dEscherichia coli sequence types (STs) 69, 73, 95, and 131 are collectively responsible for a large proportion of E. coli urinary tract and bloodstream infections, and they differ markedly in their antibiotic susceptibilities. Here, we describe a novel PCR method to rapidly detect and distinguish these lineages. Three hundred eighteen published E. coli genomes were compared in order to identify signature sequences unique to each of the four major STs. The specificities of these sequences were assessed in silico by seeking them in an additional 98 genomes. A PCR assay was designed to amplify size-distinguishable fragments unique to the four lineages and was validated using 515 E. coli isolates of known STs. Genome comparisons identified 22 regions ranging in size from 335 bp to 26.5 kb that are unique to one or more of the four predominant E. coli STs, with two to 10 specific regions per ST. These regions predominantly harbor genes encoding hypothetical proteins and are within or adjacent to prophage sequences. Most (13/22) were highly conserved (>96.5% identity) in the genomes of their respective ST. The new assay correctly identified all 142 representatives of the four major STs in the validation set (n ‫؍‬ 515), with only two ST12 isolates misidentified as ST95. Compared with MLST, the assay has 100% sensitivity and 99.5% specificity. The rapid identification of major extraintestinal E. coli STs will benefit future epidemiological studies and could be developed to tailor antibiotic therapy to the different susceptibilities of these dominant lineages. Extraintestinal pathogenic Escherichia coli (ExPEC) strains are frequent pathogens, causing infections spanning a great range of severity (1, 2). They are responsible for 70 to 90% of acute community-acquired uncomplicated urinary infections, 85% of asymptomatic bacteriuria cases, and Ͼ60% of recurrent cystitis infections (3). E. coli is also one of the major pathogens of bloodstream infections, with mortality rates of 10 to 30%; in the United Kingdom, it has been the most common cause of bacteremia in most years since 1990, showing year-on year increases and now accounting for almost one-third of all bacteremias (see www.hpa .org.uk) (4). Successful treatment has been complicated by a rise in the prevalence of antibiotic-resistant strains.DNA profiling, e.g., by multilocus sequence typing (MLST), has advanced our understanding of ExPEC lineages, and several international studies have reported the predominance of sequence types (STs) 69, 73, 95, and 131 among large collections of ExPEC from human infections (5-8). In the United Kingdom, recent regional studies reported the consistent prevalences of these four STs among ExPEC from urinary and bloodstream infections. Collectively, they comprised 45% of the ExPEC strains from community and hospital urine samples recovered in 2007 to 2009 and 2007 to 2008 in Northwest (NW) England and the East Midlands, respectively, as well as 58% of those from bacteremias in northern England in 2010 to 2012 (9-11). The antibiotic ...

Amplification and sequence analysis of the 16S rRNA gene can be applied to detect and identify bacteria in clinical samples. We examined 75 clinical samples (17 culture-positive, 58 culturenegative) prospectively by two different PCR protocols, amplifying either a single fragment (1343 bp) or two fragments (762/598 bp) of the 16S rRNA gene. The 1343 bp PCR and 762/ 598 bp PCRs detected and identified the bacterial 16S rRNA gene in 23 (31 %) and 38 (51 %) of the 75 samples, respectively. The 1343 bp PCR identified 19 of 23 (83 %) PCR-positive samples to species level while the 762/598 bp PCR identified 14 of 38 (37 %) bacterial 16S rRNA gene fragments to species level and 24 to the genus level only. Amplification of shorter fragments of the bacterial 16S rRNA gene (762 and 598 bp) resulted in a more sensitive assay; however, analysis of a large fragment (1343 bp) improved species discrimination. Although not statistically significant, the 762/598 bp PCR detected the bacterial 16S rRNA gene in more samples than the 1343 bp PCR, making it more likely to be a more suitable method for the primary detection of the bacterial 16S rRNA gene in the clinical setting. The 1343 bp PCR may be used in combination with the 762/598 bp PCR when identification of the bacterial rRNA gene to species level is required.

The objective of this study was to develop and validate an expanded multiplex PCR assay for the simultaneous detection of eight plasmid-mediated quinolone-resistance determinants in Enterobacteriaceae. Primers were designed to amplify conserved fragments of qnrABCDS, qepA, oqxAB and aac(69)-Ib-cr genes and were optimized in uniplex and multiplex PCR assays with control template DNA. The assay was used to determine the prevalence of plasmid-mediated quinolone resistance (PMQR) genes in 174 ciprofloxacin-resistant and 43 ciprofloxacinsusceptible extraintestinal pathogenic Escherichia coli isolates. Each resistance gene could be detected alone and in combination. PMQR determinants were detected in 65 ciprofloxacinresistant isolates (37 %) and one ciprofloxacin-susceptible isolate (2 %). Prevalences of the identified determinants were: aac(69)-Ib-cr, 34.5 %; qnrS, 1.1 %; qepA, 1.1 %; and oqxAB, 0.6 %. In conclusion, we developed an eight-target multiplex PCR for the accurate detection of PMQR genes and confirmed that PMQR prevalence remains low among human Escherichia coli clinical isolates in the UK. INTRODUCTIONResistance to fluoroquinolones in Enterobacteriaceae is most commonly mediated by mutations in the DNA gyrase and topoisomerase IV genes, gyrA and parC, which are responsible for binding fluoroquinolones during DNA replication (Pan et al., 1996). Plasmid-mediated quinolone resistance (PMQR) genes are alternative mediators of quinolone resistance or reduced susceptibility that encode DNA gyrase protection proteins (qnrA, B, C, D and S), efflux pumps (oqxAB and qepA) and a variant of a common aminoglycoside-modifying enzyme [aac(69)-Ib-cr]. These proteins reduce the susceptibility of Enterobacteriaceae to quinolones, while oqxAB and aac(69)-Ib-cr also reduce susceptibility to chloramphenicol and aminoglycosides, respectively (Strahilevitz et al., 2009).In some geographical regions, including Argentina, the USA and Southeast Asia, PMQR determinants have been reported at high prevalence in both human and animal isolates and are often associated with the expression of extended-spectrum b-lactamases (ESBLs) (Andres et al., 2013; Deepak et al., 2009;Liu et al., 2013;Park et al., 2006;Shaheen et al., 2013;Zhang et al., 2012). By contrast, most PMQR determinants have been rarely found in the UK, with only a few reports of qnrA, qnrB and qnrS genes in Enterobacteriaceae isolated from humans and cattle with infections (Corkill et al., 2005;Hopkins et al., 2007;Kirchner et al., 2011;Younes et al., 2011). The aac(69)-Ib-cr allele is more common, however, and is often found on IncFII plasmids in conjunction with the gene encoding the cefotaxime (CTX)-M-15 ESBL (Amin & Wareham, 2009 PMQR multiplex PCR assay. PCR was performed using the Qiagen Multiplex PCR kit for amplification and resolution of all targets. PMQR primers used in this multiplex PCR are listed in Table 2. Thermocycling conditions were as follows: initial denaturation at 95 uC for 15 min; 30 cycles of 94 uC for 30 s, optimized annealing temperature for 90 s ...