Life Science Journal 2012;9(4) http://www.lifesciencesite.com http://www.americanscience.org editor@americanscience.org 2472 Detection of Plasmid-Mediated 16S rRNA Methylase Conferring High-Level Resistance to Aminoglycosides in Gram negative bacilli from Egypt Aisha Abu Aitta 1 ; Manal El Said 1 , Ehab El Dabaa 2 ; Mohamed Abd El Salam 3 and Hesham Mohamed Mahdy 3 1 Microbiology Department, Theodor Bilharz Research Institute, Giza, Egypt. 2 Biochemistry Department, Theodor Bilharz Research Institute, Giza, Egypt 3 Medical Microbiology Department, Faculty of Science, Al-Azhar University, Cairo, Egypt manalmicrobiology@yahoo.com Abstract: Background: Recently, production of 16S rRNA methylases by Gram-negative bacilli has emerged as a novel mechanism for high-level resistance to aminoglycosides by these organisms in a variety of geographic locations. Therefore, the spread of high-level aminoglycoside resistance determinants has become a great concern. Objective: to determine the prevalence of the occurrence of 16S rRNA methylases genes in aminoglycoside- resistant gram-negative bacteria isolates. Methods: 30 Gram-negative bacilli amikacin resistant isolates were collected from inpatients and outpatients at Theodor Bilharz Research Institute (TBRI). Biochemical identification of bacterial species was performed with API 20E system. Antimicrobial susceptibilities of amikacin, gentamicin, tobramycin, neomycin, kanamycin, and netilmicin, were determined by the disk diffusion method and the MICs of amikacin were detected by the E test method. PCR was used to identify 16S rRNA methylas armA and rmtB genes. Result: E.coli, (15/30; 50%), Klebsiella pneumonia (7/30; 23.3%), Pseudomonas aeruginosa (4/30; 13.3%), Enterobacter cloaca and Acinetobacter baumanii (2/30; 6.7% each) were isolated. Extended spectrum β-lactamases (ESBLs) were detected in 7 (23.6%) isolates. Among the 30 isolates, 13 (43.3%) isolates showed a high level of resistance to amikacin. Seven out of 30 (23.3%) amikacin-resistant isolates were positive for 16S rRNA methylase genes. Six isolates (20%) were positive for rmtB gene and one (3.3%) Enterobacter cloaca ESBLs producer isolate was positive for armA gene. rmtB was detected in one (3.3%) Pseudomonas aeruginosae isolate and five (16.7%) E. coli isolates, in which, 3 of them were ESBLs producers. ArmA and rmtB genes were not detected simultaneously. Conclusions: 16S rRNA methylase genes were detected in gram negative bacilli in TBRI. RmtB was found to be more prevalent than armA. There was correlation between the detection of methylase genes and the production of ESBLs. [Aisha Abu Aitta; Manal El Said, Ehab El Dabaa; Mohamed Abd El Salam and Hesham Mohamed Mahdy. Detection of Plasmid-Mediated 16S rRNA Methylase Conferring High-Level Resistance to Aminoglycosides in Gram negative bacilli from Egypt. Life Sci J 2012;9(4):2472-2479]. (ISSN: 1097-8135). http://www.lifesciencesite.com . 365 Keywords: Aminoglycosides; Gram negative bacilli; RmtB; armA; PCR; ESBLs 1. Introduction Aminoglycosides are clinically effective agents for treating a broad range of life-threatening infections caused by Gram-negative pathogens, usually in combination with β-lactam agents (Yu et al., 2010). These agents bind to the A site of the 16S rRNA of prokaryotic 30S ribosomal subunits and subsequently block bacterial growth through interference with protein synthesis (Wachino et al., 2006). Over the past few decades, there have been many studies conducted regarding the mechanisms of resistance to aminoglycosides. Amikacin was developed to suppress a variety of aminoglycoside- modifying enzymes from their accessing target sites, and therefore rare, amikacin-resistant bacteria could be expected (Kang et al., 2008). The emergence of pan-aminoglycoside– resistant, 16S rRNA methylase–producing, gram negative bacteria has been increasingly reported in recent years (Yamane et al., 2007). Alteration of the 16S rRNA A site by these enzymes (designated ArmA, RmtA, RmtB, RmtC, RmtD, and NpmA) confers resistance to almost all aminoglycosides, by limiting the binding of these agents to ribosomal target sites following methylation of specific nucleotides. ArmA (Enterobacteriaceae, Acinetobacter spp.) and rmtB (Enterobacteriaceae) appear to be the most widespread and have been detected primarily in Asia and Europe (Fritche et al., 2008). During the last years, bacterial identification based on molecular methods, especially those including the sequencing of genes coding for ribosomal 16S rDNA, has become a very important tool in studying bacterial communities in samples (Head et al., 1998). Nucleic acid-based detection systems including polymerase chain reaction (PCR) offer rapid and sensitive methods to detect the