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we selected 222 mrsa isolates that were not classified as the new york / japan ( ny / jp ) clone on the basis of the absence of sccmec kdpc ( 9 ) . in addition , 37 ny / jp clone - like isolates were used .
of these 259 isolates , 227 were collected during 20042010 at nagoya medical center , and 32 isolates were collected in 20062009 at kyoto university hospital , including 9 usa300 outbreak isolates ( 6 ) .
atcc baa1556 ( usa300 fpr3757 ) ( american type culture collection , manassas , va , usa ) and atcc baa1717 ( usa300-hou - mr tch1516 ) strains were also used in our study .
susceptibilities to bacitracin and neomycin were tested by the kirby - bauer disk diffusion method ( becton dickinson , franklin lakes , nj , usa ) .
mics of bacitracin , neomycin , and pl - b for usa300 strains were determined by the agar dilution method according to the clinical and laboratory standard institute m07-a8 guidelines ( 10 ) . to observe interaction among these 3 antimicrobial drugs ,
a double - disk synergy test was performed with modification by using atcc baa1717 ( 11 ) .
* community - acquired infections were determined on the basis of patients histories according to centers for disease control and prevention ( atlanta , ga , usa ) guidelines ( www.cdc.gov/mrsa/diagnosis/index.html ) .
isolates possessing both pvl and arca ( 13 ) were analyzed by pulsed - field gel electrophoresis as described in our previous study ( 9 ) .
moreover , usa300 isolates were genotyped by mlst ( www.mlst.net ) and staphylococcal protein a ( spa ) typing ( www.spaserver.ridom.de ) .
of these 19 isolates , 18 had been collected from kyoto university hospital and 1 from nagoya medical center ( table 2 ) .
all 19 pvl- and acme - positive isolates were determined to be st8 by mlst .
these isolates showed usa300 pfge patterns identical to atcc baa1556 and were of sccmec type iva .
sccmec elements of other isolates were determined as type i ( n = 4 ) , iia ( n = 37 ) , iib ( n = 52 ) , ii untypeable ( n = 14 ) , iv ( n = 104 ) , and v ( n = 9 ) . the sccmec element of the remaining 20 isolates could not be identified .
* mrsa , methicillin resistant staphylococcus aureus ; atcc , american type culture collection ( manassas , va , usa ) ; r , resistant ; s , susceptible ; i , intermediate resistance . 
mics are expressed as units / ml for bacitracin and polymyxin b and in g / ml for neomycin .
the 18 usa300 isolates collected from kyoto university hospital showed the same spa type ( t008 ) .
however , the 1 usa300 isolate collected from nagoya medical center was of spa t190 .
atcc baa1717 and 9 usa300 isolates collected during 20072009 at kyoto university hospital were resistant to both bacitracin and neomycin .
the usa300 isolate detected at nagoya medical center in 2004 was bacitracin resistant and neomycin susceptible .
the other 9 usa300 isolates and atcc baa1556 were susceptible to both drugs ( table 2 ) .
( 14 ) found that the bacitracin- and aminoglycoside - resistant genes were located on pusa300-hou - mr , a plasmid typically observed in the usa300 strain tch1516 .
the resistance to bacitracin and neomycin may depend on the presence of the plasmid and may be absent in some usa300 clones . on the other hand , nearly all mrsa isolates that were determined to be a type other than usa300 were susceptible to bacitracin .
also , 11 ( 4.5% ) of the 240 mrsa isolates not deemed to be usa300 were resistant to neomycin , while 132 ( 55% ) demonstrated intermediate resistance ( table 2 ) .
a study performed in the 1990s reported that most mrsa strains were susceptible to bacitracin , and many were resistant to neomycin ( 8) .
mics of bacitracin , neomycin , and pl - b were 400 units / ml , 128 g / ml , and 400 units / ml , respectively , among most usa300 isolates with resistance to both bacitracin and neomycin ( table 2 ) .
the concentrations of neomycin and pl - b in the taos were 10 to 30 higher than the mics of both drugs .
in addition , neomycin and pl - b were observed to be weakly synergistic ( figure ) .
however , bearden et al . reported that despite containing antimicrobial drugs at concentrations far exceeding their mics among mrsa , pl - b and neomycin ointment , or pl - b and gramicidin ointment exhibited deficient bactericidal activity in time - kill assays ( 15 ) .
acquiring resistance to bacitracin and neomycin may be essential for survival under the selective pressure of taos .
if so , bacitracin resistance should be considered a key characteristic of the usa300 clone .
double - disk synergy test with 3 disks , bacitracin ( b10 disk ) , neomycin ( n30 disk ) , or polymyxin b ( pl - b , pb300 disk ) was performed with usa300 strain atcc baa1717 .
disks were placed at 6 mm ( a ) , 9 mm ( b ) , and 11 mm ( c ) distance from disk centers .
taos containing bacitracin , neomycin , and pl - b are widely used in the united states ; thus , bacitracin- and neomycin - resistant strains may be selected by the selective pressure of the taos .
although bacitracin and neomycin ointments are also available as otc drugs in japan , use of the ointments is not widespread . as a result , the selective pressure that leads to bacitracin and neomycin resistance is weak in japan
the emergence of mrsa usa300 depends partly on the virulence of mrsa usa300 , but it may be influenced by usage of otc drugs . in each country , susceptibilities of mrsa usa300 to bacitracin and neomycin should be thoroughly investigated , and relationships between the dissemination of mrsa usa300 and the usage of otc drugs should be clarified
. such an investigation will provide valuable information regarding the emergence of organisms resistant to otc topical antibiotics and likely a warning against the indiscriminate use of antimicrobial drugs .
we tested 259 methicillin - resistant staphylococcus aureus isolates and 2 usa300 atcc type strains for susceptibility to bacitracin and neomycin contained in over - the - counter antibacterial ointments . 
 resistance to both bacitracin and neomycin was found only in usa300 . 
 the use of over - the counter antimicrobial drugs may select for the usa300 clone .