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during august 2012april 2014 , respiratory specimens that tested positive for m. pneumoniae by molecular methods were collected from 6 medical centers throughout the united states .
these sites were located in chicago , illinois ; kansas city , missouri ; hackensack , new jersey ; new york , new york ; seattle , washington ; and birmingham , alabama .
five medical centers tested for m. pneumoniae dna using the filmarray respiratory pathogen panel ( biofire diagnostics , salt lake city , ut , usa ) .
the university of alabama at birmingham used a laboratory - developed real - time pcr targeting repmp1 . testing for 23s rrna mutations conferring macrolide resistance
was performed on original specimens by real - time pcr melt curve analysis ( 6 ) and confirmed by dna sequencing at the lurie children s hospital ( chicago ) . subculture and phenotypic
antimicrobial susceptibility testing was performed by using a broth microdilution method approved by the clinical and laboratory standards institute at the university of alabama at birmingham .
patients ages ranged from 10 months to 66 years ; 83 ( 91.2% ) samples were from patients < 18 years of age .
specimen types were nasopharyngeal or nasal swabs ( 72 samples ) , nasal aspirate / washes ( 12 samples ) , bronchoalveolar lavage ( 5 samples ) , and throat swab and tracheal aspirate samples ( 1 sample each ) .
we found a 23s rrna point mutation a2063 g known to confer macrolide resistance in 10 ( 10.9% ) of 91 specimens by direct real - time pcr with melting curve analysis .
pcr and sequencing on those subcultures confirmed macrolide resistance in 6 specimens that had been previously identified by direct real - time pcr and in 2 additional specimens that had not previously been detected by pcr .
the 2 specimens most likely tested negative for mutations by direct pcr because of a low number of organisms in the original sample .
macrolide resistance was not significantly correlated with patient age or specimen type ( data not shown ) .
mrmp was detected in a total of 12 ( 13.2% ) samples ( table ) .
we conducted phenotypic antimicrobial susceptibility testing on all 62 m. pneumoniae isolates obtained by subculture .
all 54 macrolide - susceptible isolates showed very low erythromycin mics ( < 0.008 g / ml ) , whereas the 8 isolates that had the a2063 g mutation showed uniformly high erythromycin mics ( > 256 g / ml ) .
all 62 isolates were susceptible to tetracycline ( mic 0.0311 g / ml ) and levofloxacin ( mic 0.0311 g / ml ) .
of the 80 respiratory specimens for which co - infection data were available , 26 ( 32.5% ) had a viral pathogen detected along with m. pneumoniae .
these viruses were 15 ( 18.8% ) rhinovirus / enterovirus group viruses , 3 ( 3.8% ) respiratory syncytial viruses , 4 ( 5% ) parainfluenza viruses , 1 ( 1.3% ) human metapneumoviruses , and 3 ( 3.8% ) coronaviruses .
mrmp prevalence has been reported to range from 2% to 26% in european countries ( 7,8 ) and is 30% in israel ( 9 ) . the prevalence is much higher in asia , where mrmp first emerged in 2000 , and now exceeds 90% in some areas of china and japan ( 10,11 ) .
the centers for disease control and prevention recently published results from 199 specimens obtained from case - patients , small clusters , and outbreaks that occurred during 20062013 but did not specify geographic locations from which specimens were derived .
louis , missouri , usa , during 20072010 and found 4 ( 8.2% ) that contained the a2063 g mutation ( 14 ) .
detected mrmp in 12.1% specimens collected in ontario , canada , during 20102011 ( 15 ) .  
our finding of high - level macrolide resistance in 13.2% of specimens from all 6 centers throughout a broad geographic area in the united states proves this problem has emerged in all regions of the nation and might increase over time , as it has in other countries .
the design of our epidemiologic study provides the most accurate estimate of the point prevalence of mrmp available thus far .
previous studies reported from the united states were limited primarily to individual case reports , clusters , outbreaks , or single geographic locations .
the mechanism for macrolide resistance in m. pneumoniae is point mutations in a few positions of domain v of the peptidyl transferase loop of 23s rrna , the location of macrolide binding to the 50s bacterial ribosome subunit ( 3 ) .
the a2063 g transition mutation has been the most common one detected in most studies and was the only mutation found in our study .
although less common , other mutations conferring macrolide resistance , but not found in our study , include a2063 t or c , a2064 g , a2067 g , and c2617a or g ( 3,10,15 ) .
unlike previous studies , our study used both molecular and phenotypic techniques to detect macrolide resistance .
thus , we were able to correlate erythromycin mics with the presence of 23s rrna mutations .
our results showed striking differences in erythromycin mics between the susceptible and resistant isolates and confirmed high - level resistance to erythromycin in every isolate .
erythromycin is usually used for testing because mutations in a2063 and a2064 consistently have been shown to cause high resistance to both erythromycin and azithromycin ( 3,5,10 ) .
inclusion of patient information , especially antimicrobial therapy , will enhance data interpretation in prospectively conducted future studies .
one notable observation from our study is the co - infection of m. pneumoniae with various viral pathogens .
a viral co - infection rate > 30% supports the use of multiplex testing for viral and bacterial pathogens in children with respiratory infections of uncertain etiology .
although our study has confirmed mrmp in 6 geographically diverse us states , macrolides should remain the drugs of choice in children with m. pneumoniae respiratory infections .
clinicians should be vigilant for macrolide treatment failures and consider using alternative drugs if necessary .
countries such as japan and china that have a very high macrolide resistance rate for m. pneumoniae and other respiratory pathogens often have high consumption of macrolides .
( 5 ) reported that macrolides account for 30% of all oral antibacterial drugs in japan and concluded that the increase in macrolide - resistant bacteria during the past several years in that country was closely related to selective pressure resulting from widespread antimicrobial use .
given the common use of macrolides in the united states to treat pediatric respiratory infections , judicious use of antimicrobial drugs should be emphasized .
reevaluation of existing classes and investigation of new classes of antimicrobial agents may be prudent to have additional treatment alternatives for mrmp infections beyond tetracyclines and fluoroquinolones .
macrolide - resistant mycoplasma pneumoniae ( mrmp ) is highly prevalent in asia and is now being reported from europe . 
 few data on mrmp are available in the united states . using genotypic and phenotypic methods 
 , we detected high - level mrmp in 13.2% of 91 m. pneumoniaepositive specimens from 6 us locations .