On January 24, 2007, S. sonnei strain UCN59 was isolated from a 4-year-old girl admitted to Robert Debré 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. All patients lived in the Paris area and attended 8 religious schools.

Figure 1

Figure 1. Pulsed-field gel electrophoresis–generated dendrogram for 43 Shigella sonnei isolates obtained from sporadic or outbreak cases during 1996–2007 in the Paris area. Profile 1) representative isolates from the 2007 outbreak, including 32...

Figure 2

Figure 2. Repetitive sequence–based, PCR–generated dendrogram for 43 Shigella sonnei isolates obtained from sporadic or outbreak cases during 1996–2007 in the Paris area. Isolates with >97% similarity were considered to be closely...

Typing by pulsed-field gel electrophoresis (PFGE) and repetitive sequence–based PCR (rep-PCR) by using the automated DiversiLab system (bioMérieux, 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 1996–2007 in the Paris area. Five different PFGE patterns were obtained by using the enzyme BlnI. 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 2002–2006 outbreak isolates (data not shown), showing the persistence of this clonal type over 10 years in this area of Paris. Other isolates displayed PFGE types 2 to 5. 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.

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^–3 per donor cell-forming unit after the mating period. A single plasmid was extracted from a transconjugant E. coli AG100A/pUV21. After restriction analysis, its size was estimated at ≈90 kb. PCR experiments showed that this plasmid belonged to incompatibility group I (12). 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/pUC18Ωmph(A). Sequence of the inserted DNA was determined. 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 Salmonellaenterica serotype Typhimurium (C.Y. Chen et al., unpub. data, GenBank accession no. NC_006816), of Shigella flexneri transposon TnSF1 (J.H. Chen and J.Y. Chen, unpub. data, GenBank accession no. AF188331), and of plasmids pRSB101 and pSRB107 (13,14).

Few data are available on azithromycin resistance in Shigella spp. 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. Chen JY, unpub. data, GenBank accession no. 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.