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

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.

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.

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.

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 Fisher's 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). The ultrastructural analysis of biofilm was performed by a Field Emission Scanning Electron Microscope (FESEM) (Zeiss, Germany). Briefly, adjusted inocula (200 μl, 0.5 McF) of each strain diluted with 1.8 ml of fresh LB supplemented with 1% (v/v) glucose were added to 24-well plates with round glass coverslips (1 cm diameter) put into each well and incubated at 37°C for 24 h. The content of each well was removed and the round coverslips were washed with PBS (1%) twice. Biofilms grown on coverslips were fixed with 2,5% glutaraldehyde in Na-cacodylate 0,1 M (pH 7.4) buffer solution (AppliChem, Germany) for 2 h at room temperature. Following three washing steps with the same buffer solution, samples were dehydrated through graded ethanol (30°, 50°, 70°, 85°, 95°, 100°) and dried with hexamethyldisilazane (Alfa Aesar, USA) for 1 h30'. Samples were air dried overnight and coated by sputtering with a gold target [19].

Intraclonal diversity of ST69 isolates
Thirteen isolates corresponding to 7 PFGE types were classified in different serogroups (O11, O17, O73, O77), and clustered in two groups on the basis of the similarity of the XbaI restriction profiles. Cluster I comprised closely related isolates (n = 10, 73.8% homology) causing hospital or  community acquired infections that exhibited a common virulence gene profile (80%, fimH-iha-iutA-kpsMTII-K5-traT-sat-ompT-papA-papEF-papGII-papC). Cluster II (n = 3, 71.8% homology) included two indistinguishable isolates recovered from different samples of ready-to-eat salads in Portugal and from poultry meat in Norway. They differ in the presence of iroN, iss, bmaE (n = 2/3) and gafD (n = 2/3), and the lack of iha, sat and papGII, observed for isolates of cluster I. All ST69 isolates exhibited resistance to streptomycin and trimethoprim-sulfamethoxazole, and they were frequently resistant to tetracycline (85%), and to chloramphenicol (46%). None of the isolates produced ESBL, but one encoded CMY-2.
Isolates belonging to cluster I seem to have been circulating among different continents since at least 1999, as reflects this and other studies [31][32][33]. Despite of the small sample analysed, differences among ST69 isolates from human and non-human origins suggest independent evolution of particular E. coli variants in different hosts.
The study highlights the spread of ST393 isolates of biotype C with highly similar virulence gene profile in different continents over almost three decades, supporting previous observations in specific countries [5,8]. Unfortunately, clonal relatedness among different strains could not be analysed due to the spontaneous lysis of DNA, also reported by other groups [6,34].

Conclusion
Factors responsible for the increased ability of particular E. coli clones to successfully spread and persist are poorly understood, and our work represents one of the few studies exploring the phenotypic traits involved in the increased epidemicity of emerging antibiotic resistant E. coli clonal groups [28,36]. The results highlight the inter and intraclonal diversity of E. coli clones of phylogroup D and further suggest the circulation of highly transmissible ST69, ST393 and ST405 variants, some of them being particularly widespread in different geographic areas and settings. The lack of association between the ability to produce biofilm exhibited by a few strains and specific virulence gene or virulence gene profiles points out the need to further explore factors involved in the selection of particular epidemic variants with enhanced ability to colonize and persist for extended periods of time.