f Escherichia coli sequence type 131 (ST131), a widely disseminated multidrug-resistant extraintestinal pathogen, typically exhibits serotype O25b:H4. However, certain ST131 isolates exhibit serotype O16:H5 and derive from a phylogenetic clade that is distinct from the classic O25b:H4 ST131 clade. Both clades are assigned to ST131 by the Achtman multilocus sequence typing (MLST) system and a screening PCR assay that targets ST131-specific sequence polymorphisms in the mdh and gyrB genes. However, they are classified as separate STs by the Pasteur Institute MLST system, and an ST131 PCR method that targets the O25b rfb region and an ST131-specific polymorphism in pabB detects only the O25b-associated clade. Here, we describe a novel PCRbased method that allows for rapid and specific detection of the O16-associated ST131 clade. The clade members uniformly contained allele 41 of fimH (type 1 fimbrial adhesin) and a narrow range of alleles of gyrA and parC (fluoroquinolone target genes). The virulence genotypes of the clade members resembled those of classic O25b:H4 ST131 isolates; representative isolates were variably lethal in a mouse subcutaneous sepsis model. Several pulsotypes spanned multiple sources (adults, children, pets, and human fecal samples) and locales. An analysis of recent clinical E. coli collections showed that the O16 ST131 clade is globally distributed, accounts for 1 to 5% of E. coli isolates overall, and, when compared with other ST131 isolates, it is associated with resistance to ampicillin, gentamicin, and trimethoprim-sulfamethoxazole and with susceptibility to fluoroquinolones and extended-spectrum cephalosporins. Attention to this O16-associated ST131 clade, which is facilitated by our novel PCR-based assay, is warranted in future epidemiological studies of ST131 and, conceivably, in clinical applications. E scherichia coli sequence type 131 (ST131), designated according to the Achtman multilocus sequence typing (MLST) system, has emerged dramatically over the past decade to become the single most prevalent human extraintestinal E. coli strain in many regions, especially among isolates resistant to fluoroquinolones and/or extended-spectrum cephalosporins (1-6). Although ST131 classically exhibits serotype O25b:H4, multiple studies have documented a minor subset of ST131 isolates with serotype O16:H5 or O-type O16 (7-12). This O16 subset within ST131 remains poorly characterized.Among the extended-spectrum -lactamase (ESBL)-producing E. coli isolates from Japan, the O16 ST131 subset was recently shown to represent a different Pasteur Institute sequence type (PST506) than classic O25b ST131 isolates (PST43) and to constitute a distinct phylogenetic clade (10, 11). Additionally, compared with classic O25b ST131 clade members, the studied O16 ST131 clade members exhibited a relatively greater prevalence of CTX-M-14 over (ST131-associated) CTX-M-15, were less commonly fluoroquinolone resistant but more commonly trimethoprimsulfamethoxazole resistant, and escaped detection by a widely used PCR...