Acquisition of 16S rRNA methylase gene in Pseudomonas aeruginosa
Keiko Yokoyama1, Yohei Doi, Kunikazu Yamane
1Department of Bacterial Pathogenesis and Infection Control, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashi-Murayama, Tokyo 208-0011, Japan.
Background:
Bacteria develop resistance to aminoglycosides by producing aminoglycoside-modifying enzymes such as acetyltransferase, phosphorylase, and adenyltransferase. These enzymes, however, cannot confer consistent resistance to various aminoglycosides because of their substrate specificity. Notwithstanding, a Pseudomonas aeruginosa strain AR-2 showing high-level resistance (minimum inhibitory concentration >1024 mg/L) to various aminoglycosides was isolated clinically. We aimed to clone and characterise the genetic determinant of this resistance.
Methods:
We used conventional methods for DNA manipulation, susceptibility testing, and gene analyses to clone and characterise the genetic determinant of the resistance seen. PCR detection of the gene was also done on a stock of P aeruginosa strains that were isolated clinically since 1997.
Findings:
An aminoglycoside-resistance gene, designated rmtA, was identified in P aeruginosa AR-2. The Escherichia coli transformant and transconjugant harbouring the rmtA gene showed very high-level resistance to various aminoglycosides, including amikacin, tobramycin, isepamicin, arbekacin, kanamycin, and gentamicin. The 756-bp nucleotide rmtA gene encoded a protein, RmtA. This protein showed considerable similarity to the 16S rRNA methylases of aminoglycoside-producing actinomycetes, which protect bacterial 16S rRNA from intrinsic aminoglycosides by methylation. Incorporation of radiolabelled methyl groups into the 30S ribosome was detected in the presence of RmtA. Of 1113 clinically isolated P aeruginosa strains, nine carried the rmtA gene, as shown by PCR analyses.
Interpretation:
Our findings strongly suggest intergeneric lateral gene transfer of 16S rRNA methylase gene from some aminoglycoside-producing microorganisms to P aeruginosa. Further dissemination of the rmtA gene in nosocomial bacteria could be a matter of concern in the future.
Insights
A novel aminoglycoside resistance gene, rmtA, was identified in Pseudomonas aeruginosa. This gene confers high-level resistance to multiple antibiotics, suggesting potential for widespread dissemination in clinical settings.
Area of Science:
- Microbiology
- Genetics
- Molecular Biology
Background:
- Aminoglycoside resistance in bacteria is often mediated by modifying enzymes with limited substrate specificity.
- A Pseudomonas aeruginosa strain (AR-2) exhibited exceptionally high resistance to diverse aminoglycosides.
- The genetic basis for this broad-spectrum resistance was investigated.
Purpose of the Study:
- To clone and characterize the genetic determinant responsible for high-level aminoglycoside resistance in Pseudomonas aeruginosa AR-2.
- To understand the mechanism of resistance conferred by the identified gene.
Main Methods:
- Conventional DNA manipulation techniques.
- Bacterial susceptibility testing and gene analysis.
- Polymerase Chain Reaction (PCR) for gene detection in clinical isolates.
Main Results:
- An aminoglycoside resistance gene, designated rmtA, was identified and cloned.
- Expression of rmtA in Escherichia coli resulted in high-level resistance to multiple aminoglycosides.
- The rmtA gene encodes a 16S rRNA methylase, RmtA, which methylates the 30S ribosome.
- PCR analysis revealed the presence of rmtA in 9 out of 1113 clinical Pseudomonas aeruginosa isolates.
Conclusions:
- The findings suggest intergeneric lateral gene transfer of the 16S rRNA methylase gene (rmtA) to Pseudomonas aeruginosa.
- The potential for further dissemination of rmtA in nosocomial bacteria raises significant clinical concerns.
