Diversity and biofilm-production ability among isolates of Escherichia coli phylogroup D belonging to ST69, ST393 and ST405 clonal groups

BMC Microbiology Diversity and biofilm-production ability among isolates of Escherichia coli phylogroup D belonging to ST69, ST393 and ST405 clonal groups ngela Novais 0 3 Claudia Vuotto 2 Joo Pires 0 3 Carolina Montenegro 0 3 Gianfranco Donelli 2 Teresa M Coque 1 Lusa Peixe 0 3 0 Departamento de Microbiologia, REQUIMTE, Faculdade Farmacia Universidade Porto , Rua Jorge Viterbo Ferreira no 228, 4050-313 Porto, Porto , Portugal 1 Servicio de Microbiologia and CIBER en Epidemiologia y Salud Publica (CIBERESP), Instituto Ramon y Cajal de Investigacion Sanitaria (IRYCIS), Hospital Universitario Ramon y Cajal , Madrid , Spain 2 Microbial Biofilm Laboratory (LABIM), IRCCS Fondazione Santa Lucia , Rome , Italy 3 Departamento de Microbiologia, REQUIMTE, Faculdade Farmacia Universidade Porto , Rua Jorge Viterbo Ferreira no 228, 4050-313 Porto, Porto , Portugal Background: Phylogenetic group D Escherichia coli clones (ST69, ST393, ST405) are increasingly reported as multidrug resistant strains causing extra-intestinal infections. We aim to characterize inter- and intraclonal diversity of a broad sample (isolates from different geographic locations and origins with variable antibiotic resistance profiles, 1980-2010) and their ability to adhere and form biofilm by both a modified quantitative biofilm producing assay and Field Emission Scanning Electron Microscopy (FESEM). Results: High virulence scores were observed among ST69 (median 14/range 9-15) and ST393 (median 14/range 8-15) clones, particularly enriched in pap alleles, iha, kpsMTII-K5 and ompT, in contrast with ST405 (median 6/range 2-14) isolates, exhibiting frequently fyuA, malX and traT. All ST69 and ST393 and only two ST405 isolates were classified as ExPEC. Biofilm production was detected in two non-clinical ST69 and three ST393 isolates from different origins showing variable virulence profiles. Within each clonal group, and despite the high diversity of PFGE-types observed, isolates from different countries and recovered over large periods of time were clustered in a few groups sharing common virulence gene profiles among ST69 (n = 10 isolates) and ST393 (n = 9 isolates) (fimH-iha-iutA-kpsMTII-K5-(traT)-sat-(ompT)-papA-papEF-papGII-papC) or ST405 (n = 6 isolates) (fimH-traT-fyuA-malX). Conclusions: This study highlights the circulation of highly transmissible ST69, ST393 and ST405 variants among different settings. Biofilm production seems not to be directly correlated with their epidemiological success. ExPEC; High-risk clones; ESBL; Virulence; Adhesion; Biofilm - Background Multidrug resistant Escherichia coli clones of the phylogenetic group D causing extraintestinal human infections are increasingly reported all over the world [1-4]. Among them, E. coli clonal groups D-ST69 (also recognized as clonal group A or CGA) and D-ST393 (also known as O15:K52:H1 clonal group) are widely spread among different hosts, often causing urinary tract infections (UTI) and conferring resistance to antibiotics [5-10]. Isolates belonging to the D-ST405 have been involved in the spread of genes encoding extended spectrum -lactamases (ESBLs) (mainly CTX-M enzymes), cephamycinases (AmpC), carbapenemases (NDM) or methylases (AmrA, RmtB) [2,11-15]. Recent studies have reported variants of ST69, ST393 and ST405 among ESBL and non-ESBL-producing strains from specific locations [4,5,8,9,13,16]. The presence of genes possibly involved in biofilm production detected in some of these surveys (fimH, papC, papG, fyuA or kpsMT II) suggests the ability of these clones to adhere and form biofilm, which could be favouring their persistence; however, such ability has not been specifically evaluated [5,8,13,17,18]. Biofilm growing ability of bacteria is commonly assessed by a quantitative measure of their adherence to microtiter plates, although electron microscopy analyses provide more accurate information on the biofilm structure and presence of matrix [17-19]. In this study, we aim to characterize the intraclonal diversity of extraintestinal pathogenic E. coli (ExPEC) isolates from phylogenetic group D (ST69, ST393, ST405) isolated from different geographic locations and sources, and to assess their ability to adhere and form biofilm on abiotic surfaces in order to evidence a possible contribution of biofilm formation to their persistence and epidemicity. Methods Bacterial isolates We analysed thirty-five E. coli isolates belonging to ST69 (n = 13), ST393 complex (10 ST393, 1 ST2321) and ST405 complex (10 ST405, 1 ST964) isolated from multiple sources and countries. They include either isolates associated with nosocomial or community outbreaks in different countries [2,4,9,12,20] or isolates collected from non-clinical sources from distinct countries and showing variable antibiotic resistance profiles, selected from published papers by the end of 2010. They were recovered from nosocomial (66%) and community-acquired infections (17%), healthy volunteers (8%), food products (6%) or animals (3%) and produced diverse ESBL or AmpC enzymes. Their epidemiological features are shown in Table 1. Isolates were taken as part of standard patient care and no ethical approval was required for their use. Clonal diversity Relatedness among isolates was established by XbaIpulsed-field gel electrophoresis (PFGE), multi-locus sequence typing (MLST, http://mlst.ucc.ie/mlst/dbs/Ecoli), and identification of E. coli phylogenetic groups and serogroups by PCR [28]. Isolates exhibiting 85% homology were considered to belong to the same PFGE-type. XbaI-profiles were compared using InfoQuest FP version 5.4 software (BioRad Laboratories), by applying the UPGMA algorithm based on the Dice coefficient (1.0% band tolerance; 1.0% optimization). Virulence genes profile Screening of 38 virulence factors (VFs) including adhesins, toxins, siderophores, polysaccharide coatings and others (malX, usp, ibeA, iss, tsh) presumptively associated with ExPEC isolates was performed by PCR as previously described [8,28]. The Fishers exact test was used for each comparison, a p value <0.05 being considered to reveal significant differences. A strain satisfied the criteria for being ExPEC if it carried two or more of the following genes: papA, papC, sfa/focDE, afa/draBC, iutA and kpsMII [8]. Adhesion and biofilm-producing assays The ability of D-E.coli strains to in vitro adhere was investigated by a modified quantitative biofilm production assay, as previously described [28]. The E. coli strain CFT073 and the culture medium supplemented with 1% (v/v) glucose were used as positive and negative controls, respectively. Assays were performed in quintuplicate and repeated at least 4 times. The cut-off optical density (ODc) was defined as three standard deviations above the mean OD of the negative control (culture medium), and strains were classified as non-adherent (OD ODc), weakly adherent (ODc < OD 2 ODc), moderately adherent (2 ODc < OD 4 ODc), or strongly adherent (OD > 4 ODc). (...truncated)