Bacteria within biofilms secrete and surround themselves with an extracellular matrix, which serves as a first line of defense against antibiotic attack. Polysaccharides constitute major elements of the biofilm matrix and are implied in surface adhesion and biofilm organization, but their contributions to the resistance properties of biofilms remain largely elusive. Using a combination of static and continuous-flow biofilm experiments we show that Psl, one major polysaccharide in the Pseudomonas aeruginosa biofilm matrix, provides a generic first line of defense toward antibiotics with diverse biochemical properties during the initial stages of biofilm development. Furthermore, we show with mixed-strain experiments that antibiotic-sensitive “non-producing” cells lacking Psl can gain tolerance by integrating into Psl-containing biofilms. However, non-producers dilute the protective capacity of the matrix and hence, excessive incorporation can result in the collapse of resistance of the entire community. Our data also reveal that Psl mediated protection is extendible to E. coli and S. aureus in co-culture biofilms. Together, our study shows that Psl represents a critical first bottleneck to the antibiotic attack of a biofilm community early in biofilm development.

Microorganisms can form biofilms, which are multicellular communities surrounded by a hydrated extracellular matrix of polymers. Central properties of the biofilm are governed by this extracellular matrix, which provides mechanical stability to the three-dimensional biofilm structure, regulates the ability of the biofilm to adhere to surfaces, and determines the ability of the biofilm to adsorb gasses, solutes, and foreign cells. Despite their critical relevance for understanding and eliminating of biofilms, the materials properties of the extracellular matrix are understudied. Here, we offer the reader a guide to current technologies that can be utilized to specifically assess the permeability and mechanical properties of the biofilm matrix and its interacting components. In particular, we highlight technological advances in instrumentation and interactions between multiple disciplines that have broadened the spectrum of methods available to conduct these studies. We review pioneering work that furthers our understanding of the material properties of biofilms.

Biofilms are communities of surface-adherent bacteria surrounded by secreted polymers known as the extracellular polymeric substance (EPS). Biofilms are harmful in many industries, and thus it is of great interest to understand their mechanical properties and structure to determine ways to destabilize them. By performing single particle tracking with beads of varying surface functionalization it was found that charge interactions play a key role in mediating mobility within biofilms. With a combination of single particle tracking and microrheological concepts, it was found that Escherichia coli biofilms display height dependent charge density that evolves over time. Statistical analyses of bead trajectories and confocal microscopy showed inter-connecting micron scale channels that penetrate throughout the biofilm, which may be important for nutrient transfer through the system. This methodology provides significant insight into a particular biofilm system and can be applied to many others to provide comparisons of biofilm structure. The elucidation of structure provides evidence for the permeability of biofilms to microscale objects, and the ability of a biofilm to mature and change properties over time.

Mucus is a biological gel that lines all wet epithelia in the body, including the mouth, lungs, and digestive tract, and has evolved to protect the body from pathogenic infection. However, microbial pathogenesis is often studied in mucus-free environments that lack the geometric constraints and microbial interactions in physiological three-dimensional mucus gels. We developed fluid-flow and static test systems based on purified mucin polymers, the major gel-forming constituents of the mucus barrier, to understand how the mucus barrier influences bacterial virulence, particularly the integrity of Pseudomonas aeruginosa biofilms, which can become resistant to immune clearance and antimicrobial agents. We found that mucins separate the cells in P. aeruginosa biofilms and disperse them into suspension. Other viscous polymer solutions did not match the biofilm disruption caused by mucins, suggesting that mucin-specific properties mediate the phenomenon. Cellular dispersion depended on functional flagella, indicating a role for swimming motility. Taken together, our observations support a model in which host mucins are key players in the regulation of microbial virulence. These mucins should be considered in studies of mucosal pathogenesis and during the development of novel strategies to treat biofilms.

Swimming motility is considered a beneficial trait among bacterial species as it enables movement across fluid environments and augments invasion of tissues within the host. However, non-swimming bacteria also flourish in fluid habitats, but how they effectively spread and colonize distant ecological niches remains unclear. We show that non-motile staphylococci can gain motility by hitchhiking on swimming bacteria, leading to extended and directed motion with increased velocity. This phoretic interaction was observed between Staphylococcus aureus and Pseudomonas aeruginosa, Staphylococcus epidermidis and P. aeruginosa, as well as S. aureus and Escherichia coli, suggesting hitchhiking as a general translocation mechanism for non-motile staphylococcal species. By leveraging the motility of swimming bacteria, it was observed that staphylococci can colonize new niches that are less available in the absence of swimming carriers. This work highlights the importance of considering interactions between species within polymicrobial communities, in which bacteria can utilize each other as resources.

In many environments, bacteria favor a sessile, surface-attached community lifestyle. These communities, termed biofilms, are ubiquitous among many species of bacteria. In some cases, biofilms form under flow conditions. Flow chambers, and in particular microfluidic channels, can be used to observe biofilm development and physiological effects while varying nutrient conditions, flow velocities, or introducing antimicrobials to the biofilm in real time. Here, we describe a microfluidic-based kill-kinetics assay for the observation of antimicrobial effects on biofilms under flowing conditions.

Background Blood stream infections (BSI) are among the leading cause of morbidity and mortality, yet gold standard culture-based diagnostics have limited ability to guide therapeutic intervention due to multi-day turnaround time and low sensitivity. Day Zero Diagnostics has developed Blood2Bac™, a culture-free, species agnostic process to enrich bacteria direct from whole blood. Coupled with whole genome sequencing (WGS) and Day Zero Diagnostics’ Keynome® algorithmic tools for species ID and antimicrobial resistance (AMR), we conducted the first proof-of-concept feasibility study in an inpatient clinical setting. Methods Study participants were enrolled and specimens collected from Boston Medical Center. Eligibility criteria included hospitalized adults with suspected and/or documented BSI, irrespective of empiric antibiotic therapy duration. Whole blood samples were processed with Blood2Bac, sequenced on a nanopore platform, and bacterial ID determined with Keynome ID. Keynome ID results were compared with blood culture results to measure concordance. Results Specimens from 21 participants were processed with Blood2Bac and nanopore sequencing. For 20/21 samples, Keynome ID calls were concordant with clinical blood culture, where 6 concordant positive and 14 were concordant negative. In 3 concordant samples, Keynome ID called positive while concurrent blood cultures were negative. However, all IDs corresponded with positive blood culture results from the day prior, suggesting potentially higher sensitivity for the Blood2Bac compared to blood culture. Two concordant positive IDs, resulted in >95% of the genome recovered and Keynome concomitantly resulted in AMR predictions with 100% accuracy compared to pathogen phenotype. In 1 discordant specimen, the Keynome ID result was negative while blood cultures 8 hours before were positive. In this case, the patient was on empiric therapy for 8 days prior to samples collection and cultures were negative 19-hours post specimen collection. Conclusion These results highlight the sensitivity of a real-time blood WGS approach to identify BSI and its utility as a diagnostic to minimize unnecessary antibiotic exposure contributing to the antibiotic resistance crisis. Disclosures Archana Asundi, MD, Gilead (Scientific Research Study Investigator)Merck (Scientific Research Study Investigator)ViiV (Scientific Research Study Investigator) Nicole Billings, PhD, Day Zero Diagnostics (Employee) Zoe H. Rogers, MPH, Day Zero Diagnostics (Employee, Shareholder) Lisa S. Cunden, PhD, Day Zero Diagnostics (Shareholder) Imaly A. Nanayakkara, PhD, Day Zero Diagnostics (Employee, Shareholder) Chiahao Tsui, n/a, Day Zero Diagnostics (Employee, Shareholder) Paul Knysh, PhD, Day Zero Diagnostics (Employee) Cabell Maddux, n/a, Day Zero Diagnostics (Employee, Shareholder) Zachary Munro, n/a, Day Zero Diagnostics Inc. (Employee, Shareholder) Ian Herriott, BS, Day Zero Diagnostics (Employee, Shareholder) Miriam Huntley, PhD, Day Zero Diagnostics (Employee, Shareholder) Nina H. Lin, MD, Gilead Sciences (Scientific Research Study Investigator)ViiV (Scientific Research Study Investigator)

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