we conducted a preliminary screening of the 16s rrna methylase - producing bacilli on our gram - negative microbial stock of 2,877 strains isolated from japanese hospitals within the past several years .
arbekacin , a semisynthetic aminoglycoside belonging to the kanamycin group , requires 2 modifications at the ( 6  ) aminogroup and the ( 2   ) hydroxyl group for inactivation , so this agent is not inactivated by known plasmid - mediated aminoglycoside - modifying enzymes .
512 mg / l ) was used as a marker for screening the 16s rrna methylase - producing strains .
all arbekacin - resistant strains were subjected to polymerase chain reaction ( pcr ) analysis to detect rmta , rmtb , or arma , and all strains were pcr positive , except for a strain of acinetobacter demonstrating a very high level of resistance to arbekacin ( mic 1,024 mg / l ) .
this strain was later shown to produce both aminoglycoside 6-acetyltransferase and 2-adenyltransferase ( 12 ) , so arbekacin was inactivated in this strain by both 6-acetylation and 2-adenylation .
each pcr primer set was used to detect rmta and rmtb genes as in our previous reports ( 6,7 ) .
the pcr primers for amplification of arma were newly designed ( forward : 5-agg ttg ttt cca ttt ctg ag-3  , reverse : 5-tct ctt cca ttc cct tct cc-3  ) , and the predicted size of the amplicon was 590 bp .
these 3 sets of pcr primers were very reliable in detecting rmta , rmtb , and arma genes , respectively .
each pcr amplicon was then subjected to sequencing analyses on both strands to confirm its nucleotide sequences for detecting mutations in the methylase genes .
as reported in our previous study , rmta and rmtb genes had been found in p. aeruginosa isolates ( 6,10 ) and in 1 strain of s. marcescens ( 7 ) , respectively . as shown in the table , 5 p. aeruginosa strains isolated after our previous report ( 6 ) were rmta positive .
the rmtb gene was additionally identified in 4 k. pneumoniae , 2 e. coli , and 1 k. oxytoca strains in japan . to our surprise ,
the arma gene , which had been found in various gram - negative microbial species belonging to the family enterobacteriaceae exclusively in europe as reported by galimand et al .
( 13 ) , was also identified in japan in 1 strain each of e. coli , s. marcescens , and acinetobacter sp .
notably , the arma and rmtb genes were also recently identified in k. pneumoniae and e. coli in taiwan ( 9 ) .
furthermore , the genetic environment of the arma gene found in c. freundii isolated in poland was similar to that of k. pneumoniae isolated in france .
the genetic environments of the arma gene found in the 3 japanese microbial species , e. coli , s. marcescens , and acinetobacter sp . , ( genbank accession nos . ab116388 and ab117519 ) , were also similar to those found in europe ( genbank accession nos . af550415 and ay220558 ) .
these findings suggest that the arma - producing gram - negative nosocomial microbes that harbor a very similar genetic environment carrying the arma gene have spread globally .
as described previously , arbekacin still shows a very broad antimicrobial spectrum from gram - positive to gram - negative nosocomial microbes and has been approved solely to treat methicillin - resistant staphylococcus aureus ( mrsa ) infections in japan since 1990 to ensure the prudent use of this agent .
the emergence and presence of the 16s rrna methylase - producing gram - negative bacilli , however , has not been well recognized in japan to date ; arbekacin has not been listed among the antimicrobial agents for daily antimicrobial susceptibility testing of gram - negative microbes .
the use of semisynthetic aminoglycosides , including arbekacin , in japanese clinical settings for > 10 years may have promoted the emergence and dissemination of the 16s rrna methylase - producing gram - negative microbes in japan .
the large amount of various aminoglycosides used in livestock - farming environments could have also been a selective pressure for the emergence and spread of pathogenic microbes that harbor genetic determinants for the newly identified 16s rrna methylases , as exemplified by recent isolation of arma - producing e. coli from swine in spain ( genbank accession no .
since acquisition of multidrug resistance against clinically important antimicrobial agents such as carbapenems and fluoroquinolones has been developing rapidly worldwide , the acceleration of even greater aminoglycoside resistance among gram - negative bacilli promises to become an actual clinical concern in the near future , just as vancomycin - resistant enterococci ( vre ) did in the 1990s ( 14 ) .
the emergence of gram - positive cocci including mrsa and vre that acquire the 16s rrna methylase could also be a grave clinical matter , although fortunately no such hazardous microbes have been identified .
thus , steps must be taken to further block proliferation of these multidrug - resistant gram - negative super microbes , including p. aeruginosa , k. pneumoniae , and acinetobacter spp .
, as well as multidrug - resistant cocci such as mrsa and vre , which have acquired an extraordinarily high level of resistance to various aminoglycosides through production of 16s rrna methylases , especially in clinical environments .
 emergence of the newly identified 16s rrna methylases rmta , rmtb , and arma in pathogenic gram - negative bacilli has been a growing concern . 
 arma , which had been identified exclusively in europe , was also found in several gram - negative pathogenic bacilli isolated in japan , suggesting global dissemination of hazardous multiple aminoglycoside resistance genes . 