Single-dose azithromycin therapy has recently been used in Uruguay for the treatment of uncomplicated gonococcal infections. As part of an active surveillance study to monitor the emergence of antibiotic resistance in gonococcal isolates, we examined the levels of azithromycin susceptibility in 51 consecutive isolates obtained from males with uncomplicated gonococcal urethritis. Isolates with decreased susceptibility to azithromycin (MICs, 0.25 to 0.5 μg/ml) were common, and these isolates often displayed cross-resistance to hydrophobic antimicrobial agents (erythromycin and Triton X-100). Resistance to erythromycin and Triton X-100 is frequently due to overexpression of the mtrCDE-encoded efflux pump mediated by mutations in themtrR gene, which encodes a transcriptional repressor that modulates expression of the mtrCDE operon. Accordingly, we questioned whether clinical isolates that express decreased azithromycin susceptibility harbor mtrR mutations. Promoter mutations that would decrease the level of expression ofmtrR as well as a missense mutation at codon 45 in themtrR-coding region that would result in a radical amino acid replacement within the DNA-binding motif of MtrR were found in these strains. When these mutations were transferred into azithromycin-susceptible strain FA19 by transformation, the susceptibility of gonococci to azithromycin was decreased by nearly 10-fold. The mtrCDE-encoded efflux pump system was responsible for this property since insertional inactivation of themtrC gene resulted in enhanced susceptibility of gonococci to azithromycin. We conclude that the mtrCDE-encoded efflux pump can recognize azithromycin and that the emergence of gonococcal strains with decreased susceptibility to azithromycin can, in part, be explained by mtrR mutations.

During a screen of Neisseria gonorrhoeae clinical isolates obtained in Uruguay for susceptibility to azithromycin, we noticed that approximately 10% of the strains examined displayed decreased susceptibility to azithromycin and erythromycin due to the mtr(CDE)-encoded efflux pump system, but remained susceptible to Triton X-100. We now report that the mtr(R) promoter region of one of these isolates contains a dinucleotide insertion (TT) that mediates this resistance phenotype.

One hundred four bacterial strains mediating urinary tract infections in separate individuals from a Uruguayan community were isolated. Forty-six strains conferred a multidrug resistance phenotype. All 104 strains were examined for the presence of class 1, 2, and 3 integrons. Class 1 integrons were found in 21 isolates across four distinct bacterial genera. A large class 1 integron in a Klebsiella pneumoniae strain was fully sequenced and was 29,093 bp in length. This integron probably arose by homologous recombination since it was embedded in a hybrid Tn21-like transposon backbone which comprised a Tn5036-like tnp transposition module at the IRi integron end and a Tn21 mer module at the IRt integron end. The parent integron/transposon that contributed the Tn5036 module was not related to Tn1696 since the integron insertion points in the transposon backbones were 16 bases apart. Examination of the other 20 class 1 integron-containing strains revealed further evidence of genetic exchange. This included a strain that possessed a Tn5036 module at the IRt end but not at the IRi end and another that possessed a tnp module beyond IRi that was a hybrid of Tn21 and Tn5051 and that is presumed to have arisen by site-specific recombination. This study highlights the ability of different genetic elements to act cooperatively to spread and rearrange antibiotic resistance in a community.

Two Neisseria gonorrhoeae isolates from Seattle and two isolates from Uruguay were resistant to erythromycin (MIC, 4 to 16 g/ml) and had reduced susceptibility to azithromycin (MIC, 1 to 4 g/ml) due to the presence of the self-mobile rRNA methylase gene(s) ermF or ermB and ermF. The two Seattle isolates and one isolate from Uruguay were multiresistant, carrying either the 25.2-MDa tetM-containing plasmid (Seattle) or a ␤lactamase plasmid (Uruguay). Sixteen commensal Neisseria isolates (10 Neisseria perflava-N. sicca, 2 N. flava, and 4 N. mucosa) for which erythromycin MICs were 4 to 16 g/ml were shown to carry one or more known rRNA methylase genes, including ermB, ermC, and/or ermF. Many of these isolates also were multiresistant and carried the tetM gene. This is the first time that a complete transposon or a complete conjugative transposon carrying an antibiotic resistance gene has been described for the genus Neisseria. MATERIALS AND METHODSBacterial isolates. Erythromycin-resistant (Em r ) Pro Ϫ /IA-1,2 N. gonorrhoeae isolates were isolated in Seattle during the 1994-1995 gonococcal outbreak (Table 1). Both Seattle isolates (94-965 and 95-1) were also tetracycline resistant. The 1995 Uruguay isolate, 581, was Pro Ϫ /IB-3, and the 1991 Uruguay isolate, 1101, was nonrequiring Proto/IB-3. Strain 1101 carried the 3.2-MDa ␤-lactamase plasmid and was resistant to penicillin in addition to erythromycin, while all the other N. gonorrhoeae isolates did not carry a ␤-lactamase plasmid. Six other Tc r Pro Ϫ /IA-1,2 N. gonorrhoeae isolates that were from the Seattle outbreak but that were not resistant to erythromycin were available for comparison with the two Em r Tc r N. gonorrhoeae isolates. The NRL (Neisseria Reference Laboratory, University of Washington, Seattle) strains were isolated prior to 1986. The N. gonorrhoeae isolates were confirmed by biochemical methods (11). Auxotypes, protein I serovars, and plasmid contents of the gonococcal isolates were determined by established methods (5,6,9,32).We also examined 16 isolates of commensal Neisseria spp., including 10 N. perflava-N. sicca, 2 N. flava, and 4 N. mucosa (Table 1). These isolates were confirmed by biochemical methods. The commensal Neisseria spp. were clinical isolates collected from the periodontal pockets of seven periodontitis patients seen at the Graduate Periodontics Clinic at the University of Washington, Seattle, between 1991 and 1995, isolates collected from oropharyngeal specimens from six patients attending the DeKalb County Sexually Transmitted Disease Clinic in 1986 (designated CTM before the number [7]), and type strains (NRL strains) obtained from Joan Knapp (22,23).Media. GC base or GCP broth (Difco Laboratories, Detroit, Mich.) supplemented as previously described (9, 20) was used for routine culturing of N. gonorrhoeae, Neisseria spp., and Enterococcus faecalis.Antimicrobial susceptibilities. Mueller-Hinton medium (Difco) was used to determine the MICs for the commensal Neisseria spp., N. meningiditis, and E. faecalis transconjugants, a...

The report of ceftriaxone-resistant isolates coupled with the emergence and spread of resistance to ciprofloxacin and azithromycin in Latin America and the Caribbean in the 2000s indicates the importance of active surveillance of N. gonorrhoeae antimicrobial susceptibility to determine antimicrobial resistance emerging trends so as to promptly inform and guide the development of effective treatment options for gonococcal infections.