) has listed MDR P. aeruginosa as one of the most threatening human pathogens [40]. In this study, we identified an MDR P. aeruginosa strain, Pa150, that contain a conjugative plasmid, pTJPa150. The pTJPa150 plasmid harbors an rmtB gene, which contributes significantly to aminoglycoside antibiotic resistance (Table 1). As far as we know, this is the first report of an rmtB-carrying plasmid in P. aeruginosa.
Plasmid conjugation is a major route in the spread of ARGs [41]. In this study, we found that the transconjugant carrying the pTJPa150 plasmid displayed elevated MICs of β-lactam, quinolone, and aminoglycoside antibiotics, compared with the parental recipient strain (PAO1-lacZ) (Table 1). In the pTJPa150 plasmid, a large number of ARGs form gene clusters at specific locations (Figure 2). Site-specific recombinases and transposases that may mediate horizontal transfer also exist in the pTJPa150 plasmid and are located close to the ARGs (Figure 2). The rmtB gene was found in the pTJPa150 plasmid containing multiple MGEs, which might promote the spread of rmtB in P. aeruginosa. In addition, the pTJPa150 plasmid contains an RND efflux pump tnfxB-tmexCD-toprJ. The pump has been identified on plasmids in P. aeruginosa and K. pneumonia, which significantly increased the bacterial resistance to tigecycline [42,43]. Further studies are warranted to understand its role in bacterial resistance to various types of antibiotics.
AMEs and 16S rRNA methylase are the main causes of bacterial resistance to aminoglycoside antibiotics. Gram-negative bacteria containing rmtB also carry other enzymes that are responsible for different kinds of antibiotic resistance, such as β-lactamase [44]. In this study, the nucleotide sequences of rmtB adjacent to blaTEM in the pTJPa150 plasmid was 100% identical to that in pS95B2 in S. marcescens S-95, which was first identified as the rmtB-carrying strain [27]. It has been reported that rmtB in K. pneumoniae might be transferred through an IS26-mediated homologous recombination, although no IS26 recognition repeat sequence has been found [16]. In the pTJPa150 plasmid, the IS26 sequence exists only on one side of rmtB, while on the other side is IS91, part of which is actually an ISCR15b sequence. Wang et al. also showed the diversity of MGEs on both sides of the tet(X6) gene [43]. Based on this observation and the finding in this study, we speculated that this diversity might be more conducive to the transfer of resistance genes. Strains containing the rmtB gene are widely distributed worldwide and have been isolated in 32 countries. Almost all the known rmtB-carrying strains are Enterobacteriaceae strains. Most of the rmtB genes exist on plasmids, which may be an important reason for the rapid spread of the rmtB gene since its discovery in 2003. In addition to clinical samples, edible animals or animal feeding environments are also the main sources of the rmtB-carrying strains of the reported 620 strains, which may promote the transfer of rmtB to humans through the food chain.
In summary, we reported for the first time an rmtB gene in a plasmid of an MDR P. aeruginosa strain. The transfer of the rmtB gene might be mediated by IS26 and ISCR15b. In addition, we carried out statistical analysis on all strains containing the rmtB gene and found that rmtB-carrying strains are widely distributed throughout the world. Most of them exist on the plasmids in the strains.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/microorganisms10091818/s1, Table S1: Strains and plasmids used in this study; Table S2: Primers used in this study; Table S3: General features of the P. aeruginosa Pa150 genome; Table S4: Antibiotic resistance genes identified in P. aeruginosa Pa150; Table S5: Information of 620 rmtB-positive isolates obtained from the NCBI database.

Author Contributions

Conceptualization, J.G. and W.W.; methodology, J.G., X.W. and L.Y.; software, J.G.; validation, Y.J., F.B. and Z.C.; investigation, J.G.; data curation, W.W.; writing—original draft preparation, J.G. and W.W.; writing—review and editing, J.G. and W.W.; visualization, J.G.; supervision, W.W.; funding acquisition, W.W. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Key Research and Development Project of China (2021YFE0101700, 82061148018), the National Science Foundation of China (32170177, 32170199, 31970179, 31970680, and 31900115), and the Fundamental Research Funds for the Central Universities (63223030). The funders had no role in the study design, the data collection, the interpretation of results, or the decision to submit the work for publication.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. García-Ulloa, M., 2nd; Ponce-Soto, G.Y.; González-Valdez, A.; González-Pedrajo, B.; Díaz-Guerrero, M.; Souza, V.; Soberón-Chávez, G. Two Pseudomonas aeruginosa clonal groups belonging to the PA14 clade are indigenous to the Churince system in Cuatro Ciénegas Coahuila, México. Environ. Microbiol. 2019, 218, 2964–2976. [Google Scholar] [CrossRef] [PubMed]
  2. Stover, C.K.; Pham, X.Q.; Erwin, A.L.; Mizoguchi, S.D.; Warrener, P.; Hickey, M.J.; Brinkman, F.S.; Hufnagle, W.O.; Kowalik, D.J.; Lagrou, M.; et al. Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen. Nature 2000, 406, 959–964. [Google Scholar] [CrossRef] [PubMed]
  3. Azam, M.W.; Khan, A.U. Updates on the pathogenicity status of Pseudomonas aeruginosa. Drug Discov. Today 2019, 24, 350–359. [Google Scholar] [CrossRef] [PubMed]
  4. Kotra, L.P.; Haddad, J.; Mobashery, S. Aminoglycosides: Perspectives on mechanisms of action and resistance and strategies to counter resistance. Antimicrob. Agents Chemother. 2000, 44, 3249–3256. [Google Scholar] [CrossRef] [PubMed]
  5. Wargo, K.A.; Edwards, J.D. Aminoglycoside-induced nephrotoxicity. J. Pharm. Pract. 2014, 27, 573–577. [Google Scholar] [CrossRef] [PubMed]
  6. Ramirez, M.S.; Tolmasky, M.E. Amikacin: Uses, resistance, and prospects for inhibition. Molecules 2017, 22, 2267. [Google Scholar] [CrossRef]
  7. Mangiaterra, G.; Cedraro, N.; Citterio, B.; Simoni, S.; Vignaroli, C.; Biavasco, F. Diffusion and characterization of Pseudomonas aeruginosa aminoglycoside resistance in an Italian regional cystic fibrosis centre. Adv. Exp. Med. Biol. 2021, 1323, 71–80. [Google Scholar]
  8. Odumosu, B.T.; Adeniyi, B.A.; Chandra, R. Occurrence of aminoglycoside-modifying enzymes genes (aac(6′)-I and ant(2″)-I) in clinical isolates of Pseudomonas aeruginosa from Southwest Nigeria. Afr. Health Sci. 2015, 15, 1277–1281. [Google Scholar] [CrossRef]
  9. Tenover, F.C. Mechanisms of antimicrobial resistance in bacteria. Am. J. Med. 2006, 119, S3–S10. [Google Scholar] [CrossRef]
  10. Mulcahy, L.R.; Isabella, V.M.; Lewis, K. Pseudomonas aeruginosa biofilms in disease. Microb. Ecol. 2014, 68, 1–12. [Google Scholar] [CrossRef]
  11. Belaynehe, K.M.; Shin, S.W.; Hong-Tae, P.; Yoo, H.S. Occurrence of aminoglycoside-modifying enzymes among isolates of Escherichia coli exhibiting high levels of aminoglycoside resistance isolated from Korean cattle farms. FEMS Microbiol. Lett. 2017, 364. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  12. Thirumalmuthu, K.; Devarajan, B.; Prajna, L.; Mohankumar, V. Mechanisms of fluoroquinolone and aminoglycoside resistance in keratitis-associated Pseudomonas aeruginosa. Microb. Drug Resist. 2019, 25, 813–823. [Google Scholar] [CrossRef] [PubMed]
  13. Ramirez, M.S.; Tolmasky, M.E. Aminoglycoside modifying enzymes. Drug Resist. Updat. 2010, 13, 151–171. [Google Scholar] [CrossRef] [PubMed]
  14. Doi, Y.; Wachino, J.I.; Arakawa, Y. Aminoglycoside resistance: The emergence of acquired 16S ribosomal RNA methyltransferases. Infect. Dis. Clin. N. Am. 2016, 30, 523–537. [Google Scholar] [CrossRef]
  15. Yokoyama, K.; Doi, Y.; Yamane, K.; Kurokawa, H.; Shibata, N.; Shibayama, K.; Yagi, T.; Kato, H.; Arakawa, Y. Acquisition of 16S rRNA methylase gene in Pseudomonas aeruginosa. Lancet 2003, 362, 1888–1893. [Google Scholar] [CrossRef]
  16. Wang, J.; Wang, Z.Y.; Wang, Y.; Sun, F.; Li, W.; Wu, H.; Shen, P.C.; Pan, Z.M.; Jiao, X. Emergence of 16S rRNA methylase gene rmtB in Salmonella Enterica Serovar London and evolution of RmtB-producing plasmid mediated by IS26. Front. Microbiol. 2021, 11, 604278. [Google Scholar] [CrossRef]
  17. Smillie, C.; Garcillán-Barcia, M.P.; Francia, M.V.; Rocha, E.P.; de la Cruz, F. Mobility of plasmids. Microbiol. Mol. Biol. Rev. 2010, 74, 434–452. [Google Scholar] [CrossRef]
  18. Grohmann, E.; Muth, G.; Espinosa, M. Conjugative plasmid transfer in gram-positive bacteria. Microbiol. Mol. Biol. Rev. 2003, 67, 277–301. [Google Scholar] [CrossRef]
  19. Partridge, S.R.; Kwong, S.M.; Firth, N.; Jensen, S.O. Mobile genetic elements associated with antimicrobial resistance. Clin. Microbiol. Rev. 2018, 31, e00088-17. [Google Scholar] [CrossRef]
  20. Zechner, E.L.; Moncalián, G.; de la Cruz, F. Relaxases and plasmid transfer in gram-negative bacteria. Curr. Top. Microbiol. Immunol. 2017, 413, 93–113. [Google Scholar]
  21. Lerminiaux, N.A.; Cameron, A.D.S. Horizontal transfer of antibiotic resistance genes in clinical environments. Can. J. Microbiol. 2019, 65, 34–44. [Google Scholar] [CrossRef] [PubMed]
  22. McInnes, R.S.; McCallum, G.E.; Lamberte, L.E.; van Schaik, W. Horizontal transfer of antibiotic resistance genes in the human gut microbiome. Curr. Opin. Microbiol. 2020, 53, 35–43. [Google Scholar] [CrossRef] [PubMed]
  23. Li, B.; Qiu, Y.; Song, Y.; Lin, H.; Yin, H. Dissecting horizontal and vertical gene transfer of antibiotic resistance plasmid in bacterial community using microfluidics. Environ. Int. 2019, 131, 105007. [Google Scholar] [CrossRef] [PubMed]
  24. Andam, C.P.; Fournier, G.P.; Gogarten, J.P. Multilevel populations and the evolution of antibiotic resistance through horizontal gene transfer. FEMS Microbiol. Rev. 2011, 35, 756–767. [Google Scholar] [CrossRef] [PubMed]
  25. Tartari, D.C.; Zamparette, C.P.; Martini, G.; Christakis, S.; Costa, L.H.; Silveira, A.C.O.; Sincero, T.C.M. Genomic analysis of an extensively drug-resistant Pseudomonas aeruginosa ST312 harbouring IncU plasmid-mediated blaKPC-2 isolated from ascitic fluid. J. Glob. Antimicrob. Resist. 2021, 25, 151–153. [Google Scholar] [CrossRef] [PubMed]
  26. Jeannot, K.; Danassie, M.; Triponney, P.; Bour, M.; Gueudet, T.; Beyrouthy, R.; Bonnet, R.; Plésiat, P. A novel IncQ plasmid carrying gene blaCTX-M-3 in Pseudomonas aeruginosa. J. Antimicrob. Chemother. 2019, 74, 823–825. [Google Scholar] [CrossRef]
  27. Ho, B.T.; Basler, M.; Mekalanos, J.J. Type 6 secretion system-mediated immunity to type 4 secretion system-mediated gene transfer. Science 2013, 342, 250–253. [Google Scholar] [CrossRef]
  28. Shun-Mei, E.; Zeng, J.M.; Yuan, H.; Lu, Y.; Cai, R.X.; Chen, C. Sub-inhibitory concentrations of fluoroquinolones increase conjugation frequency. Microb. Pathog. 2018, 114, 57–62. [Google Scholar] [CrossRef]
  29. Miyoshi-Akiyama, T.; Tada, T.; Ohmagari, N.; Viet Hung, N.; Tharavichitkul, P.; Pokhrel, B.M.; Gniadkowski, M.; Shimojima, M.; Kirikae, T. Emergence and spread of epidemic multidrug-resistant Pseudomonas aeruginosa. Genome Biol. Evol. 2017, 9, 3238–3245. [Google Scholar] [CrossRef]
  30. Recio, R.; Sánchez-Diener, I.; Viedma, E.; Meléndez-Carmona, M.Á.; Villa, J.; Orellana, M.Á.; Mancheño, M.; Juan, C.; Zamorano, L.; Lora-Tamayo, J.; et al. Pathogenic characteristics of Pseudomonas aeruginosa bacteraemia isolates in a high-endemicity setting for ST175 and ST235 high-risk clones. Eur. J. Clin. Microbiol. Infect. Dis. 2020, 39, 671–678. [Google Scholar] [CrossRef]
  31. Pragasam, A.K.; Veeraraghavan, B.; Anandan, S.; Narasiman, V.; Sistla, S.; Kapil, A.; Mathur, P.; Ray, P.; Wattal, C.; Bhattacharya, S.; et al. Dominance of international high-risk clones in carbapenemase-producing Pseudomonas aeruginosa: Multicentric molecular epidemiology report from India. Indian J. Med. Microbiol. 2018, 36, 344–351. [Google Scholar] [CrossRef] [PubMed]
  32. Goessweiner-Mohr, N.; Arends, K.; Keller, W.; Grohmann, E. Conjugation in gram-positive bacteria. Microbiol. Spectr. 2014, 2, PLAS-0004-2013. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  33. Macé, K.; Vadakkepat, A.K.; Redzej, A.; Lukoyanova, N.; Oomen, C.; Braun, N.; Ukleja, M.; Lu, F.; Costa, T.R.D.; Orlova, E.V.; et al. Cryo-EM structure of a type IV secretion system. Nature 2022, 607, 191–196. [Google Scholar] [CrossRef]
  34. Lee, M.; Choi, T.J. Antimicrobial resistance caused by KPC-2 encoded by promiscuous plasmids of the Klebsiella pneumoniae ST307 strain. Ann. Lab. Med. 2021, 41, 86–94. [Google Scholar] [CrossRef]
  35. Lee, Y.S.; Kim, G.H.; Koh, Y.J.; Jung, J.S. Identification of strA-strB genes in streptomycin-resistant Pseudomonas syringae pv. actinidiae Biovar 2 strains isolated in Korea. Plant Pathol. J. 2021, 37, 489–493. [Google Scholar] [CrossRef] [PubMed]
  36. Han, H.S.; Koh, Y.J.; Hur, J.S.; Jung, J.S. Occurrence of the strA-strB streptomycin resistance genes in Pseudomonas species isolated from kiwifruit plants. J. Microbiol. 2004, 42, 365–368. [Google Scholar] [PubMed]
  37. Doi, Y.; Yokoyama, K.; Yamane, K.; Wachino, J.; Shibata, N.; Yagi, T.; Shibayama, K.; Kato, H.; Arakawa, Y. Plasmid-mediated 16S rRNA methylase in Serratia marcescens conferring high-level resistance to aminoglycosides. Antimicrob. Agents Chemother. 2004, 48, 491–496. [Google Scholar] [CrossRef]
  38. Rozwandowicz, M.; Brouwer, M.S.M.; Fischer, J.; Wagenaar, J.; Gonzalez-Zorn, B.; Guerra, B.; Mevius, D.J.; Hordijk, J. Plasmids carrying antimicrobial resistance genes in Enterobacteriaceae. J. Antimicrob. Chemother. 2018, 73, 1121–1137. [Google Scholar] [CrossRef]
  39. Al-Wrafy, F.; Brzozowska, E.; Górska, S.; Gamian, A. Pathogenic factors of Pseudomonas aeruginosa—The role of biofilm in pathogenicity and as a target for phage therapy. Postepy Hig. Med. Dosw. 2017, 71, 78–91. [Google Scholar] [CrossRef]
  40. Cłapa, T.; Michalski, J.; Syguda, A.; Narożna, D.; van Oostrum, P.; Reimhult, E. Morpholinium-based ionic liquids show antimicrobial activity against clinical isolates of Pseudomonas aeruginosa. Res. Microbiol. 2021, 172, 103817. [Google Scholar] [CrossRef]
  41. Alanis, A.J. Resistance to antibiotics: Are we in the post-antibiotic era? Arch. Med. Res. 2005, 36, 697–705. [Google Scholar] [CrossRef] [PubMed]
  42. Lv, L.; Wan, M.; Wang, C.; Gao, X.; Yang, Q.; Partridge, S.R.; Wang, Y.; Zong, Z.; Doi, Y.; Shen, J.; et al. Emergence of a plasmid-encoded resistance-nodulation-division efflux pump conferring resistance to multiple drugs, including Tigecycline, in Klebsiella pneumonia. MBio 2020, 11, e02930-19. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  43. Wang, C.Z.; Gao, X.; Lv, L.C.; Cai, Z.P.; Yang, J.; Liu, J.H. Novel tigecycline resistance gene cluster tnfxB3-tmexCD3-toprJ1b in Proteus spp. and Pseudomonas aeruginosa, co-existing with tet(X6) on an SXT/R391 integrative and conjugative element. J. Antimicrob. Chemother. 2021, 76, 3159–3167. [Google Scholar] [CrossRef] [PubMed]
  44. Cassu-Corsi, D.; Martins, W.M.; Nicoletti, A.G.; Almeida, L.G.; Vasconcelos, A.T.; Gales, A.C. Characterisation of plasmid-mediated rmtB-1 in Enterobacteriaceae clinical isolates from São Paulo, Brazil. Mem. Inst. Oswaldo Cruz 2018, 113, e180392. [Google Scholar] [CrossRef] [Green Version]
Microorganisms 10 01818 g001 550
Figure 1. Graphical circular schematic map of the clinical isolate Pa150 from circle 1 (outmost) to circle 7 (innermost). Circles 1 and 2, coding sequences (CDS), forward and reverse frames, respectively; circle 3, tRNAs (orange) and rRNAs (purple); circle 4, CRISPR-related genes (blue); circle 5, GC content; circle 6, GC-Skew; circle 7, coverage.
Figure 1. Graphical circular schematic map of the clinical isolate Pa150 from circle 1 (outmost) to circle 7 (innermost). Circles 1 and 2, coding sequences (CDS), forward and reverse frames, respectively; circle 3, tRNAs (orange) and rRNAs (purple); circle 4, CRISPR-related genes (blue); circle 5, GC content; circle 6, GC-Skew; circle 7, coverage.
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Microorganisms 10 01818 g002 550
Figure 2. Schematic map of the pTJPa150 plasmid. The innermost circle is the scale. The GC content is illustrated in the second circle. The third circle shows GC skew (+: green, −: purple). The outermost circle indicates the CDs, in which antibiotic resistance genes are highlighted in red and mobile genetic elements in blue.
Figure 2. Schematic map of the pTJPa150 plasmid. The innermost circle is the scale. The GC content is illustrated in the second circle. The third circle shows GC skew (+: green, −: purple). The outermost circle indicates the CDs, in which antibiotic resistance genes are highlighted in red and mobile genetic elements in blue.
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Microorganisms 10 01818 g003 550
Figure 3. Phylogenetic tree and genetic information of rmtB. (A) Phylogenetic tree of RmtB and RmtB-like proteins. Neighbor-joining tree based on the amino acid sequences of RmtB and RmtB-like proteins (obtained from the NCBI databases) generated using MEGA7; the bootstrap was 1000. The bacteria species are indicated by the colors as follows: P. aeruginosa, green; K. pneumonia, orange; E. coli, blue; others, black. (B) Comparison of the genetic environment of rmtB with those of closely related sequences. The mobile genetic elements are shown in blue.
Figure 3. Phylogenetic tree and genetic information of rmtB. (A) Phylogenetic tree of RmtB and RmtB-like proteins. Neighbor-joining tree based on the amino acid sequences of RmtB and RmtB-like proteins (obtained from the NCBI databases) generated using MEGA7; the bootstrap was 1000. The bacteria species are indicated by the colors as follows: P. aeruginosa, green; K. pneumonia, orange; E. coli, blue; others, black. (B) Comparison of the genetic environment of rmtB with those of closely related sequences. The mobile genetic elements are shown in blue.
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Microorganisms 10 01818 g004 550
Figure 4. Prevalence and distribution of rmtB-positive isolates. (A) Species classification of rmtB positive isolates. (B) Source statistics of rmtB-positive isolates (C) Geographical distribution of rmtB-positive isolates in China. The color depth correlates with the quantity. (D) Distribution of rmtB-positive isolates worldwide. (E) Isolation of rmtB-positive isolates from 2003 to 2021.
Figure 4. Prevalence and distribution of rmtB-positive isolates. (A) Species classification of rmtB positive isolates. (B) Source statistics of rmtB-positive isolates (C) Geographical distribution of rmtB-positive isolates in China. The color depth correlates with the quantity. (D) Distribution of rmtB-positive isolates worldwide. (E) Isolation of rmtB-positive isolates from 2003 to 2021.
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Table
Table 1. MICs (μg/mL) for indicated strains.
Table 1. MICs (μg/mL) for indicated strains.
StrainStrain InformationMEMAZTCTZAZT
+AVI		CTZ
+AVI		AZMCIPTBRGENAMKCOL
Pa150Isolate from Hebei646464641612832>128>128>1280.25
Pa150+PAβN 882162164>128>128>128
PAO1-lacZPAO1 expressing lacZ0.542241280.1250.250.51
PAO1-lacZ+PAβN 0.510.520.580.1250.250.51
PAO1-lacZ-pTJPa150PAO1 conjugates Pa150 plasmid1168168>1280.25>128>128>128
PAO1-lacZ pTJPa150+PAβN 0.50.50.521160.0625>128>128>128
DH5α-pPUCP20DH5α transformants with the empty vector 0.50.250.125
DH5α-pPUCP20-rmtBDH5α transformants expressing rmtB >128>128>128
PAO1-pPUCP20PAO1 transformants with the empty vector 10.50.25
PAO1-pPUCP20-rmtBPAO1 transformants expressing rmtB >128>128>128
MEM, meropenem; AZT, aztreonam; CTZ, ceftazidime; AVI, avibactam; AZM, azithromycin; CIP, ciprofloxacin; TBR, tobramycin; GEN, gentamicin; AMK, amikacin; COL, colistin.
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Gao, J.; Wei, X.; Yin, L.; Jin, Y.; Bai, F.; Cheng, Z.; Wu, W. Emergence and Transfer of Plasmid-Harbored rmtB in a Clinical Multidrug-Resistant Pseudomonas aeruginosa Strain. Microorganisms 2022, 10, 1818. https://doi.org/10.3390/microorganisms10091818

AMA Style

Gao J, Wei X, Yin L, Jin Y, Bai F, Cheng Z, Wu W. Emergence and Transfer of Plasmid-Harbored rmtB in a Clinical Multidrug-Resistant Pseudomonas aeruginosa Strain. Microorganisms. 2022; 10(9):1818. https://doi.org/10.3390/microorganisms10091818

Chicago/Turabian Style

Gao, Jiacong, Xiaoya Wei, Liwen Yin, Yongxin Jin, Fang Bai, Zhihui Cheng, and Weihui Wu. 2022. "Emergence and Transfer of Plasmid-Harbored rmtB in a Clinical Multidrug-Resistant Pseudomonas aeruginosa Strain" Microorganisms 10, no. 9: 1818. https://doi.org/10.3390/microorganisms10091818

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