An update on faecal carriage of ESBL-producing Enterobacteriaceae by Portuguese healthy humans: detection of theH30 subclone of B2-ST131Escherichia coliproducing CTX-M-27: Table 1.

Sir, Multiple studies have documented increasing rates of intestinal colonization by ESBL-producing Enterobacteriaceae (ESBL-E) in the community, with remarkable heterogeneity according to the geographical region [Asia (2%–70%) and Africa (7%–31%) versus Europe (2%–12%)].1 CTX-M enzymes (mainly CTX-M-15 or CTX-M-14) are predominant and observed mainly in diverse Escherichia coli genetic backgrounds.1 However, detailed characterization of E. coli isolates at clonal and subclonal levels is missing, which is important to understand ecological and host colonization differences of specific pathogenic and/or MDR E. coli lineages.2,3 In Portugal, a study conducted in healthy humans 10 years ago revealed an ESBL-E carriage rate of 1.8%.4 In the context of current endemicity of ESBLs (especially CTX-M-15) and emergence of different carbapenemases in clinical settings,5,6 we aimed to assess the current faecal carriage rate and provide detailed molecular data (clonal and plasmid subtyping) of ESBL- and/or carbapenemase-producing Enterobacteriaceae in Portuguese healthy humans. Faecal samples from 199 randomly selected healthy humans (aged >18 years) living in northern, central and southern regions of Portugal were collected between December 2013 and May 2014. All participants provided written informed consent and completed a standardized questionnaire (Table S1, available as Supplementary data at JAC Online). Samples (200 μL of a 2 mL saline suspension of the rectal swab) were seeded onto CHROMagar™ Orientation plates supplemented with vancomycin (4 mg/L) plus ceftazidime (1 mg/L) or ertapenem (0.25 mg/L) and incubated (37°C for 24 h). All presumptive Enterobacteriaceae isolates (oxidase negative, approximately one to five unique morphotypes per plate) were further characterized. ESBL or carbapenemase production was assessed by the double-disc synergy test or Blue-Carba, respectively, and further characterized by PCR and sequencing.5 Susceptibility to non-β-lactam antibiotics was determined by the disc diffusion method (http://www.eucast.org/clinical_breakpoints/) and presumptive E. coli ESBL producers were confirmed by species-specific PCR. Population structure analysis performed on representative ESBL-producing E. coli (according to morphotype and antibiotic susceptibility pattern) included identification of E. coli phylogroups and virulence gene markers [extraintestinal E. coli and enteroaggregative E. coli (EAEC)] by PCR, XbaI-PFGE, MLST (http://mlst.warwick.ac.uk/mlst/dbs/Ecoli)5,7,8 and subclonal typing of B2-ST131 isolates (screening of O25b:H4, O16:H5, fimHTR allele and Rx clade by PCR or PCR and sequencing).5,7,9 Plasmid analysis included replicon typing and subtyping (IncF plasmids), S1/I-CeuI-PFGE and hybridization (http://pubmlst.org/plasmid/primers/incF.shtml).4