More Like this

1. Antibiotic Susceptibility of Escherichia coli Cells during Early-Stage Biofilm Formation

   https://doi.org/10.1128/JB.00034-19  

   Gu, Huan; Lee, Sang Won; Carnicelli, Joseph; Jiang, Zhaowei; Ren, Dacheng; O'Toole, George (September 2019, Journal of Bacteriology)

   ABSTRACT Bacteria form complex multicellular structures on solid surfaces known as biofilms, which allow them to survive in harsh environments. A hallmark characteristic of mature biofilms is the high-level antibiotic tolerance (up to 1,000 times) compared with that of planktonic cells. Here, we report our new findings that biofilm cells are not always more tolerant to antibiotics than planktonic cells in the same culture. Specifically, Escherichia coli RP437 exhibited a dynamic change in antibiotic susceptibility during its early-stage biofilm formation. This phenomenon was not strain specific. Upon initial attachment, surface-associated cells became more sensitive to antibiotics than planktonic cells. By controlling the cell adhesion and cluster size using patterned E. coli biofilms, cells involved in the interaction between cell clusters during microcolony formation were found to be more susceptible to ampicillin than cells within clusters, suggesting a role of cell-cell interactions in biofilm-associated antibiotic tolerance. After this stage, biofilm cells became less susceptible to ampicillin and ofloxacin than planktonic cells. However, when the cells were detached by sonication, both antibiotics were more effective in killing the detached biofilm cells than the planktonic cells. Collectively, these results indicate that biofilm formation involves active cellular activities in adaption to the attached life form and interactions between cell clusters to build the complex structure of a biofilm, which can render these cells more susceptible to antibiotics. These findings shed new light on bacterial antibiotic susceptibility during biofilm formation and can guide the design of better antifouling surfaces, e.g., those with micron-scale topographic structures to interrupt cell-cell interactions. IMPORTANCE Mature biofilms are known for their high-level tolerance to antibiotics; however, antibiotic susceptibility of sessile cells during early-stage biofilm formation is not well understood. In this study, we aim to fill this knowledge gap by following bacterial antibiotic susceptibility during early-stage biofilm formation. We found that the attached cells have a dynamic change in antibiotic susceptibility, and during certain phases, they can be more sensitive to antibiotics than planktonic counterparts in the same culture. Using surface chemistry-controlled patterned biofilm formation, cell-surface and cell-cell interactions were found to affect the antibiotic susceptibility of attached cells. Collectively, these findings provide new insights into biofilm physiology and reveal how adaptation to the attached life form may influence antibiotic susceptibility of bacterial cells. 

   more » « less
2. Non-disruptive matrix turnover is a conserved feature of biofilm aggregate growth in paradigm pathogenic species

   https://doi.org/10.1128/mbio.03935-24  

   Reichhardt, Courtney; Matwichuk, Michael L; Lewerke, Lincoln T; Jacobs, Holly M; Yan, Jing; Parsek, Matthew R (February 2025, mBio)

   Whiteley, Marvin (Ed.)

   ABSTRACT Bacteria form multicellular aggregates called biofilms. A crucial component of these aggregates is a protective matrix that holds the community together. Biofilm matrix composition varies depending upon bacterial species but typically includes exopolysaccharides (EPS), proteins, and extracellular DNA.Pseudomonas aeruginosais a model organism for the study of biofilms, and in non-mucoid biofilms, it uses the structurally distinct EPS Psl and Pel, the EPS-binding protein CdrA, and eDNA as key matrix components. An interesting phenomenon that we and others have observed is that the periphery of a biofilm aggregate can be EPS-rich and contain very few cells. In this study, we investigated two possible models of assembly and dynamics of this EPS-rich peripheral region: (i) newly synthesized EPS is inserted and incorporated into the existing EPS-rich region at the periphery during biofilm aggregate growth or (ii) EPS is continuously turned over and newly synthesized EPS is deposited at the outermost edge of the aggregate. Our results support the latter model. Specifically, we observed that new EPS is continually deposited at the aggregate periphery, which is necessary for continued aggregate growth but not aggregate stability. We made similar observations in another paradigm biofilm-forming species,Vibrio cholerae. This pattern of deposition raises the question of how EPS is retained. Specifically, forP. aeruginosabiofilms, the matrix adhesin CdrA is thought to retain EPS. However, current thinking is that cell-associated CdrA is responsible for this retention, and it is not clear how CdrA might function in the relatively cell-free aggregate periphery. We observed that CdrA is enzymatically degraded during aggregate growth without negatively impacting biofilm stability and that cell-free CdrA can partially maintain aggregation and Psl retention. Overall, this study shows that the matrix ofP. aeruginosabiofilms undergoes both continuous synthesis of matrix material and matrix turnover to accommodate biofilm aggregate growth and that cell-free matrix can at least partially maintain biofilm aggregation and EPS localization. Furthermore, our similar observations forV. choleraebiofilms suggest that our findings may represent basic principles of aggregate assembly in bacteria. IMPORTANCEHere, we show that, to accommodate growing cellular biomass, newly produced Psl is deposited over existing Psl at the periphery of biofilm aggregates. We demonstrated thatV. choleraeemploys a similar mechanism with its biofilm matrix EPS, VPS. In addition, we found that the protease LasB is present in the biofilm matrix, resulting in degradation of CdrA to lower molecular weight cell-free forms. We then show that the released forms of CdrA are retained in the matrix and remain functional. Together, our findings support that theP. aeruginosabiofilm matrix is dynamic during the course of aggregate growth and that other species may employ similar mechanisms to remodel their matrix. 

   more » « less
3. Let-7b-5p in vesicles secreted by human airway cells reduces biofilm formation and increases antibiotic sensitivity of P. aeruginosa

   https://doi.org/10.1073/pnas.2105370118  

   Koeppen, Katja; Nymon, Amanda; Barnaby, Roxanna; Bashor, Laura; Li, Zhongyou; Hampton, Thomas H.; Liefeld, Amanda E.; Kolling, Fred W.; LaCroix, Ian S.; Gerber, Scott A.; et al (July 2021, Proceedings of the National Academy of Sciences)

   Pseudomonas aeruginosais an opportunistic pathogen that forms antibiotic-resistant biofilms, which facilitate chronic infections in immunocompromised hosts. We have previously shown thatP. aeruginosasecretes outer-membrane vesicles that deliver a small RNA to human airway epithelial cells (AECs), in which it suppresses the innate immune response. Here, we demonstrate that interdomain communication through small RNA–containing membrane vesicles is bidirectional and that microRNAs (miRNAs) in extracellular vesicles (EVs) secreted by human AECs regulate protein expression, antibiotic sensitivity, and biofilm formation byP. aeruginosa. Specifically, human EVs deliver miRNA let-7b-5p toP. aeruginosa, which systematically decreases the abundance of proteins essential for biofilm formation, including PpkA and ClpV1-3, and increases the ability of beta-lactam antibiotics to reduce biofilm formation by targeting the beta-lactamase AmpC. Let-7b-5p is bioinformatically predicted to target not only PpkA, ClpV1, and AmpC inP. aeruginosabut also the corresponding orthologs inBurkholderia cenocepacia, another notorious opportunistic lung pathogen, suggesting that the ability of let-7b-5p to reduce biofilm formation and increase beta-lactam sensitivity is not limited toP. aeruginosa. Here, we provide direct evidence for transfer of miRNAs in EVs secreted by eukaryotic cells to a prokaryote, resulting in subsequent phenotypic alterations in the prokaryote as a result of this interdomain communication. Since let-7–family miRNAs are in clinical trials to reduce inflammation and because chronicP. aeruginosalung infections are associated with a hyperinflammatory state, treatment with let-7b-5p and a beta-lactam antibiotic in nanoparticles or EVs may benefit patients with antibiotic-resistantP. aeruginosainfections. 

   more » « less
4. Applying a polysaccharide lyase from Stenotrophomonas maltophilia to disrupt alginate exopolysaccharide produced by Pseudomonas aeruginosa clinical isolates

   https://doi.org/10.1128/aem.01853-24  

   Felton, Samantha M; Akula, Nikki; Kolling, Glynis L; Azadi, Parastoo; Black, Ian; Kumar, Ambrish; Heiss, Christian; Capobianco, Joseph; Uknalis, Joseph; Papin, Jason A; et al (January 2025, Applied and Environmental Microbiology)

   Zhou, Ning-Yi (Ed.)

   ABSTRACT Pseudomonas aeruginosais considered one of the most challenging, drug-resistant, opportunistic pathogens partly due to its ability to synthesize robust biofilms. Biofilm is a mixture of extracellular polymeric substances (EPS) that encapsulates microbial cells, leading to immune evasion, antibiotic resistance, and thus higher risk of infection. In the cystic fibrosis lung environment,P. aeruginosaundergoes a mucoid transition, defined by overproduction of the exopolysaccharide alginate. Alginate encapsulation results in bacterial resistance to antibiotics and the host immune system. Given its role in airway inflammation and chronic infection, alginate is an obvious target to improve treatment forP. aeruginosainfection. Previously, we demonstrated polysaccharide lyase Smlt1473 fromStenotrophomonas maltophiliastrain k279a can catalyze the degradation of multiple polyuronidesin vitro, including D-mannuronic acid (poly-ManA). Poly-ManA is a major constituent ofP. aeruginosaalginate, suggesting that Smlt1473 could have potential application against multidrug-resistantP. aeruginosaand perhaps other microbes with related biofilm composition. In this study, we demonstrate that Smlt1473 can inhibit and degrade alginate fromP. aeruginosa. Additionally, we show that testedP. aeruginosastrains are dominant in acetylated alginate and that all but one have similar M-to-G ratios. These results indicate that variation in enzyme efficacy among the isolates is not primarily due to differences in total EPS or alginate chemical composition. Overall, these results demonstrate Smlt1473 can inhibit and degradeP. aeruginosaalginate and suggest that other factors including rate of EPS production, alginate sequence/chain length, or non-EPS components may explain differences in enzyme efficacy. IMPORTANCEPseudomonas aeruginosais a major opportunistic human pathogen in part due to its ability to synthesize biofilms that confer antibiotic resistance. Biofilm is a mixture of polysaccharides, DNA, and proteins that encapsulate cells, protecting them from antibiotics, disinfectants, and other cleaning agents. Due to its ability to increase antibiotic and immune resistance, the exopolysaccharide alginate plays a large role in airway inflammation and chronicP. aeruginosainfection. As a result, colonization withP. aeruginosais the leading cause of morbidity and mortality in CF patients. Thus, it is an obvious target to improve the treatment regimen forP. aeruginosainfection. In this study, we demonstrate that polysaccharide lyase, Smlt1473, inhibits alginate secretion and degrades established alginate from a variety of mucoidP. aeruginosaclinical isolates. Additionally, Smlt1473 differs from other alginate lyases in that it is active against acetylated alginate, which is secreted during chronic lung infection. These results suggest that Smlt1473 may be useful in treating infections associated with alginate-producingP. aeruginosa, as well as have the potential to reduceP. aeruginosaEPS in non-clinical settings. 

   more » « less
5. Flagellum-driven motility enhances Pseudomonas aeruginosa biofilm formation by altering cell orientation

   https://doi.org/10.1128/aem.00821-25  

   Wei, Guanju; Palalay, Jessica-Jae S; Sanfilippo, Joseph E; Yang, Judy Q (July 2025, Applied and Environmental Microbiology)

   van_Kessel, Julia C (Ed.)

   ABSTRACT Bacterial motility plays a crucial role in biofilm development, yet the underlying mechanism remains not fully understood. Here, we demonstrate that the flagellum-driven motility ofPseudomonas aeruginosaenhances biofilm formation by altering the orientation of bacterial cells, an effect controlled by shear stress rather than shear rate. By tracking wild-typeP. aeruginosaand its non-motile mutants in a microfluidic channel, we demonstrate that while non-motile cells align with the flow, many motile cells can orient toward the channel sidewalls, enhancing cell surface attachment and increasing biofilm cell density by up to 10-fold. Experiments with varying fluid viscosities further demonstrate that bacterial swimming speed decreases with increasing fluid viscosity, and the cell orientation scales with the shear stress rather than shear rate. Our results provide a quantitative framework to predict the role of motility in the orientation and biofilm development under different flow conditions and viscosities.IMPORTANCEBiofilms are ubiquitous in rivers, water pipes, and medical devices, impacting the environment and human health. While bacterial motility plays a crucial role in biofilm development, a mechanistic understanding remains limited, hindering our ability to predict and control biofilms. Here, we reveal how the motility ofPseudomonas aeruginosa, a common pathogen, influences biofilm formation through systematically controlled microfluidic experiments with confocal and high-speed microscopy. We demonstrate that the orientation of bacterial cells is controlled by shear stress. While non-motile cells primarily align with the flow, many motile cells overcome the fluid shear forces and reorient toward the channel sidewalls, increasing biofilm cell density by up to 10-fold. Our findings provide insights into how bacterial transition from free-swimming to surface-attached states under varying flow conditions, emphasizing the role of cell orientation in biofilm establishment. These results enhance our understanding of bacterial behavior in flow environments, informing strategies for biofilm management and control. 

   more » « less