Rapid and specific detection, molecular epidemiology, and experimental virulence of the O16 subgroup within Escherichia coli sequence type 131

Abstract

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 PCR-based 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.

FIG 1

Allele-specific PCR for detection of the ST131-O16 and ST131-O25b clades. (A)), clade-specific PCR; (B), PCR O typing. M, molecular weight marker (1 Kb Plus DNA Ladder; Invitrogen). (A and B) Upper band corresponds to the amplification of the internal control uidA gene. (A) Middle band and lower bands correspond to allele-specific amplifications of the ST131-O25b clade pabB and ST131-O16 clade trpA variants, respectively. (B) Middle band and lower bands correspond to allele-specific amplifications of the O16 and O25b rfb regions, respectively. (A and B) Lanes 1 to 4, strains JJ30, JJ38, JJ59, and SE15 (all ST131-O16 clade, O16 type), respectively; lanes 5 and 6, strains JJ14 and JJ20 (both ST131-O25b clade, O nontypeable), respectively; lanes 7 and 8, strains TN03 and TU (both ST131-O25b clade, O25b type), respectively; lane 9, strain ECOR66 (non-ST131, O4 type); lane 10, strain LBC24a (non-ST131, O43 type), noting the amplification of the ST131-O16 clade trpA gene due to presumed horizontal gene transfer; lane 11, strain M1139 (non-ST131, O16 type).

FIG 2

Phylogenetic tree of 36 E. coli strains from phylogenetic group B2, subgroup I. Subgroup I of phylogenetic group B2 was defined as in Le Gall et al. (16). The tree was inferred from the DNA sequences of 12 housekeeping genes from the Achtman and Pasteur Institute schemes (8,928 nucleotides), using the maximum likelihood procedure. E. coli strain CFT073 (group B2, subgroup II [16]) served as an outgroup. Bootstrap values of >50% are indicated adjacent to the nodes. The strains in bold correspond to sequenced strains. PST, Pasteur Institute sequence type (ST). The other ST labels correspond to the Achtman MLST scheme.