Detecting 16S rRNA Methyltransferases in Enterobacteriaceae by Use of Arbekacin

Abstract

c 16S rRNA methyltransferases confer resistance to most aminoglycosides, but discriminating their activity from that of aminoglycoside-modifying enzymes (AMEs) is challenging using phenotypic methods. We demonstrate that arbekacin, an aminoglycoside refractory to most AMEs, can rapidly detect 16S methyltransferase activity in Enterobacteriaceae with high specificity using the standard disk susceptibility test. Aminoglycosides play an important role in treating serious infections caused by Gram-positive and -negative organisms, with amikacin, gentamicin, and tobramycin being of particular clinical value (1). In Gram-negative organisms, resistance is primarily mediated by aminoglycoside-modifying enzymes (AMEs), which inactivate the antibiotic through modification of the -OH or -NH 2 groups (2). AMEs are divided into three classes: the ATP (and/or GDP)-dependent aminoglycoside phosphotransferases (APHs), the acetyl-coenzyme A (CoA)-dependent aminoglycoside acetyltransferases (AACs), and the ATP-dependent aminoglycoside nucleotidyltransferases (ANTs) (2). Different classes have different antibiotic substrates, but no single AME confers resistance to all aminoglycosides. In the past decade, transmissible 16S rRNA methyltransferases (16S RMTases) have emerged as a new and worrisome source of aminoglycoside resistance that operates by reducing the affinity of nearly all aminoglycosides for 16S rRNA via ribosomal methylation (3). Since the presence of multiple AMEs can confer resistance to all clinically relevant aminoglycosides, discriminating between AME RMTase activity and 16S RMTase activity using phenotypic methods can be challenging (3).Arbekacin is a semisynthetic aminoglycoside derived from dibekacin (4). It is licensed only in Japan, where it is used to treat septicemia and pneumonia caused by methicillin-resistant Staphylococcus aureus (MRSA). However, it also has broad antibacterial activity against Gram-negative bacteria, including strains that are resistant to amikacin, gentamicin, and tobramycin (5). Recently, the U.S. Food and Drug Administration approved arbekacin for use under a protocol at the Walter Reed National Military Medical Center to treat infections for which no other antibiotic is available. Importantly, arbekacin is refractory to nearly all AMEs with the exception of some members of the AAC(6=)-I family and the bifunctional enzyme AAC(6=)/APH(2==) produced by some strains of S. aureus and enterococci (3,4,(6)(7)(8). However, even though these enzymes acetylate arbekacin, the antibiotic still retains some antimicrobial activity (9). We exploited this characteristic to develop a rapid method to detect 16S RMTase activity in Enterobacteriaceae using arbekacin-impregnated susceptibility disks.125 Escherichia coli, 74 Klebsiella pneumoniae, and 22 other Enterobacteriaceae isolates, including Enterobacter (n ϭ 4), Morganella (n ϭ 2), Proteus (n ϭ 3), Providencia (n ϭ 6), Serratia (n ϭ

High distribution of 16S rRNA methylase genes rmtB and armA among Enterobacter cloacae strains isolated from an Ahvaz teaching hospital, Iran

“…Our data indicated that the armA gene was more prevalent than the rmtB gene in E. cloacae strains. This result is consistent with other reports indicating that armA had a higher prevalence relative to rmtB among the Enterobacteriaceae family [11,[19][20][21]. Although the presence of rmtA, rmtD, and rmtC genes has been confirmed by PCR among the Enterobacteriaceae family in India [22] and Saudi Arabia [12], these genes were not detected in this study and some other countries [11,19,20,23].…”

“…In this study, the overall prevalence of the 16S rRNA methylase genes (armA and rmtB) among E. cloacae strains isolated from the various clinical samples was 42.64%. The frequency rates of the 16S rRNA methylase genes among Enterobacteriaceae family in other studies ranged from 0.66% to 46.34% [11,[18][19][20][21][22].…”

Molecular mechanisms of antibiotic co-resistance among carbapenem resistant Acinetobacter baumannii

“…The aminoglycoside resistance among A. baumannii involves the production of various types of aminoglycosides modifying enzymes (AMEs), including acetyltransferases, nucleotidyltransferases, and phosphotransferases that vary in their antibiotic substrates and no single AME is able to modify all types of aminoglycosides [12]. The ribosomal methylation is another mechanism described during the past few years through the production of 16S rRNA methyltransferases that reduce the affinity of almost all aminoglycosides [13,14] The resistance to the tetracyclines is mainly ascribed to the acquisition of efflux pumps belonging to major facilitator superfamily (MFS) i.e. tetA and tetB and the resistance nodulation division family (RND) such as adeABC, adeIJK, adeFGH, adeM, adeDE in A. baumannii isolates.…”

The First Saudi Study Investigating the Plasmid-borne Aminoglycoside and Sulfonamide Resistance among Acinetobacter baumannii Clinical Isolates Genotyped by RAPD-PCR: the Declaration of a Novel Allelic Variant Called aac(6ʹ)-SL and Three Novel Mutations in the sul1 Gene in the Acinetobacter Plasmid (s)

“…Resistance to aminoglycosides can also be achieved through ribosomal target modification via methylation of 16S rRNA by methyltransferases (MT) such as aminoglycosides resistance methyltransferase A (armA) 14 and 16S rRNA methyltransferases B (rmtB). 13 , 19 …”