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In previous studies, Propionibacterium acnes was cultured from intervertebral disc tissue of ~25% of patients undergoing microdiscectomy, suggesting a possible link between chronic bacterial infection and disc degeneration. However, given the prominence of P. acnes as a skin commensal, such analyses often struggled to exclude the alternate possibility that these organisms represent perioperative microbiologic contamination. This investigation seeks to validate P. acnes prevalence in resected disc cultures, while providing microscopic evidence of P. acnes biofilm in the intervertebral discs.Specimens from 368 patients undergoing microdiscectomy for disc herniation were divided into several fragments, one being homogenized, subjected to quantitative anaerobic culture, and assessed for bacterial growth, and a second fragment frozen for additional analyses. Colonies were identified by MALDI-TOF mass spectrometry and P. acnes phylotyping was conducted by multiplex PCR. For a sub-set of specimens, bacteria localization within the disc was assessed by microscopy using confocal laser scanning and FISH.Bacteria were cultured from 162 discs (44%), including 119 cases (32.3%) with P. acnes. In 89 cases, P. acnes was cultured exclusively; in 30 cases, it was isolated in combination with other bacteria (primarily coagulase-negative Staphylococcus spp.) Among positive specimens, the median P. acnes bacterial burden was 350 CFU/g (12 - ~20,000 CFU/g). Thirty-eight P. acnes isolates were subjected to molecular sub-typing, identifying 4 of 6 defined phylogroups: IA1, IB, IC, and II. Eight culture-positive specimens were evaluated by fluorescence microscopy and revealed P. acnes in situ. Notably, these bacteria demonstrated a biofilm distribution within the disc matrix. P. acnes bacteria were more prevalent in males than females (39% vs. 23%, p = 0.0013).This study confirms that P. acnes is prevalent in herniated disc tissue. Moreover, it provides the first visual evidence of P. acnes biofilms within such specimens, consistent with infection rather than microbiologic contamination.

The presence of microbial biofilms in many human chronic wounds led to the hypothesis that biofilms delay healing of these wounds. We tested this hypothesis in a population of 117 older individuals with venous leg ulcers who were receiving standardised therapy, including frequent debridement. Debridement specimens were analysed for the amount of bacterial biomass by two independent methods: a microscopic approach that scored the relative size and number of bacterial aggregates, interpreted as a biofilm metric, and conventional enumeration by agar plating for viable bacteria. The plating protocol yielded three distinct values: the total viable bacterial count, bleach‐tolerant bacteria, and the log reduction in viable bacteria upon bleach treatment. Wound healing rates over an 8‐week observation period were calculated as the rate of decrease of the equivalent diameter of the wound. There was no statistically significant association between wound healing and the biofilm metric in any of the three analyses performed (p ≥0.15). In all three statistical tests, wound healing was associated with the log reduction caused by bleach treatment (p ≤0.004); wounds that harboured bacteria that were more bleach‐susceptible healed more slowly. A refinement of the model of chronic wound infection pathogenesis is proposed in which dormant bacteria constitute a persistent nidus and outgrowth of metabolically active cells impairs healing. This model constitutes a new hypothesis as metabolic activity was not directly measured in this investigation. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus (S. aureus) is the leading cause of surgical site infections (SSIs) and is capable of biofilm growth on implanted foreign devices. The use of surgical irrigation solutions has become a common strategy to combat bacterial contamination events that occur during surgery. Despite their antimicrobial activity, SSI rates remain consistent, suggesting that low-level contamination persists. In these cases, circulating neutrophils must traffic from the blood to contamination sites to aid in bacterial clearance. The influence of irrigation solutions on neutrophils' ability to engage with bacteria has not been explored. The effects of three commonly used irrigation solutions: Xperience (sodium lauryl sulfate), Irrisept (chlorhexidine gluconate), and Betadine® (povidone-iodine) on nascent S. aureus biofilms alone and in the presence of human neutrophils were assessed at manufactured and diluted concentrations. All three solutions, at a 10% dilution, inhibited bacterial growth as demonstrated by culture assays and confocal video microscopy of bacterial aggregate formation. The effects of 10% dilutions of each of these solutions on neutrophil membrane integrity (by flow cytometry and propidium iodide staining) and motility (by confocal video microscopy of neutrophil track length) were investigated with differing outcomes for each irrigation solution. At this concentration only Irrisept preserved neutrophil membrane integrity and motility. Together, this study examines an overlooked aspect of surgical irrigation solutions by investigating their impact on innate immunity and highlights the feasibility of formulations wherein solution effectiveness is complemented by neutrophil function to reduce risks of infection. [ABSTRACT FROM AUTHOR]

Nontypeable Haemophilus influenzae (NTHI) bacteria are commensals in the human nasopharynx, as well as pathogens associated with a spectrum of acute and chronic infections. Two important factors that influence NTHI pathogenicity are their ability to adhere to human tissue and their ability to form biofilms. Extracellular polymeric substances (EPS) and bacterial appendages such as pili critically influence cell adhesion and intercellular cohesion during biofilm formation. Structural components in the outer cell membrane, such as lipopolysaccharides, also play a fundamental role in infection of the host organism. In spite of their importance, these pathogenic factors are not yet well characterized at the nanoscale. Here, atomic force microscopy (AFM) was used in aqueous environments to visualize structural details, including probable Hif-type pili, of live NTHI bacteria at the early stages of biofilm formation. Using single-molecule AFM-based spectroscopy, the molecular elasticities of lipooligosaccharides present on NTHI cell surfaces were analyzed and compared between two strains (PittEE and PittGG) with very different pathogenicity profiles. Furthermore, the stiffness of single cells of both strains was measured and subsequently their turgor pressure was estimated.

It has long been suspected that microbial biofilms harbor cells in a variety of activity states, but there have been few direct experimental visualizations of this physiological heterogeneity. Spatial patterns of DNA replication and protein synthetic activity were imaged and quantified in staphylococcal biofilms using immunofluorescent detection of pulse-labeled DNA and also an inducible green fluorescent protein (GFP) construct. Stratified patterns of DNA synthetic and protein synthetic activity were observed in all three biofilm systems to which the techniques were applied. In a colony biofilm system, the dimensions of the zone of anabolism at the air interface ranged from 16 to 38 [micro]m and corresponded with the depth of oxygen penetration measured with a microelectrode. A second zone of activity was observed along the nutrient interface of the biofilm. Much of the biofilm was anabolically inactive. Since dead cells constituted only 10% of the biofilm population, most of the inactive cells in the biofilm were still viable. Collectively, these results suggest that staphylococcal biofilms contain cells in at least four distinct states: growing aerobically, growing fermentatively, dead, and dormant. The variety of activity states represented in a biofilm may contribute to the special ecology and tolerance to antimicrobial agents of biofilms.

A three-dimensional computer model of bio-film dynamics was developed as a tool for investigating mechanisms of protection from antimicrobial agents in bio-films. The differences between the protective mechanisms predicted in these simulations could guide the design of experiments to discriminate antimicrobial tolerance mechanisms in biofilms.

A kinetic model is proposed to assess the feasibility of strategies for the removal of biofilms by using substances that induce detachment by affecting the cohesiveness of the matrix of extracellular polymeric substances (EPSs). The model uses a two-state description of the EPS (natural EPS and compromised EPS) to provide a unified representation of diverse mechanisms of action of detachment-promoting agents (DPAs), which include enzymes that degrade the EPS and other agents described in the literature. A biofilm-cohesiveness factor describes local increases in detachment rates resultant from losses in cohesive strength. The kinetic model was implemented in an individual-based biofilm-modelling framework, including detachment rates dependent on local cohesiveness. The efficacy of treatments with DPAs was assessed by three-dimensional model simulations. Changes in treatment efficacy were evaluated quantitatively by using a Thiele modulus, which quantifies the relationship between diffusion of the DPA through the biofilm matrix and DPA decay rate, and a Damkohler number relating the rate of EPS reaction with a DPA and the rate of EPS production by the micro-organisms in the biofilm. This study demonstrates the feasibility and limits of implementing biofilm-control strategies based on attacking the EPS.

A mathematical model of biofilm dynamics was used to investigate the protection from antimicrobial killing that could be afforded to micro-organisms in biofilms based on a mechanism of 'persister' cell or phenotypic variant formation. The persister state is a hypothetical, highly protected state adopted by a small fraction of the cells in a biofilm. Persisters were assumed to be generated at a fixed rate, independent of the presence of substrate or antimicrobial agent. Cells were assumed to revert from the persister state when exposed to the growth substrate. Persister cells were assumed to be incapable of growth. The model predicted that persister cells increased in numbers in the biofilm, even though they were unable to grow, accumulating in regions of substrate limitation. In these regions, normal cells failed to grow, but did slowly convert to the persister state. Calculations of persister formation in planktonic cultures predicted that persisters would be present in low numbers in growing cultures, but should accumulate under conditions of slow growth, e.g. very low dilution rates in continuous culture or stationary phase in batch culture. When antibiotic treatment was simulated, bacteria near the biofilm surface were killed, but persisters in the depth of the biofilm were poorly killed. After antibiotic treatment ceased, surviving persister cells quickly reverted and allowed the biofilm to regrow. This modelling study provides motivation for further investigation of the hypothetical persister cell state as an explanation for biofilm resistance to antimicrobial agents.

Author(s): Thien-Fah Mah [1]; Betsey Pitts [2]; Brett Pellock [3, 4]; Graham C. Walker [3]; Philip S. Stewart [2]; George A. O'Toole (corresponding author) [1] Biofilms are surface-attached microbial communities [...]

Research was conducted to determine the role of catalase enzymes in protecting bacterial biofilms against hydrogen peroxide biocide. Results indicate besides the protective action of the catalases, other mechanisms also appear to influence resistance of biofilms to hydrogen peroxide.

A study was conducted to analyze the function of oxygen availability on the physiological activity of Pseudomonas aeruginosa in biofilms. A drip-flow reactor was utilized to cultivate the biofilms. Experimental results indicated marked spatial physiological heterogeneity within P. aeruginosa biofilms wherein protein synthesis is hindered by the availability of oxygen to the upper 30 microns of the biofilm.