Hitchhiking Behavior in Bacteriophages Facilitates Phage Infection and Enhances Carrier Bacteria Colonization

Abstract

Interactions between bacteriophages (phages) and biofilms remain poorly understood despite the broad implications for microbial ecology, water quality, and microbiome engineering. Here, we demonstrate that lytic coliphage PHH01 can hitchhike on carrier bacteria Bacillus cereus to facilitate its infection of host bacteria, Escherichia coli, in biofilms. Specifically, PHH01 could adsorb onto the flagella of B. cereus, and thus phage motility was increased, resulting in 4.36-fold more effective infection of E. coli in biofilm relative to free PHH01 alone. Moreover, phage infection mitigated interspecies competition and enhanced B. cereus colonization; the fraction of B. cereus in the final biofilm increased from 9% without phages to 43% with phages. The mutualistic relationship between the coliphage and carrier bacteria was substantiated by migration tests on an E. coli lawn: the conjugation of PHH01 and B. cereus enhanced B. cereus colonization by 6.54-fold compared to B. cereus alone (6.15 vs 0.94 cm 2 in 24 h) and PHH01 migration by 5.15-fold compared to PHH01 alone (10.3 vs 2.0 mm in 24 h). Metagenomic and electron microscopic analysis revealed that the phages of diverse taxonomies and different morphologies could be adsorbed by the flagella of B. cereus, suggesting hitchhiking on flagellated bacteria might be a widespread strategy in aquatic phage populations. Overall, our study highlights that hitchhiking behavior in phages can facilitate phage infection of biofilm bacteria, promote carrier bacteria colonization, and thus significantly influence biofilm composition, which holds promise for mediating biofilm functions and moderating associated risks.

Phage co-transport with hyphal-riding bacteria fuels bacterial invasion in water-unsaturated microbial ecosystems

“…Flagellar mobility hence seems to be a driver for efficient phage transport by bacteria. Although a few studies have reported on preferential adsorption of phages to bacterial flagella [19], we found similar efficiencies of T4 adsorption to flagellated WT and non-flagellated △filM (Fig S1). T4 adsorption to the bacterial cell surface rather than to flagella was also confirmed by HIM imaging.…”

“…Although described for aquatic environments [19,41,42], little is known on viral co-transport with non-host microorganisms in vadose habitats; even though adsorption and subsurface cotransport of nano-colloids with organic or biological materials has been described nearly two decades ago [43]. Here, we show that Escherichia virus T4 can adsorb to the hyphal-riding soil bacterium P. putida KT2440 (Fig.…”

“…T4 adsorption to and co-transport by carrier bacteria hence may therefore lead to conservative estimates for phage adsorption and co-transport. For instance, a recent study using Bacillus cereus as a model bacterium for waste water has shown its ability to adsorb phages of different morphologies from 10 viral families [19]. T4, furthermore, was also found to adsorb to the soil bacterium P. fluorescence Lp6a despite of its highly distinct physico-chemical surface properties [47] from P. putida KT2440 (Fig S1a).…”

“…as prophages) have been suggested to serve as agents of 'bacterial warfare' [22][23][24]. In aquatic environments, lytic phages adsorbed to bacteria have been shown to facilitate phage infection of biofilm bacteria and to promote biofilm colonization by carrier bacteria [19]. In unsaturated environments like soil however, little is known about co-transport of phage with motile bacteria and its effect on bacterial population dynamics.…”

Bacterial swarm-mediated phage transportation disrupts a biofilm inherently protected from phage penetration

“…Examples include the transport of nonmotile bacteria 22 and spores 23 by swimming bacteria, gliding bacteria 24 , and zooplankton 25 . Electron microscopy images have shown that phage particles bind to bacterial flagella 26 and imaging of colonies infected by the phage suggests hitchhiking 26,27 . Do non-flagellated but motile bacteria transport phage particles and can one directly visualize phage transportation?…”