Epidemic population structure of extraintestinal pathogenic Escherichia coli determined by single nucleotide polymorphism pyrosequencing Natalia Fernández-Romero, María Pilar Romero-Gómez, María Rosa Gómez-Gil, Jesús Mingorance ⇑ Servicio de Microbiología, Hospital Universitario La Paz, IdiPAZ, Paseo de La Castellana, 261, Madrid 28046, Spain article info Article history: Received 23 February 2011 Received in revised form 10 June 2011 Accepted 16 June 2011 Available online 23 June 2011 Keywords: Bacterial population structure SNP MLST Escherichia coli Bacteremia Urinary tract infection abstract We have developed an MLST-based scheme for typing Escherichia coli isolates using pyrosequencing of single nucleotide polymorphic positions (SNP). The SNP sequences are converted into allelic patterns and analyzed using the same approach used for MLST analyses. We have tested the method in two unse- lected collections of clinical isolates of E. coli obtained from blood and urine cultures. The two collections had a similar structure, 25% of the profiles (representing 68% of the isolates) were common to both, and 62% of the profiles (nearly 20% of the isolates) were unique. The four major profiles accounted for 44% of the isolates, and among these the most frequent one was related to the pandemic ST131 clone. The method is easy to implement and might be useful for typing large microbial collections. Ó 2011 Elsevier B.V. All rights reserved. 1. Introduction Escherichia coli is a commensal of the vertebrate gut and an occasional pathogen. In humans it may produce diverse intestinal infections, it is the most frequent causative agent of urinary infec- tions, and one of the most frequent causes of bacteremia and sepsis (Kaper et al., 2004). Due to its prominent role as a model organism and a pathogen, the diversity of infections involved, and the com- mensal–pathogen duality it is a good model to study the emer- gence and the evolution of pathogens (Tenaillon et al., 2010). The genetic diversity of clinical and environmental E. coli iso- lates has been investigated using a variety of techniques. Multilo- cus enzyme electrophoresis (MLEE) has shown a clonal population structure with four major phylogenetic groups (Selander et al., 1987). This result has been refined later on by multilocus sequence typing analyses (MLST) that confirmed the main features of the MLEE analyses, and identified two new groups, E and F (Wirth et al., 2006; Touchon et al., 2009; Clermont et al., 2011). The scheme defined by MLEE was subsequently incorporated into a multiplex PCR that has become popular because of its simplicity (Clermont et al., 2000), though it has some weak points (Wirth et al., 2006; Gordon et al., 2008). In spite of the large amount of work done some aspects of the evolutionary history and popula- tion structure of E. coli are still controversial, with some studies detecting low levels of recombination (Ochman and Selander, 1984; Reid et al., 2000; Feil et al., 2001; Pérez-Losada et al., 2006) and others supporting a predominant role of recombination over mutation (Mau et al., 2006; Touchon et al., 2009). Comparisons of the whole genome sequences of a few selected strains, mostly human pathogens, show that the E. coli genome consists of a conserved core of about 2000 genes and a variable fraction of roughly 18,000 genes (Rasko et al., 2008; Touchon et al., 2009; Lukjancenko et al., 2010). The distribution of con- served and variable genes suggest that the genomes have evolved according to an open genome model in which recombination and gene exchange play a predominant role. From the epidemiological standpoint it is of interest to find out if there are relations between the phylogenetic groups and their pathogenicity. The pathogenic types, also called pathotypes, are groups of strains with well-defined and differentiated pathogenic characteristics. Several E. coli pathotypes have been defined, including at least six different intestinal pathotypes, a generic group that includes most extraintestinal pathogens (ExPEC), and the commensal (non-pathogenic) type (Kaper et al., 2004). The commensal strains and the intestinal pathogens tend to belong to phylogenetic groups A and B1, while the extra-intestinal patho- gens are more frequently found in groups B2 and D (Picard et al., 1999; Moulin-Schouleur et al., 2007; but see also Bailey et al., 2010). The different intestinal pathotypes are formed by clonal or closely related isolates, and carry specific groups of virulence genes and pathogenicity islands (Kaper et al., 2004). The ExPECs are usu- ally harmless part of the normal intestinal flora, and occasional 1567-1348/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.meegid.2011.06.010 ⇑ Corresponding author. Tel.: +34 91 2071364; fax: +34 91 727 7372. E-mail addresses: jmingorancec.hulp@salud.madrid.org, jesus.mingorance@idi- paz.es (J. Mingorance). Infection, Genetics and Evolution 11 (2011) 1655–1663 Contents lists available at ScienceDirect Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid