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

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.


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], Microorganisms 2022, 10 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.

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

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 10 6 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.

Whole Genome Sequencing (WGS) and Bioinformatics Analysis
Total DNA from Pa150 was extracted using the TIANamp Bacteria DNA Kit (TIAN-GEN, 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.

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.

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).

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

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

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 bla TEM and parB genes. Compared with the recipient strain, the MICs of β-lactam, macrolide, and quinolone antibiotics for the transconjugant were twoto 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.

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.

Possible Origin and Mobilization of rmtB
On the pTJPa150 plasmid, rmtB is in close proximity to bla TEM , 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 rmtBcarrying 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 bla TEM and Tn3. Additionally, IS91, IS30, and IS26 transposons and the rmtB-bla TEM -Tn3 cluster form an IS91-groEL-nahP-rmtB-bla TEM -Tn3-IS30-IS26 module ( Figure 3B). Sequence alignment using the rmtB-bla TEM -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-bla TEM -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-bla TEM -Tn3 cluster in all of the 11 strains. Of the 11 strains, the MGEs on both sides of the rmtB-bla TEM -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-bla TEM -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. Microorganisms 2022, 10, x FOR PEER REVIEW 7 of 11

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.

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). 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).

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 bla TEM 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.