In a Korean nationwide surveillance, the amikacin resistance rates of K. pneumoniae increased from 8% in 1997 to 13% in 2003.67 The frequency of high-level resistance to amikacin or arbekacin was 9.5% (15/158), 10.3% (13/126), and 17.1% (22/129) for Enterobacter cloacae, Citrobacter freundii and Serratia marcescens isolates, respectively.8 In another study, 24 hospitals and two Community Labs participated in collecting antimicrobial susceptibility data in Korea, and the resistance rates of K. pneumoniae, E. cloacae, S. marcescens, Acinetobacter spp., and P. aeruginosa to amikacin were high in the hospital laboratories: 15%, 5%, 10%, 48%, and 19%, respectively.9 Taken together, these results suggest that 16S RMTase-producing bacteria are becoming prevalent in Korea and highlight the need for continued surveillance to investigate the overall trend of aminoglycoside resistance among clinically important pathogens. To provide ongoing insight into aminoglycoside resistance, in this study, we investigated predominant 16S RMTases in Korea from our data and from previously published literatures.

To examine the recent predominance of aminoglycoside resistance associated with 16S RMTases in Korea, a total of 222 potential amikacin resistant Gram-negative clinical isolates from patient specimens were collected between 1999 and 2015 from three hospital banks across Korea and primarily identified by each bank through Vitek Mass Spectrometry: 73 isolates from Chonbuk National University Hospital Culture Collection for Pathogens from 2001 to 2013; 88 isolates from Gyeongsang National University Hospital Culture Collection for Pathogens from 1999 to 2015; 61 isolates from Kyungpook National University Hospital Culture Collection for Pathogens from 2012 to 2015. The specimens were from the following sources: 10 peritoneal fluid, 77 blood, 27 open wound, 6 bile acid, 51 urine, 46 sputum and bronchial washing, 3 ear discharge, and 2 pleural fluid. To further confirm amikacin-resistance and determine the minimum inhibitory concentrations (MICs) of amikacin for the collected isolates, we evaluated the susceptibility to amikacin by using the Etest. Briefly, Etest strips (bioMérieux, Marcy-l'Étoile, France) with amikacin were placed on Mueller-Hinton (MH) agar plates lawn-inoculated with suspension of isolates grown to an optical density of 0.5 McFarland units to determine the MICs of amikacin for each isolate. After incubation, MICs were read directly from the Etest strip described previously.10 We also screened the amikacin-resistant isolates using polymerase chain reaction (PCR) to detect the known 16S RMTase genes armA, rmtA, rmtB, rmtB2, rmtC, rmtD, rmtE, rmtF, rmtG, and rmtH. Briefly, DNA template for PCR were prepared from bacterial isolates cultured in LB broth containing amikacin (10 μg/mL) and previously described oligonucleotides were used to amplify gene fragments from 16S RMTases.1112 The PCR products were sequenced, and the sequences were compared with those in the GenBank nucleotide database. A total of 198 (89.2%) isolates were confirmed as amikacin-resistant (> 16 μg/mL) among the 222 collected isolates primarily confirmed as potential aminoglycoside-resistance at the three hospitals in Korea (Table 1). Among them, 190 (96.0%) were highly resistant to amikacin (> 256 μg/mL), a resistance phenotype consistent with production of 16S RMTase. By PCR, 142 (74.7%) of the high level-amikacin resistant bacteria were positive for 16S RMTase genes; 141 armA and one rmtB.

To further investigate whether aminoglycoside resistance was transferable, plasmids from clinical isolates with high-level resistance to amikacin (> 256 μg/mL) and with 16S RMTase genes (armA or rmtB) were prepared using the alkaline lysis method and were electro-transformed into Escherichia coli TOP10 competent cells (Invitrogen, Carlsbad, TX, USA). Transformants were selected on LB agar plates containing 50 μg/mL gentamicin, and gentamicin-resistant transformants were tested for susceptibility to amikacin (256 μg/mL). The plasmids from transformants with both gentamicin and amikacin resistance were isolated, used as template DNAs for the PCR-based detection of 16S RMTases, and analyzed against the GenBank nucleotide database, as described above. Of six armA and one rmtB from the high-level amikacin-resistant isolates tested, all transferred resistance to amikacin through plasmids.

We reviewed 419 articles using the keywords “16S rRNA methyltransferase” or “16S methyltransferase” or “Korea” in PubMed for articles published from 1974 to 2017 to study overall trends over time of 16S RMTases from clinical isolates in Korea. A total of 10 papers related to 16S RMTases from clinical isolates were published in Korea from 2006 to 2017 (Table 2).781314151617181920 Among 331 amikacin resistant gram-negative organism (63 E. coli; 149 K. pneumoniae; 2 K. spp.; 8 C. freundii; 27 E. cloacae; 7 E. spp.; 14 S. marcescens; 30 Acinetobacter spp.; 27 P. aeruginosa; 3 Providencia spp.; 1 Morganella morganii), armA was 192 and rmtB was 52 isolates.71415 In 145 high-level amikacin resistant isolates, armA was 110 and rmtB was 19 isolates.816 In the other 5 articles, armA was detected 30 of 44 extended spectrum β-lactamase producing K. pneumonia, 14 of 111 multi-drug resistant (MDR) P. aeruginosa, 28 of 31 MDR Acinetobacter baumannii, 4 of 264 isolates from urines.13171920 One rmtB was from urinary isolate E. coli.18 Interestingly, only one P. aeruginosa isolate with rmtA was reported in 2006.17

Taken together, this study provides recent information regarding the 16S RMTase genes responsible for aminoglycoside-resistant isolates circulating in Korean community settings. ArmA and rmtB were the predominant 16S RMTase genes, and one RmtA-producing isolate was detected back in 2006 but not since then. Given that armA and rmtB have been found worldwide and that other 16S RMTase genes (rmtA, rmtC, rmtD, rmtF, rmtG and rmtH) are regionally spread, further studies with a larger number of clinical isolates are needed to confirm the presence of a variety of 16S RMTases among aminoglycoside-resistant bacteria in Korea.