Diarrhea-associated biofilm formed by enteroaggregative Escherichia coli and aggregative Citrobacter freundii: a consortium mediated by putative F pili

Loreny G Giugliano,Ana Cmm Gomes,Ana Cg Araújo,Alex L Pereira,Thiago N Silva

doi:10.1186/1471-2180-10-57

Loreny G Giugliano, Ana Cmm Gomes + Show 3 more

Open Access

https://doi.org/10.1186/1471-2180-10-57

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Abstract

BackgroundEnteroaggregative Escherichia coli (EAEC) are enteropathogenic strains identified by the aggregative adhesion (AA) pattern that share the capability to form biofilms. Citrobacter freundii is classically considered as an indigenous intestinal species that is sporadically associated with diarrhea.ResultsDuring an epidemiologic study focusing on infantile diarrhea, aggregative C. freundii (EACF) and EAEC strains were concomitantly recovered from a severe case of mucous diarrhea. Thereby, the occurrence of synergic events involving these strains was investigated. Coinfection of HeLa cells with EACF and EAEC strains showed an 8-fold increase in the overall bacterial adhesion compared with single infections (P < 0.001). The synergic effect was mediated by physical interactions among the bacteria and primed in the absence of chemical signaling and without the participation of host cells. Thus, significant increases (2.7-fold on average) in bacterial adhesion were also observed during the formation of mixed biofilms on abiotic surfaces. Bacterial settling assays showed that EAEC strains harboring F-pili genes (traA) were capable of forming bacterial aggregates only in the presence of EACF. Scanning electronic microscopy analyses revealed that bacterial aggregates as well as enhanced biofilms formed by EACF and traA-positive EAEC were mediated by non-bundle forming, flexible pili. Moreover, mixed biofilms formed by EACF and traA-positive EAEC strains were significantly reduced using nonlethal concentration of zinc, a specific inhibitor of F pili. In addition, EAEC strains isolated from diarrheic children frequently produced single biofilms sensitive to zinc.ConclusionsPutative F pili expressed by EAEC strains boosted mixed biofilm formation when in the presence of aggregative C. freundii.

Highlights

Enteroaggregative Escherichia coli (EAEC) are enteropathogenic strains identified by the aggregative adhesion (AA) pattern that share the capability to form biofilms
Adherence assays showed that strain C. freundii strain 205 (Cf205) displayed a mannose-resistant AA phenotype (Figure 1A) indistinguishable to that developed by EAEC prototype strain 042 (Figure 1C)
The EACF 205 population adhered to HeLa cells and inactivated by antibiotics still held the capability to boost the adhesion of the EAEC strain 3401 (Figure 3, frame B). These results showed that the increase in the bacterial adherence developed by EACF 205-EAEC combinations were supported by physical interactions, which were triggered by EAEC strains, independently of chemical signals or the influence of host cells

Summary

Introduction

Enteroaggregative Escherichia coli (EAEC) are enteropathogenic strains identified by the aggregative adhesion (AA) pattern that share the capability to form biofilms. Enteroaggregative E. coli (EAEC) is defined by its adhesion to cultured cells in a stacked brick-like formation [1]. This phenotype, termed aggregative adherence (AA), is mediated by specific fimbriae (AAF) encoded by plasmids (pAA) [2,3]. Elucidation of the mechanisms involved in EAEC pathogenesis has been limited because of the heterogeneity displayed by wild-type strains [6,11]. Given this genetic heterogeneity, expression of biofilms has been considered a consensual virulence factor among EAEC isolates [1,12,13]. An AAF-independent mechanism for biofilm formation, which is mediated by plasmid-encoded type IV pili, was described in the atypical EAEC strain C1096 [14]

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