scholarly journals Plasmid-Mediated 16S rRNA Methylase in Serratia marcescens Conferring High-Level Resistance to Aminoglycosides

2004 ◽  
Vol 48 (2) ◽  
pp. 491-496 ◽  
Author(s):  
Yohei Doi ◽  
Keiko Yokoyama ◽  
Kunikazu Yamane ◽  
Jun-ichi Wachino ◽  
Naohiro Shibata ◽  
...  
Keyword(s):  
16S Rrna ◽  
Serratia Marcescens ◽  
Resistance Gene ◽  
Pathogenic Bacteria ◽  
Treatment Options ◽  
Resistance Mechanism ◽  
Aminoglycoside Resistance ◽  
Reading Frame ◽  
High Level ◽  
16S Rrna Methylase

ABSTRACT Serratia marcescens S-95, which displayed an unusually high degree of resistance to aminoglycosides, including kanamycins and gentamicins, was isolated in 2002 from a patient in Japan. The resistance was mediated by a large plasmid which was nonconjugative but transferable to an Escherichia coli recipient by transformation. The gene responsible for the aminoglycoside resistance was cloned and sequenced. The deduced amino acid sequence of the resistance gene shared 82% identity with RmtA, which was recently identified as 16S rRNA methylase conferring high-level aminoglycoside resistance in Pseudomonas aeruginosa. Histidine-tagged recombinant protein showed methylation activity against E. coli 16S rRNA. The novel aminoglycoside resistance gene was therefore designated rmtB. The genetic environment of rmtB was further investigated. The sequence immediately upstream of rmtB contained the right end of transposon Tn3, including bla TEM, while an open reading frame possibly encoding a transposase was identified downstream of the gene. This is the first report describing 16S rRNA methylase production in S. marcescens. The aminoglycoside resistance mechanism mediated by production of 16S rRNA methylase and subsequent ribosomal protection used to be confined to aminoglycoside-producing actinomycetes. However, it is now identified among pathogenic bacteria, including Enterobacteriaceae and P. aeruginosa in Japan. This is a cause for concern since other treatment options are often limited in patients requiring highly potent aminoglycosides such as amikacin and tobramycin.

scholarly journals Fitness Cost and Interference of Arm/Rmt Aminoglycoside Resistance with the RsmF Housekeeping Methyltransferases

2012 ◽  
Vol 56 (5) ◽  
pp. 2335-2341 ◽  
Author(s):  
Belen Gutierrez ◽  
Jose A. Escudero ◽  
Alvaro San Millan ◽  
Laura Hidalgo ◽  
Laura Carrilero ◽  
...  
Keyword(s):  
16S Rrna ◽  
Pathogenic Bacteria ◽  
Binding Pocket ◽  
Maldi Mass Spectrometry ◽  
Fitness Cost ◽  
Resistance Pattern ◽  
Aminoglycoside Resistance ◽  
Content Type ◽  
E Coli ◽  
High Level

ABSTRACTArm/Rmt methyltransferases have emerged recently in pathogenic bacteria as enzymes that confer high-level resistance to 4,6-disubstituted aminoglycosides through methylation of the G1405 residue in the 16S rRNA (like ArmA and RmtA to -E). In prokaryotes, nucleotide methylations are the most common type of rRNA modification, and they are introduced posttranscriptionally by a variety of site-specific housekeeping enzymes to optimize ribosomal function. Here we show that while the aminoglycoside resistance methyltransferase RmtC methylates G1405, it impedes methylation of the housekeeping methyltransferase RsmF at position C1407, a nucleotide that, like G1405, forms part of the aminoglycoside binding pocket of the 16S rRNA. To understand the origin and consequences of this phenomenon, we constructed a series of in-frame knockout and knock-in mutants ofEscherichia coli, corresponding to the genotypesrsmF+, ΔrsmF,rsmF+rmtC+, and ΔrsmF rmtC+. When analyzed for the antimicrobial resistance pattern, the ΔrsmFbacteria had a decreased susceptibility to aminoglycosides, including 4,6- and 4,5-deoxystreptamine aminoglycosides, showing that the housekeeping methylation at C1407 is involved in intrinsic aminoglycoside susceptibility inE. coli. Competition experiments between the isogenicE. colistrains showed that, contrary to expectation, acquisition ofrmtCdoes not entail a fitness cost for the bacterium. Finally, matrix-assisted laser desorption ionization (MALDI) mass spectrometry allowed us to determine that RmtC methylates the G1405 residue not only in presence but also in the absence of aminoglycoside antibiotics. Thus, the coupling between housekeeping and acquired methyltransferases subverts the methylation architecture of the 16S rRNA but elicits Arm/Rmt methyltransferases to be selected and retained, posing an important threat to the usefulness of aminoglycosides worldwide.

scholarly journals 30S Subunit-Dependent Activation of the Sorangium cellulosum So ce56 Aminoglycoside Resistance-Conferring 16S rRNA Methyltransferase Kmr

2015 ◽  
Vol 59 (5) ◽  
pp. 2807-2816 ◽  
Author(s):  
Miloje Savic ◽  
S. Sunita ◽  
Natalia Zelinskaya ◽  
Pooja M. Desai ◽  
Rachel Macmaster ◽  
...  
Keyword(s):  
16S Rrna ◽  
Pathogenic Bacteria ◽  
Resistance Mechanism ◽  
Binding Pocket ◽  
Product Inhibition ◽  
Sorangium Cellulosum ◽  
Aminoglycoside Resistance ◽  
Content Type ◽  
30S Subunit ◽  
Self Protection

ABSTRACTMethylation of bacterial 16S rRNA within the ribosomal decoding center confers exceptionally high resistance to aminoglycoside antibiotics. This resistance mechanism is exploited by aminoglycoside producers for self-protection while functionally equivalent methyltransferases have been acquired by human and animal pathogenic bacteria. Here, we report structural and functional analyses of theSorangium cellulosumSo ce56 aminoglycoside resistance-conferring methyltransferase Kmr. Our results demonstrate that Kmr is a 16S rRNA methyltransferase acting at residue A1408 to confer a canonical aminoglycoside resistance spectrum inEscherichia coli. Kmr possesses a class I methyltransferase core fold but with dramatic differences in the regions which augment this structure to confer substrate specificity in functionally related enzymes. Most strikingly, the region linking core β-strands 6 and 7, which forms part of theS-adenosyl-l-methionine (SAM) binding pocket and contributes to base flipping by the m1A1408 methyltransferase NpmA, is disordered in Kmr, correlating with an exceptionally weak affinity for SAM. Kmr is unexpectedly insensitive to substitutions of residues critical for activity of other 16S rRNA (A1408) methyltransferases and also to the effects of by-product inhibition byS-adenosylhomocysteine (SAH). Collectively, our results indicate that adoption of a catalytically competent Kmr conformation and binding of the obligatory cosubstrate SAM must be induced by interaction with the 30S subunit substrate.

scholarly journals Coproduction of Novel 16S rRNA Methylase RmtD and Metallo-β-Lactamase SPM-1 in a Panresistant Pseudomonas aeruginosa Isolate from Brazil

2006 ◽  
Vol 51 (3) ◽  
pp. 852-856 ◽  
Author(s):  
Yohei Doi ◽  
Doroti de Oliveira Garcia ◽  
Jennifer Adams ◽  
David L. Paterson
Keyword(s):  
Pseudomonas Aeruginosa ◽  
16S Rrna ◽  
Moderate Degree ◽  
Aminoglycoside Resistance ◽  
Disk Diffusion Test ◽  
Serious Infections ◽  
High Level ◽  
16S Rrna Methylase ◽  
Cloning Experiment ◽  
Level Resistance

ABSTRACT Serious infections with Pseudomonas aeruginosa are frequently treated with the combination of a β-lactam antimicrobial and an aminoglycoside. P. aeruginosa strain PA0905 was isolated in 2005 from an inpatient in Brazil. It showed a panresistant phenotype that included resistance to β-lactams, aminoglycosides, and fluoroquinolones. The β-lactam resistance was conferred by the production of the metallo-β-lactamase SPM-1. No inhibitory zone was observed when a disk diffusion test was performed with the semisynthetic aminoglycoside arbekacin, raising suspicion of 16S rRNA methylase production. A cloning experiment subsequently revealed the presence of a novel 16S rRNA methylase, RmtD, which accounted for the high-level resistance to all 4,6-disubstituted deoxystreptamine aminoglycosides, such as amikacin, tobramycin, and gentamicin. RmtD shared a moderate degree of identity with RmtA, another 16S rRNA methylase that was initially reported to occur in P. aeruginosa in Japan in 2003. This is the first identification of aminoglycoside resistance mediated by a 16S rRNA methylase in South America. This is also the first report to document coproduction of a metallo-β-lactamase and a 16S rRNA methylase, a combination that would severely compromise therapeutic options for the infected patients.

scholarly journals Discovery of a novel integron-borne aminoglycoside resistance gene present in clinical pathogens by screening environmental bacterial communities

Microbiome ◽  
2020 ◽  
Vol 8 (1) ◽  
Author(s):  
Maria-Elisabeth Böhm ◽  
Mohammad Razavi ◽  
Nachiket P. Marathe ◽  
Carl-Fredrik Flach ◽  
D. G. Joakim Larsson
Keyword(s):  
Resistance Gene ◽  
Resistance Genes ◽  
Bacterial Infections ◽  
Treatment Options ◽  
Gene Cassette ◽  
Resistance Mechanisms ◽  
Clinical Isolates ◽  
Aminoglycoside Resistance ◽  
The Usa ◽  
High Level

Abstract Background New antibiotic resistance determinants are generally discovered too late, long after they have irreversibly emerged in pathogens and spread widely. Early discovery of resistance genes, before or soon after their transfer to pathogens could allow more effective measures to monitor and reduce spread, and facilitate genetics-based diagnostics. Results We modified a functional metagenomics approach followed by in silico filtering of known resistance genes to discover novel, mobilised resistance genes in class 1 integrons in wastewater-impacted environments. We identified an integron-borne gene cassette encoding a protein that conveys high-level resistance against aminoglycosides with a garosamine moiety when expressed in E. coli. The gene is named gar (garosamine-specific aminoglycoside resistance) after its specificity. It contains none of the functional domains of known aminoglycoside modifying enzymes, but bears characteristics of a kinase. By searching public databases, we found that the gene occurs in three sequenced, multi-resistant clinical isolates (two Pseudomonas aeruginosa and one Luteimonas sp.) from Italy and China, respectively, as well as in two food-borne Salmonella enterica isolates from the USA. In all cases, gar has escaped discovery until now. Conclusion To the best of our knowledge, this is the first time a novel resistance gene, present in clinical isolates, has been discovered by exploring the environmental microbiome. The gar gene has spread horizontally to different species on at least three continents, further limiting treatment options for bacterial infections. Its specificity to garosamine-containing aminoglycosides may reduce the usefulness of the newest semisynthetic aminoglycoside plazomicin, which is designed to avoid common aminoglycoside resistance mechanisms. Since the gene appears to be not yet common in the clinics, the data presented here enables early surveillance and maybe even mitigation of its spread.

scholarly journals High Prevalence of 16S rRNA methylase genes High Prevalence of 16S rRNA methylase genes Among Carbapenem-resistant Hypervirulent Klebsiella pneumoniae Isolates in China

2019 ◽  
Author(s):  
Wenjian Liao ◽  
Dan Li ◽  
Dan Dan Wei ◽  
Fang-lin Du ◽  
Dan Long ◽  
...  
Keyword(s):  
16S Rrna ◽  
Klebsiella Pneumoniae ◽  
Clinical Isolates ◽  
Aminoglycoside Resistance ◽  
Pfge Typing ◽  
Carbapenem Resistant ◽  
Carbapenemase Gene ◽  
High Prevalence ◽  
High Level ◽  
16S Rrna Methylase

Abstract Background : the existence of 16S rRNA methylase genes would increase treatment difficulty of patients infected with CR-hvKP strains, this study was aimed to testify the prevalence of the 16S rRNA methylase genes genes in the CR-hvKP strains in China.Methods : Thirty-nine carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) isolates collected from a Chinese hospital during the whole year of 2018 were evaluated to characterize the prevalence of 16S rRNA methylase genes. Results : In tatal 66.7% (26/39) of the CR-hvKP isolates were found to carry 16S rRNA methylase genes, and the most frequently detected gene was armA (11,42.3%), followed by rmtB (8,30.8%),and 7 CR-hvKP strains were found to carry both armA and rmtB (26.9%). All the clinical isolates were found to carry at least one carbapenemase gene,with KPC-2 (79.5%,31/39), NDM-1 (10.3%,4/39), and cocarrying KPC-2 and NDM-1 (10.3%,4/39). A total of 89.7% (35/39) isolates carried ESBL genes, including 61.5% (24/39) blaSHV-1 ,71.8% (28/39) blaTEM-1 and 89.7% (35/39) blaCTX-M-1 4. All except four isolates (89.7%,35/39) harbored PMQR genes,with qnrS (82.1%,32/39), aac(6’)-Ib-cr (79.5%,31/39), qnrB (2.6%,1/39).All the 16S rRNA methylase genes-positive CR-hvKP strains were firstly found to cocarry carbapenemase genes, ESBL genes and PMQR genes simultaneously. The most prevalent virulence genes were rmpA2 and entB (100%, 39/39),followed by silS (97.4%, 38/39), ybtS (94.9%, 37/39), iutA (92.3%, 36/39), kpn (92.3%, 36/39), rmpA (87.2%, 34/39), terW (84.6%, 33/39), aerobactin (23.1%, 9/39), repA (17.9%, 7/39), magA (10.3%, 4/39), kfuB C (10.3%, 4/39), w ca G (10.3%, 4/39), allS (10.3%, 4/39). Multilocus sequence typing (MLST) analysis assigned the 39 CR-hvKP isolates into 4 sequence types (STs), with ST11 encompassing 79.5% of the strains. Pulsed field gel electrophoresis (PFGE) typing showed that strains closely related by MLST clustered in major PFGE clusters, of which cluster A accounts for 31 ST11 isolates.The analysis of the transconjugants showed a high-level aminoglycoside resistance and a popular cotransfer of bla KPC-2 with the 16S rRNA methylase genes.Conclusions : 16S rRNA methylase genes are highly prevalent in CR-hvKP clinical isolates especially for ST11, it is therefore critical to continuously monitor the 16S rRNA methylase-producing CR-hvKP epidemiology and minimize potential risks from aminoglycoside -resistant CR-hvKP.

scholarly journals Functionally critical residues in the aminoglycoside resistance-associated methyltransferase RmtC play distinct roles in 30S substrate recognition

2019 ◽  
Vol 294 (46) ◽  
pp. 17642-17653 ◽  
Author(s):  
Meisam Nosrati ◽  
Debayan Dey ◽  
Atousa Mehrani ◽  
Sarah E. Strassler ◽  
Natalia Zelinskaya ◽  
...  
Keyword(s):  
16S Rrna ◽  
Binding Affinity ◽  
Pathogenic Bacteria ◽  
Substrate Recognition ◽  
Functional Study ◽  
Aminoglycoside Resistance ◽  
Terminal Domain ◽  
Critical Residues ◽  
High Level ◽  
Potential Exploitation

Methylation of the small ribosome subunit rRNA in the ribosomal decoding center results in exceptionally high-level aminoglycoside resistance in bacteria. Enzymes that methylate 16S rRNA on N7 of nucleotide G1405 (m7G1405) have been identified in both aminoglycoside-producing and clinically drug-resistant pathogenic bacteria. Using a fluorescence polarization 30S-binding assay and a new crystal structure of the methyltransferase RmtC at 3.14 Å resolution, here we report a structure-guided functional study of 30S substrate recognition by the aminoglycoside resistance-associated 16S rRNA (m7G1405) methyltransferases. We found that the binding site for these enzymes in the 30S subunit directly overlaps with that of a second family of aminoglycoside resistance-associated 16S rRNA (m1A1408) methyltransferases, suggesting that both groups of enzymes may exploit the same conserved rRNA tertiary surface for docking to the 30S. Within RmtC, we defined an N-terminal domain surface, comprising basic residues from both the N1 and N2 subdomains, that directly contributes to 30S-binding affinity. In contrast, additional residues lining a contiguous adjacent surface on the C-terminal domain were critical for 16S rRNA modification but did not directly contribute to the binding affinity. The results from our experiments define the critical features of m7G1405 methyltransferase–substrate recognition and distinguish at least two distinct, functionally critical contributions of the tested enzyme residues: 30S-binding affinity and stabilizing a binding-induced 16S rRNA conformation necessary for G1405 modification. Our study sets the scene for future high-resolution structural studies of the 30S-methyltransferase complex and for potential exploitation of unique aspects of substrate recognition in future therapeutic strategies.

scholarly journals Detection of the High-Level Aminoglycoside Resistance Gene aph(2")-Ib in Enterococcus faecium

2000 ◽  
Vol 44 (10) ◽  
pp. 2876-2879 ◽  
Author(s):  
Susan J. Kao ◽  
Il You ◽  
Don B. Clewell ◽  
Susan M. Donabedian ◽  
Marcus J. Zervos ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Resistance Gene ◽  
Enterococcus Faecium ◽  
Open Reading Frame ◽  
Aminoglycoside Resistance ◽  
Reading Frame ◽  
Gentamicin Resistance ◽  
High Level

ABSTRACT A new high-level gentamicin resistance gene, designatedaph(2")-Ib, was cloned from Enterococcus faecium SF11770. The deduced amino acid sequence of the 897-bp open reading frame of aph(2")-Ib shares homology with the aminoglycoside-modifying enzymes AAC(6′)-APH(2"), APH(2")-Ic, and APH(2")-Id. The observed phosphotransferase activity is designated APH(2")-Ib.

scholarly journals Functionally critical residues in the aminoglycoside resistance-associated methyltransferase RmtC play distinct roles in 30S substrate recognition

2019 ◽  
Author(s):  
Meisam Nosrati ◽  
Debayan Dey ◽  
Atousa Mehrani ◽  
Sarah E. Strassler ◽  
Natalia Zelinskaya ◽  
...  
Keyword(s):  
16S Rrna ◽  
Binding Affinity ◽  
Pathogenic Bacteria ◽  
Substrate Recognition ◽  
Functional Study ◽  
Aminoglycoside Resistance ◽  
Terminal Domain ◽  
Critical Residues ◽  
High Level ◽  
Potential Exploitation

ABSTRACTMethylation of the small ribosome subunit rRNA in the ribosomal decoding center results in exceptionally high-level aminoglycoside resistance in bacteria. Enzymes that methylate 16S rRNA on N7 of nucleotide G1405 (m7G1405) have been identified in both aminoglycoside-producing and clinically drug-resistant pathogenic bacteria. Using a fluorescence polarization 30S-binding assay and a new crystal structure of the methyltransferase RmtC at 3.14 Å resolution, here we report a structure-guided functional study of 30S substrate recognition by the aminoglycoside resistance–associated 16S rRNA (m7G1405) methyltransferases. We found that the binding site for these enzymes in the 30S subunit directly overlaps with that of a second family of aminoglycoside resistance–associated 16S rRNA (m1A1408) methyltransferases, suggesting both groups of enzymes may exploit the same conserved rRNA tertiary surface for docking to the 30S. Within RmtC, we defined an N-terminal domain surface, comprising basic residues from both the N1 and N2 subdomains, that directly contributes to 30S-binding affinity. In contrast, additional residues lining a contiguous adjacent surface on the C-terminal domain were critical for 16S rRNA modification, but did not directly contribute to the binding affinity. The results from our experiments define the critical features of m7G1405 methyltransferase–substrate recognition and distinguish at least two distinct, functionally critical contributions of the tested enzyme residues: 30S-binding affinity and stabilizing a binding-induced 16S rRNA conformation necessary for G1405 modification. Our study sets the scene for future high-resolution structural studies of the 30S–methyltransferase complex and for potential exploitation of unique aspects of substrate recognition in future therapeutic strategies.

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