on january 24 , 2007 , s. sonnei strain ucn59 was isolated from a 4-year - old girl admitted to robert debr hospital , paris , for bloody diarrhea and fever .
the strain was resistant to ampicillin , trimethoprim , sulfonamides , and cotrimoxazole but susceptible to quinolones , third - generation cephalosporins , and doxycycline according to the disk - diffusion technique .
mics of macrolides were markedly increased for s. sonnei ucn59 compared with those for a susceptible control s. sonnei ucn62 ( table ) . from january to april 23 , 2007 , a total of 50 cases of laboratory - confirmed shigellosis were identified .
isolates included , in addition to ucn59 , 31 s. sonnei that had an azithromycin mic > 64 mg / l from 31 children < 15 years of age , who had each been prescribed azithromycin for diarrhea .
typing by pulsed - field gel electrophoresis ( pfge ) and repetitive sequence  based pcr ( rep - pcr ) by using the automated diversilab system ( biomrieux , la - balme - les - grottes , france ) ( 10 ) was performed on the 32 azithromycin - resistant and on 11 azithromycin - susceptible ( mic < 16 mg / l ) sporadic or outbreak isolates obtained during 19962007 in the paris area .
all the 2007 outbreak isolates , including the 32 azithromycin - resistant isolates and 2 azithromycin - susceptible isolates , were clustered into a single profile , profile 1 ( figure 1 ) .
the presence of azithromycin - susceptible isolates with profile 1 was detected among the 1996 and 20022006 outbreak isolates ( data not shown ) , showing the persistence of this clonal type over 10 years in this area of paris .
low diversity of pfge profiles was consistent with isolation of strains in the same area and for most of them from the same community .
four different patterns with < 97% similarity were distinguished by rep - pcr ( figure 2 ) .
again , all the 2007 azithromycin - resistant isolates were clustered ; however , they could be distinguished from the 2007 azithromycin - susceptible isolates .
in contrast to pfge findings , rep - pcr showed that isolates representative of the 1996 , 2002 , and 2003 outbreaks were genetically related .
pulsed - field gel electrophoresis  generated dendrogram for 43 shigella sonnei isolates obtained from sporadic or outbreak cases during 19962007 in the paris area .
profile 1 ) representative isolates from the 2007 outbreak , including 32 isolates with azithromycin mic > 256 mg /
l by etest and 2 isolates with azithromycin mic < 16 mg / l . profile 2 ) 6 representative isolates from sporadic cases ( 20032006 ) with azithromycin mic <
16 mg / l . profile 3 ) representative isolate shi 03 - 3580 from 2003 outbreak with azithromycin mic < 16 mg / l by etest . profile 4 )
representative isolate shi 02 - 9633 from 2002 outbreak with azithromycin mic < 16 mg / l by etest . profile 5 )
representative isolate shi 96 1420 from 1996 outbreak with azithromycin mic < 16 mg / l by etest
. repetitive sequence  based , pcr  generated dendrogram for 43 shigella sonnei isolates obtained from sporadic or outbreak cases during 19962007 in the paris area . isolates with > 97% similarity were considered to be closely genetically related .
profile 1 ) representative of the 32 isolates of the 2007outbreak with azithromycin ( mic > 256 mg / l by etest ) .
profile 2 ) 1 of 6 isolates from sporadic cases ( 20032006 ) with azithromycin mic < 16 mg / l . profile 3 ) representative isolate shi 03 - 3580 from 2003 outbreak with azithromycin mic < 16 mg / l by etest . profile 4 )
representative isolate shi 02 - 9633 from 2002 outbreak with azithromycin mic < 16 mg / l by etest . profile 5 )
representative isolate shi 96 1420 from 1996 outbreak with azithromycin mic < 16 mg / l by etest .
profile 6 ) isolate from 2007 with azithromycin mic < 16 mg / l by etest ; another azm s 2007 isolate had an identical profile .
azm , azithromycin ; r , resistant ; s , sensitive ; rep , repetitive sequence - based pcr . the mph(a ) gene , which encodes a macrolide 2-phosphotransferase that inactivates macrolide antimicrobial drugs , was amplified from s. sonnei ucn59 dna by pcr ( 11 ) .
pcr was negative for the erm(a ) , erm(tr ) , erm(b ) , erm(c ) , and erm(x ) methylase genes ; the ere(a ) , ere(b ) genes encoding esterases ; the mph(b ) gene encoding a phosphotransferase ; and the efflux genes mef(a ) and msr(a ) .
sequence of the genes that encode ribosomal structures composing the target of macrolides , rrl , rpld , and rplv genes in s. sonnei ucn59 , did not display any mutation in the critical bases of resistance to macrolides .
the genes conferring resistance to ampicillin and erythromycin were transferred en bloc by conjugation from s. sonnei ucn59 to a macrolide - susceptible mutant escherichia coli ag100a at a frequency of 10 per donor cell - forming unit after the mating period .
a single plasmid was extracted from a transconjugant e. coli ag100a / puv21 . after restriction analysis ,
mics of macrolides for e. coli ag100a / puv21 confirmed that this plasmid conferred cross - resistance to macrolides ( table ) .
ecori - restricted fragments of plasmid puv21 were transferred to a nylon membrane and hybridized to an mph(a ) probe .
the mph(a ) gene was borne by an 20-kb ecori fragment , confirming that resistance to azithromycin was plasmid mediated .
after plasmid digestion with psti enzyme , a dna fragment that conferred resistance to erythromycin was cloned in plasmid puc18 and introduced by transformation into e. coli ag100a to generate e. coli k12 ag100a / puc18mph(a ) .
the fragment contained 4 open reading frames ( orfs ) in the same orientation : mph(a ) , mrx that putatively encodes a membrane protein , mphr(a ) that regulates the expression of mph(a ) , and an orf of unknown function .
this series of orfs was flanked by a copy of is26 at the 5 end and a copy of is6100 at the 3 end .
blast analysis ( www.ncbi.nlm.nih.gov/blast/blast.cgi ) showed that the nucleotide sequence was nearly identical to that of fragments of plasmid pu302l from salmonella enterica serotype typhimurium ( c.y .
a recent report from bangladesh mentioned that 16% of shigella isolates were resistant to azithromycin and that 62% had intermediate resistance according to the clinical laboratory standards institute breakpoints recommended for streptococci ( > 1 mg / l , resistant ; < 0.25 mg / l , susceptible ) ( 15 ) .
however , the mic90 ( mic at which 90% are susceptible ) of 8 mg / l displayed by the microorganisms was within the normal range of mics for this microorganism ; no isolate had an azithromycin mic > 24 mg / l , which suggests that none had acquired resistance to azithromycin .
surveillance for resistance to azithromycin in shigella spp . requires specific breakpoints for this species ( 3 ) .
the mph(a ) gene has been detected in the sequence of transposon tnsf1 isolated from s. flexneri ( j.h . chen and j.y .
af188331 ) . the mph(a ) gene was first reported in an e. coli isolate from japan ( 10 ) . since then , the gene has been found in aeromonas hydrophila , pseudomonas spp .
, stenotrophomonas spp . , and a variety of enterobacteria ( listed at http://faculty.washington.edu/marilynr/ermweb4.pdf ) .
azithromycin was used to treat shigellosis in france only after the release of the french recommendations in 2004 .
subsequent rapid emergence of azithromycin - resistant isolates may be a limitation for the use of macrolides in shigellosis .
because use of azithromycin is proposed for treatment of shigellosis , susceptibility of the isolates to azithromycin should be routinely tested .
 shigella sonnei ucn59 , isolated during an outbreak of s. sonnei in january 2007 , was resistant to azithromycin ( mic 64 mg / l ) . 
 the isolate contained a plasmid - borne mph(a ) gene encoding a macrolide 2-phosphotransferase that inactivates macrolides . 
 emergence of the mph(a ) gene in s. sonnei may limit usefulness of azithromycin for treatment of shigellosis . 
The Study
Conclusions