Coaggregation occurs amongst bacteria within and between biofilms in domestic showerheads

Abstract

Showerheads support the development of multi-species biofilms that can be unsightly, produce malodor, and may harbor pathogens. The outer-surface spray-plates of many showerheads support visible biofilms that likely contain a mixture of bacteria from freshwater and potentially from human users. Coaggregation, a mechanism by which genetically distinct bacteria specifically recognize one another, may contribute to the retention and enrichment of different species within these biofilms. The aim of this work was to describe the bacterial composition of outer spray-plate biofilms of three domestic showerheads and to determine the intra- and inter-biofilm coaggregation ability of each culturable isolate. The bacterial composition of the three biofilms was determined by using bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP) and by culturing on R2A medium. An average of 31 genera per biofilm were identified using bTEFAP and a total of 30 isolates were cultured. Even though the microbial diversity of each showerhead biofilm differed, every cultured isolate was able to coaggregate with at least one other isolate from the same or different showerhead biofilm. Promiscuous coaggregating isolates belonged to the genera Brevundimonas, Micrococcus, and Lysobacter. This work suggests that coaggregation may be a common feature of showerhead biofilms. Characterization of the mechanisms mediating coaggregation, and the inter-species interactions they facilitate, may allow for novel strategies to inhibit biofilm development.

Fig. 1

Neighbor-joining phylogenetic tree of the partial 16S rRNA gene sequences from isolates cultured from the three showerhead biofilms. Colors relate to the showerhead biofilm from which the isolate was cultured. Scale bar represents one substitution for every 10 nucleotides. The outgroup sequence is from Thermus thermophilus (accession number AJ251638).

Fig. 2

Representative confocal laser scanning microscope images showing the ability of two autoaggregating species to coaggregate. Cells have been falsely colored and surface rendered using Imaris® Surpass to aid differentiation of cells in coaggregates. Cell suspensions of (A) M. hispanicum AH007 and, (B) M. trichothecenolyticum HM016, and (C) coaggregating autoaggregates of M. hispanicum AH007 and M. trichothecenolyticum HM016 are shown. Bar represents 15 μm.

Fig. 3

Representative confocal laser scanning microscope images from multiple experiments demonstrating the interdigitated nature of coaggregation between three showerhead biofilm species that coaggregated with one another. Cells have been falsely colored and surface rendered using Imaris® Surpass to aid differentiation of cells in coaggregates. Cell suspensions of (A) M. luteus AH004, (B) B. lenta HM006, (C) L. gummosus HM010, and coaggregates of (D) B. lenta HM006 and L. gummosus HM010 (visual score of 4), (E) M. luteus AH004 and B. lenta HM006 (visual score of 4), and (F) M. luteus AH004 and L. gummosus HM010 (visual score of 2). Bar represents 15 μm.

Fig. 4

A diagrammatic representation of the inter- and intra-biofilm specificity of coaggregation between the showerhead biofilm isolates after growth in batch culture for 48h. Visual coaggregation scores <2 are not shown. Colors highlight the showerhead from which the isolates were actually harvested. Cells are not to scale and visual scores are depicted as connecting lines of different thickness. Thickest line (⚊) represents a visual coaggregation score of 4, line of intermediate thickness represents score of 3 (―), and thinnest dotted line (----) represents a score of 2.