Aeromonas flagella (polar and lateral) are enterocyte adhesins that contribute to biofilm formation on surfaces

Abstract

Aeromonas spp. (gram-negative, aquatic bacteria which include enteropathogenic strains) have two distinct flagellar systems, namely a polar flagellum for swimming in liquid and multiple lateral flagella for swarming over surfaces. Only approximately 60% of mesophilic strains can produce lateral flagella. To evaluate flagellar contributions to Aeromonas intestinal colonization, we compared polar and lateral flagellar mutant strains of a diarrheal isolate of Aeromonas caviae for the ability to adhere to the intestinal cell lines Henle 407 and Caco-2, which have the characteristic features of human intestinal enterocytes. Strains lacking polar flagella were virtually nonadherent to these cell lines, while loss of the lateral flagellum decreased adherence by approximately 60% in comparison to the wild-type level. Motility mutants (unable to swim or swarm in agar assays) had adhesion levels of approximately 50% of wild-type values, irrespective of their flagellar expression. Flagellar mutant strains were also evaluated for the ability to form biofilms in a borosilicate glass tube model which was optimized for Aeromonas spp. (broth inoculum, with a 16- to 20-h incubation at 37 degrees C). All flagellar mutants showed a decreased ability to form biofilms (at least 30% lower than the wild type). For the chemotactic motility mutant cheA, biofilm formation decreased >80% from the wild-type level. The complementation of flagellar phenotypes (polar flagellar mutants) restored biofilms to wild-type levels. We concluded that both flagellar types are enterocyte adhesins and need to be fully functional for optimal biofilm formation.

FIG. 1.

Adherence of A. caviae strain Sch3 wild-type and flagellar mutant strains to Caco-2 cells (May-Grünwald- and Giemsa-stained coverslip culture cell monolayers). (A) Wild-type strain (Fla⁺ Laf⁺); (B) lateral flagellin double mutant strain AAR6 (Fla⁺ Laf⁻); (C) polar flagellin double mutant strain AAR31 (Fla⁻ Laf^±); (D) complemented mutant strain AAR31C (Fla⁺ Laf⁺). Bars, ∼10 μm.

FIG. 2.

Biofilm development on borosilicate glass (37°C) by the A. caviae strain Sch3 wild type (•) and flagellar mutant strains AAR6 (Fla⁺ Laf⁻) (▪) and AAR31 (Fla⁻ Laf^±) (▴) over time. Values shown are the means of three replicate tubes ± SD from a representative experiment. The values at 18 h were significantly different from that of the wild-type strain (P < 0.01) for both mutant strains.

FIG. 3.

Biofilm formation on borosilicate glass by polar (Fla) (A) and lateral (Laf) (B) flagellar mutant strains (18 h at 37°C). Each histogram represents the means ± SD of four experiments, with three to six replicate tubes per strain evaluated in each experiment. NM, nonmotile in swimming and swarming agar assays.