Aims:  In this study, we have evaluated the impact of methodological approaches in the determination of biofilm formation by four clinical isolates of Escherichia coli in static assays. Methods and Results:  The assays were performed in microtitre plates with two minimal and two enriched broths, with one‐ or two‐steps protocol, and using three different mathematical formulas to quantify adherent bacteria. Different biofilm formation patterns were found depending on the E. coli strain, culture medium and reading optical density on one‐ and two‐steps protocol. Strong or moderate biofilm formation occurred mostly in minimal media. The mathematical formulas used to quantify biofilm formation also gave different results and bacterial growth rate should be taken into account to quantify biofilm. Conclusions:  Escherichia coli forms biofilms on static assays in a method‐dependent fashion, depending on strain, and it is strongly modulated by culture conditions. Significance and Impact of the Study:  As verified in the studied E. coli strains, biofilm formation by any organism should be cautiously interpreted, considering all variables in the experimental settings.

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BackgroundCrohn's disease (CD) is a high morbidity chronic inflammatory disorder of unknown aetiology. Adherent-invasive Escherichia coli (AIEC) has been recently implicated in the origin and perpetuation of CD. Because bacterial biofilms in the gut mucosa are suspected to play a role in CD and biofilm formation is a feature of certain pathogenic E. coli strains, we compared the biofilm formation capacity of 27 AIEC and 38 non-AIEC strains isolated from the intestinal mucosa. Biofilm formation capacity was then contrasted with the AIEC phenotype, the serotype, the phylotype, and the presence of virulence genes.ResultsSpecific biofilm formation (SBF) indices were higher amongst AIEC than non-AIEC strains (P = 0.012). In addition, 65.4% of moderate to strong biofilms producers were AIEC, whereas 74.4% of weak biofilm producers were non-AIEC (P = 0.002). These data indicate that AIEC strains were more efficient biofilm producers than non-AIEC strains. Moreover, adhesion (P = 0.009) and invasion (P = 0.003) indices correlated positively with higher SBF indices. Additionally, motility (100%, P < 0.001), H1 type flagellin (53.8%, P < 0.001), serogroups O83 (19.2%, P = 0.008) and O22 (26.9%, P = 0.001), the presence of virulence genes such as sfa/focDE (38.5%, P = 0.003) and ibeA (26.9%, P = 0.017), and B2 phylotype (80.8%, P < 0.001) were frequent characteristics amongst biofilm producers.ConclusionThe principal contribution of the present work is the finding that biofilm formation capacity is a novel, complementary pathogenic feature of the recently described AIEC pathovar. Characterization of AIEC specific genetic determinants, and the regulatory pathways, involved in biofilm formation will likely bring new insights into AIEC pathogenesis.