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Divvying up the labor: subpopulation development during biofilm maturation

The changes that occur within the bacterial community as the biofilm matures modulate outcomes of biofilm-associated infection within the human host. Phenotypic diversification through the development of distinct bacterial subpopulations within the biofilm can significantly increase persistence and complicate treatment (Rani et al.,

2007). As described in Sections 3.2 and 3.3, bacteria utilize a variety of mechanisms to respond to their environment, including small molecule signaling pathways (i.e., quorum sensing and intracellular cyclic dinucleotide signaling) (Rutherford and Bassler, 2012; Camilli and Bassler, 2006; Hammer and Bassler, 2003; Miller and Bassler, 2001; Waters and Bassler, 2005; Le and Otto, 2015), transmembrane receptors (i.e., two-component systems) (Hadjifrangiskou et al., 2011; Kostakioti et al., 2009; Capra and Laub, 2012; Habdas et al., 2010; Herrera et al., 2014), and chemotaxis proteins (Gueriri et al., 2008; Jones and Armitage, 2015; Bi and Lai, 2015). These pathways allow for prompt adaptation to slight differences in the surrounding microenvironment of bacteria within a biofilm community (Frederick et al., 2011).

Sensing these alterations in the environment typically result in changes in gene expression within the bacteria, which are then reflected in variations in protein expression and/or function (Fig. 3.2(e)). Therefore, within a single-species biofilm, variations in the microenvironment can lead to alterations in gene expression within distinct regions of the community, even though the biofilm population is genetically identical (Tolker-Nielsen, 2015). The development of subpopulations is demonstrated in Fig. 3.2(e) by the variations in the color of the bacteria as the biofilm matures. The existence of distinct subpopulations has been elegantly demonstrated in single-species biofilms formed by B. subtilis (Vlamakis et al., 2008), S. aureus (Savage et al., 2013), C. jejuni (Turonova et al., 2015), and uropathogenic E. coli (UPEC) (Floyd et al., 2015). Fig. 3.3 demonstrates the differential stratification of bacterial subpopulations expressing adhesive curli amyloid fibers and type 1 pili in surface-associated 72 h-old UPEC biofilms. Given that type 1 pili localize to the air-exposed surface of the biofilm and curli localize to the air-liquid interface, it is likely that different environmental or nutrient signals induce the expression of each adhesive fiber in that particular location (Fig. 3.3). Floyd et al. (2015) went on to demonstrate that indeed stratification of type 1 pili in surface-associated UPEC biofilms results from the direct regulation of type 1 pili expression by the presence or absence of oxygen.

In 2015, the Suel group published two elegant studies in B. subtilis that are beginning to deconvolute bacterial communication between subpopulations within colony biofilms. The first study demonstrated the presence of oscillations in growing biofilm colonies due to metabolic codependence between cells in the interior and cells in the periphery of a colony biofilm (Prindle et al., 2015; Liu et al., 2015). These studies demonstrated that “pausing” in the growth of peripheral cells was necessary to allow cells at the center of the colony to obtain required nutrients and survive. Loss of this metabolic codependence compromised the resilience of the biofilm. Secondly, the Suel group has uncovered that a potassium channel facilitates electrical communication between the center and the periphery of the colony biofilm, orchestrating the growth oscillation pattern in response to the metabolic requirements of the two subpopulations (Prindle et al., 2015).

The presence of subpopulations within the biofilm also contributes to persistence of infection within the host. In many bacterial biofilms, persister cell subpopulations arise that are metabolically inactive and can thus tolerate high doses of antimicrobial treatment (Lewis, 2005, 2007, 2008, 2010, 2012; Keren et al., 2004a). These persister cell populations can regain metabolic activity and the ability to reestablish infection upon cessation of the antibiotic treatment that has abolished their metabolically active brethren. Persisters and “persister-like” bacterial subpopulations have been identified for pathogenic E. coli (Balaban et al., 2004; Keren et al., 2004b; Shah et al., 2006), P. aeruginosa (Moker et al., 2010; Harrison et al., 2005; Mulcahy et al., 2010), and S. aureus (Singh et al., 2009; Tuchscherr et al., 2011; Hammer et al., 2014). The presence of persister cell populations within nonbiofilm- and biofilm-associated bacterial infections significantly dampens the treatment and increases the risk of persistence of these infections within the host.

Spatial organizations of bacterial biofilm subpopulations visualized by imaging mass spectrometry

Figure 3.3 Spatial organizations of bacterial biofilm subpopulations visualized by imaging mass spectrometry. Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) imaging mass spectrometry (IMS) is an unbiased surface-sampling technique that can be utilized to visualize the in vivo distribution of molecules (lipids, proteins, and so on) within an intact biological sample (Norris and Caprioli, 2013). Floyd et al. (2015) previously described the use of MALDI-TOF IMS for the analysis of protein stratification within intact surface-associated biofilms formed by UPEC. Using this technique, distinct biofilm subpopulations were identified based on distinct protein expression profiles (Floyd et al., 2015). Highlighted here are two subpopulations that express differential adhesive organelles. (a-c) Images were obtained from one 72-h surface-associated UPEC biofilm, cultured as previously described (Floyd et al., 2015). The area within the dotted lines indicates the imaged area of the biofilm, where protein ion spectra were obtained at 150 pm intervals across the entire area. (a and b) Images are depicted as a heat map intensity plot, where areas of highest relative protein abundance are indicated by white/red and areas of lowest relative abundance as blue/black. (a) The major subunit of adhesive type 1 pili, FimA (m/z 16,279), was observed to localize primarily to the air-exposed region of the biofilm.

(b) The major subunit of adhesive curli amyloid fibers, CsgA (m/z 13,054), was found to primarily localize to the air-liquid interface. (c) Ion image overlay of the distributions of FimA and CsgA shown in (a) and (b), pseudo-colored on the same intensity scale and zoomed. When the distributions of these two adhesive fibers are overlaid, very little overlap is observed, suggesting that subpopulations of bacteria within the biofilm differentially express these adhesive fibers. And while no direct inverse relationship of type 1 pili and curli expression has been reported to date, the distinct localizations of these subpopulations could suggest that there may exist a feedback loop between the expressions of these fibers, keeping them from being expressed by the same subpopulations.

Lastly, many studies have demonstrated that “spores formed by” spore-forming bacterial species such as B. subtilis (Vlamakis et al., 2013), Clostridium difficile (Dapa et al., 2013), and B. cereus (Wijman et al., 2007) constitute part of biofilms formed by these bacteria and, in the case of bacterial pathogens, also lead to enhanced infection.

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