Characterisation of plasmid-mediated rmtB-1 in Enterobacteriaceae clinical isolates from São Paulo, Brazil

OBJECTIVES The emergence of 16S rRNA methyltranferases (16 RMTAses) has jeopardised the clinical use of aminoglycosides. RmtB is one of the most frequently reported in Gram-negatives worldwide. In this study, we aimed to estimate the frequency of 16S RMTAses encoding genes in Enterobacteriaceae isolated in a three-month period from a tertiary Brazilian hospital. METHODS All Gram-negatives classified as resistant to amikacin, gentamicin, and tobramycin by agar screening were selected for analysis. The presence of 16SRMTases encoding genes was verified by polymerase chain reaction (PCR). Antimicrobial susceptible profile was determined by broth microdilution. The genetic relationship among these isolates was accessed by pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Selected RmtB-producing isolates were characterised by whole genome sequencing (WGS) analysis. RESULTS Twenty-two of 1,052 (2.1%) Enterobacteriaceae were detected as producers of RmtB-1 [Klebsiella pneumoniae (n = 21) and Proteus mirabilis (n = 1)]. blaKPC-2 was identified among 20 RmtB-1-producing K. pneumoniae isolates that exhibited an identical PFGE and MLST (ST258) patterns. Two K. pneumoniae isolates, the A64216 (not harboring bla KPC-2), A64477 (harboring bla KPC-2) and one P. mirabilis isolate (A64421) were selected for WGS. rmtB-1 and bla KPC-2 genes were carried by distinct plasmids. While a plasmid belonging to the IncFIIk group harbored rmtB-1 in K. pneumoniae, this gene was carried by a non-typable plasmid in P. mirabilis. In the three analysed plasmids, rmtB-1 was inserted on a transposon, downstream a Tn2. CONCLUSION Our findings suggested that the rmtB-1 was harbored by plasmids distinct from those previously reported in Bolivia and China. It suggests that multiple mobilization events might have occurred in South America.

Enterobacteriaceae are the most frequent pathogens associated with both community-and hospital-acquired infections. In the last years, the emergence of carbapenemase production in Enterobacteriaceae has jeopardised the clinical use of carbapenems. (1) In this way, polymyxins and aminoglycosides have become therapeutic options for treatment of serious infections caused by carbapenem resistant Enterobacteriaceae (CRE). With the emergence of polymyxin resistance especially among KPC-2-producing isolates, the aminoglycosides have reached even a more important role in the treatment of CRE infections. (2) Resistance to aminoglycosides is often due to the production of aminoglycoside modifying enzymes (AMEs), which usually confer resistance to specific aminoglycoside molecules but not all aminoglycosides. (3) In contrast, the production of 16S RMTases will confer high-level of resistance to 4,6 -dissubstituted 2-deoxystreptamines aminoglycosides including plazomicin, a new antimicrobial not approved for clinical use yet. (3,4) To date, ten 16S RMTAses (ArmA, RmtA to RmtH, and NpmA) have been detected in Gram-negative isolates. (4) ArmA is the most frequently reported 16S RMTAses worldwide followed by RmtB. (4,5) The mobilisation and transfer of 16S RMTAses encoding genes by mobile genetic elements have contributed for their rapid global dissemination. (4,5) In Brazil, the production of 16S RMTases has been mainly encountered in SPM-1 producing Pseudomonas aeruginosa (RmtD). rmtD-2 has been also detected in Klebsiella pneumoniae. (4) In addition, K. pneumoniae (bla KPC-2 and rmtG; bla NDM-1 and rmtC) and Enterobacter cloacae (bla NDM-1 and armA) co-harboring carbapenemase and 16S RMTases encoding genes were also reported. (4,5,6) . The presence of rmtB-1 in Escherichia coli and Proteus mirabilis was also reported in the years 2005 and 2006, respectively. (7) F33:A-:B-plasmids carrying bla CTX-M-65 , fosA3 and rmtB are widespread in Escherichia coli isolates of animal origin from China. pHN7A8 is a representative of this plasmid group and was isolated from a Chinese dog. Curiously, the plasmids p397Kp and p477Kp, which were isolated from multi-drug resistant K. pneumoniae ST726 collected from Bolivian patients, showed to be highly related to pHN7A8 suggesting an intercontinental spread of this mobile genetic elements. (8) In this study, we aimed to estimate the frequency of 16S RMTAses encoding genes in Enterobacteriaceae isolated in a three-month period from a tertiary Brazilian hospital. In addition, the plasmids harboring rmtB-1 of selected isolates were fully sequenced.

MATERIALS AND METHODS
Bacterial strains -All Enterobacteriaceae isolated by the microbiology laboratory of a tertiary teaching hospital located in the city of São Paulo, Southeast of Brazil, were selected for this study between October and December of 2014. In order to select the resistant isolates to aminoglycosides, agar screening test was performed by testing agar plates supplemented with gentamicin (4 mg/L), tobramycin (4 mg/L), and amikacin (16 mg/L). The isolates classified as resistant to all three aminoglycosides were selected for further testing. The isolates were identified by MALDI-TOF MS (Bruker Daltonics, Germany) using BioTyper 3.1. (9) Investigation of 16S RMTAses β-lactamases encoding genes and sequencing -Detection of 16S RMTAses encoding genes was performed by two distinct multiplex polymerase chain reaction (PCR) for isolates screened as not susceptible to aminoglycosides by agar screening. PCR multiplex 1: npmA, armA, rmtB, rmtC and rmtD. PCR multiplex 2: rmtE, rmtF, rmtG e rmtH. A single PCR was tested for rmtA detection [Supplementary data (Table II)]. Investigation of bla TEM , bla SHV , bla CTX-M , bla G-ES , bla KPC was performed as previously reported (10,11). Amplicons were purified and sequenced using the Applied Biosystems 3500 genetic analyser (Applied Biosystems, PerkinElmer, USA). The obtained sequences were compared with those available at GenBank (http://www. ncbi.nlm.nih.gov/Blast.cgi).
Antimicrobial susceptibility testing -The minimal inhibitory concentrations (MICs) for kanamycin, amikacin, gentamicin, tobramycin, aztreonam, cefepime, ceftazidime, ceftriaxone, ertapenem, imipenem, meropenem, piperacillin/tazobactam, ciprofloxacin, polymyxin B, and tigecycline were determined by broth microdilution for the isolates producers of 16S RMTases. The results were interpreted according to EUCAST clinical breakpoints, except for kanamycin results, which were interpreted according to CLSI breakpoints. (12,13) Molecular typing -All K. pneumoniae isolates carrying rmtB-1 were typed by pulsed field gel electrophoresis (PFGE) using Spe-I as restriction enzyme, (14) and multilocus sequence typing (MLST). (15) Plasmid profile and transference of 16S rRNA methyltransferases and bla KPC -Total plasmid DNA extraction of the K. pneumoniae and P. mirabilis isolates harboring rmtB-1 was performed using the Kieser technique (16) and the Qiaprep spin miniprep (Qiagen, Hilden Germany). Four isolates (A64192, A64216, A64477 and A64421) carrying rmtB-1 and distinct beta-lactamases encoding genes were further selected for both conjugation and transformation. Conjugation experiments were performed using the clinical isolates as donor and E. coli J53 as the receptor strains. Cells were grown on Mac-Conkey agar plates supplemented with azide or nalidixic acid (150 mg/L) plus amikacin (8 mg/L) or ampicillin (50 mg/L) for selection of transconjugant cells carrying rmtB-1 or β-lactamase encoding genes, respectively. Additionally, the DNA plasmids obtained of the extraction by QIAprep spin miniprep were transferred by electroporation and transformation into E. coli DH5α. The transformant cells were selected according to the colony growth on Luria Bertani agar (LB agar) plates supplemented with amikacin (8 mg/L), imipenem (1 mg/L) or ampicillin (50 mg/L) for selection of colonies carrying 16S RMTases or β-lactamase encoding genes.
Southern Blot Hybridisation -Total DNA and plasmidial DNA of clinical isolates were used to perform Southern Blot Hybridisation using dioxigenin (DIG) DNA labeling and detection kit (Roche Diagnostics GmbH, Germany).
Whole genome sequencing, assembly, annotation, and analysis -Two RmtB-1-producing K. pneumoniae isolates, the A64216 (not harboring bla KPC-2 ), A64477 (harboring bla KPC-2 ) and one RmtB-1-producing P. mirabilis isolate (A64421) were selected for whole genome sequencing. To obtain a better coverage of the plasmidial against chromosomal DNA, the extraction was performed by commercial kit PerfectPrep Spin Mini Kit (5 prime, Gaithersburg, Maryland), and shipped to the National Laboratory for Scientific Computing (LNCC -Petrópolis, RJ, Brazil), where the experiments were carried out. The library was constructed by Illumina TruSeq DNA PCR-free with a fragment ~550pb and sequenced by Illumina MiniSeq 2x300 pb paired-end. The sequences were assembled using Newbler version 3.0 and Ray version 2.1. The contigs were aligned with the blast against the NT database for separation of plasmid DNA from chromosomal DNA. The coverage of the chromosomes ranged from 14x to 40x, while plasmid coverage ranged from 700x to 20,000x. The system for automated bacterial (genome) integrated annotation (SABIA) pipeline (17) was used for gene prediction and automatic annotation followed by manual validation of each predicted CDS by Uniprot (http://www.uniprot.org/), BLAST (http://blast. ncbi.nlm.nih.gov). Insertion sequences were searched by using ISfinder (https://www-is.biotoul.fr) after automatic annotation fallowed by manual validation. The comparison of the similarity genetic and Inc group analysis were performed by Multiple Genome Alignment (MAUVE) and PlasmidFinder (hhtps://cge,cbs.dtu.dk// services/PlasmidFinder/), respectively. Additionally, the investigation of the resistance genes harbored by other plasmids was evaluated by ResFinder program (https:// cge.cbs.dtu.dk/services/ResFinder/). The size confirmation of rmtB-1 were confirmed by S1-PFGE, (18) followed by radioactive hybridisation.
Susceptibility to carbapenems was observed only in K. pneumoniae (A64216) and P. mirabilis (A64421) isolates, which did not harbor bla KPC-2 . All K. pneumoniae isolates were resistant to polymyxin B, except for the A64022 and A64962 isolates. In contrast, tigecycline was the antimicrobial that exhibited the highest in vitro potency (MICs, 0.03-0.125 mg/L), and susceptibility rate with only two isolates being categorized as resistant (MICs, 2 mg/L) to this compound as shown in Table. All K. pneumoniae showed an identical PFGE pattern and belonged to the ST258. The plasmid profile obtained by Kieser methodology followed by Southern Blot/Hybridisation assay showed that rmtB-1 was located in a plasmid of similar size in all K. pneumoniae isolates. Transfer of rmtB-1 by conjugation was successfully performed only for the P. mirabilis strain (A64421). Transformation experiments testing K. pneumoniae as a donor of rmtB-1 were unsuccessful despite many attempts.
In-silico analysis showed that both pKP64477a and pKP64216a belonged to IncFIIk group of incompatibility group, while pPM64421 was a non-typable plasmid according to PlasmidFinder analysis. (19) pKP64477a and pKP26216a showed a distinct backbone, when compared with pPM464421a, except for the region in which rmtB-1 was inserted. In this region, an insertion of ISCfr1 upstream rmtB-1 region in pK-P64416a and pKP64477a was observed. While, a tnpA gene was detected in the pPM64421a. Various aminoglycoside-modifying enzymes encoding genes were detected in these plasmids, such as aadA2, aph(3'), aac(3')-IId, strA and strB. In addition, other resistance genes such as sul1, sul2, drfA12, tetG, erm(42), and catA, which confer resistance to sulphonamides, trimethroprim, tetracycline, macrolides, and phenicol, respectively, were also detected [Supplementary data (Tables I, III)]. Only two virulence encoding genes were observed on pPM64421a that carried a Fe 3+ siderophore ABC transporter permease and a Von Willenbrand factor A.
The incompatibility group of the pKP64477d, which harbors bla KPC-2 could not be fully typed. In-silico analysis suggested that this plasmid could belonged to InX3 (99.7%) or IncU (99.5%) group. pKP64477d carried only another resistance gene, sat2, which conferred resistance to streptomycin (Fig. 1). The bla KPC-2 was inserted into a transposon, flanked by an ISKpn26 and a Tn3 resolvase.

DISCUSSION
Aminoglycosides have been an important therapeutic option for treatment of serious Gram-negative multidrug resistant infections. (4) However, the emergence and spread of 16S RMTAses capable of conferring high level resistance to aminoglycosides has jeopardised the clinical use of this important class of antimicrobials. In Brazil, SPM-1-producing P. aeruginosa ST277 clone harboring rmtD-1 has been frequently detected justifying the high level of aminoglycoside resistance displayed by this MDR clone. (20) 16S RMTAse encoding genes were sporadically detected in Latin America before the year 2007, and basically restricted to the transfer of rmtD to non-P. aeruginosa species. (4) However, contemporary studies have reported the emergence of RmtG and RmtB in K. pneumoniae isolated from Brazil and Bolivia, respectively. (4,20) ArmA and RmtC were also recently identified in NDM-1-producing Enterobacter cloacae, (5) and -K. pneumoniae isolates. (6) Despite the low prevalence of 16S RMTases detected in our hospital, we observed that the increase in frequency of the 16S RMTases could be mainly attributed to the intra-hospital spread of a single RmtB-1-producing K. pneumoniae clone.
RmtB-1-producing Gram-negative isolates usually harbor distinct enzymatic mechanisms of resistance, such as β-lactamases, AMES and PMRQ. (4,5) The isolates characterised in this study also accumulated distinct mechanisms of resistance, like production of β-lactamases (KPC-2, CTX-M-14, CTX-M-15, SHV-11 and TEM-1) and diverse AMEs (AADA2, APH(3')-Ia, AAC(3)-IId, StrA, StrB). All RmtB-1-producing K. pneumoniae isolates belonged to ST258 (clonal complex CC258), which has been often detected in KPC-2-producing K. pneumoniae isolated in Brazil. (21) To date, pKP644216a and pKP64477a were distinct from other plasmids harboring rmtB-1. Both plasmids showed 99% of similarity with a partial nucleo-  . (22) This ~70 Kb region did not harbor rmtB-1, suggesting that recombination events had occurred allowing the acquisition of rmtB-1 by both Brazilian plasmids. Additionally, pKP64216a and pKP64477a belonged to IncFIIk, an uncommon group associated with rmtB-1 dissemination. (23) In contrast, the nucleotide sequences of pPM64421a showed 99% identity with a partial nucleotide sequence (~90 Kb) of a Vibrio cholareae plasmid pVC1307 (accession number KJ817377), which did not harbor rmtB-1 either, suggesting that rmtB-1 was mobilised and integrated to this genetic backbone. Although, the plasmids carrying rmtB-1 have complete conjugation machinery in their genetic backbone, (20) unsuccessful transfer of rmtB has been reported. finO was identified in the plasmids pKP644477a and pKP64216a, which is an inhibitor of traJ, and could have circumvented the genetic material transfer from K. pneumoniae to the recipient strain. (24) The genetic contexts of pKP644477a and pKP64216a were similar to previous description, (25) which showed bla TEM-1 upstream rmtB. Our results showed that ∆Tn2 [ISCfr1-∆tnpR-bla TEM-1b ] was inserted upstream rmtB-1 in pKP64216a and pKP64477, while a Tn2 [tnpA-tnpRbla TEM-1b ] was present upstream rmtB-1 in pPM64421. The region surround ∆Tn2 in pKP644477a and pK-P64216a showed high similarity with other transposons carried by pU302L (Accession number: AY333434) and pCTX-M-3 plasmids [(Accession number: AF550415)].   (26,27) In contrast to what was previously reported by Sennati and colleagues, (8) who suggested an intercontinental dissemination of plasmids harboring rmtB, the plasmids harboring rmtB-1 in Brazil were distinct from those observed in Bolivia and China [(p397Kp and p477Kp); (pHN7A8)], (8,28) respectively. The rmtB-1 genetic context was also different, since both p397Kp (Accession number: LN897474) and p477Kp (Accession number: LN897475) had an insertion sequence IS1294 upstream ΔtnpR, while both pKP644477a and pKP64216a showed an ISCfr1 upstream ΔtnpR. (8) It is curious to note that the gene encoding the Von-Willebrand factor A, a virulence factor only observed in S. aureus to date, was detected in these large plasmids. VonWillebrand factor A is responsible for protecting the bacteria from the neutrophilic attack. (29) Toxin-antitoxin system was observed in the pKP64477a and pKP64216a, the RelE-XrE-like. A few studies have evaluated the role of RelE-XrE-like system, which has been associated with cellular stress response being expressed, for example, when nutrients are limited. (30) In addition to bla KPC-2 , pKP64477d was similar to pKP13d, a previously sequenced plasmid harboring bla KPC-2, collected from another Brazilian state (accession number NZ_CP003997). The single difference observed between these two plasmids was the acquisition of 917 bp, which comprised basically the insertion of IS26. The acquisition of rmtB-1 by a KPC-2-producing K. pneumoniae ST258 clone could justify the success of rmtB-1 spread. This illustrates how dynamic is the evolution of antimicrobial resistance warranting the need for continuous surveillance.