Background Between 2006 and 2008, the prevalence of Neisseria gonorrhoeae (NG) isolates with decreased susceptibility (0.125<MIC<0.5 μg/ml) to the extended-spectrum cephalosporin (ESC) cefotaxime (CTX) among visitors of the STI clinic in Amsterdam, the Netherlands increased from 4.8 to 12.1%. The transmission patterns, clonality, phenotypic and genotypic characteristics of the NG isolates transmitted within this high-risk group were examined. Methods From 2006 to 2008, 74 NG isolates with a CTX MIC of >0.125 μg/ml (group A), 54 with a CTX MIC of 0.125 μg/ml (group B), and a control group of 74 with a CTX MIC of <0.125 μg/ml (group C), were included. All isolates were characterised using antibiograms, conventional penA mosaic gene PCR, and genotyping by NG-multi-locus variable-number tandem repeat analysis (MLVA). PenA mosaic positive isolates and a strict selection of the remaining isolates were further characterised by NG-multilocus sequence typing (MAST) and sequencing of ESC resistance determinants (penA, mtrR, and porB1b). Results The majority of the isolates in group A (n=47; 64%) but only 11% (n=6) of the isolates in group B contained a penA mosaic allele. No penA mosaic-containing isolate was identified in group C (see Abstract O1-S03.03 figure 1). All the 53 penA mosaic isolates had an identical penA sequence (type XXXIV) and were assigned to the same MLVA cluster, which additionally included three isolates that were susceptible to CTX (MIC<0.125 μg/ml). Within this MLVA cluster, 46 (87%) of the penA mosaic isolates were assigned NG-MAST ST1407, and the remaining 7 isolates had closely related STs. All these penA mosaic isolates contained a mtrR promoter deletion and in 52/53 isolates the porB1b alterations G101K and A102N were found. Decreased susceptibility to cefixime and ceftriaxone (MIC≥0.016 μg/ml) was found in 50/53 and 44/53 isolates, respectively. The mosaic penA MLVA cluster, containing ST1407 (87%) and closely related STs (13%), represented Dutch homosexual men (66%), patients ...