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 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.
The transient diffusion of fluorescent tracers into biofilm cell clusters of Staphylococcus epidermidis was visualized by time lapse confocal scanning laser microscopy. Rhodamine B diffused into the center of cell clusters that were 200 to 600 m in diameter within a few minutes. The apparent effective diffusion coefficient calculated from these data averaged 3.7 ؋ 10 ؊7 cm 2 s ؊1 or 11% of the value in pure water. Fluorescein diffused into biofilm more rapidly, with a diffusion coefficient that averaged 1.6 ؋ 10 ؊6 cm 2 s ؊1 , or 32% of the value in water. This study provides direct, visual confirmation that solutes the size of many antibiotics and biocides can diffuse rapidly into biofilms.One long-standing explanation for the antibiotic tolerance of microorganisms in biofilms is that the biofilm matrix constitutes a barrier to effective penetration of antimicrobial agents. If an antibiotic simply does not reach cells in the interior of a cluster, the failure of the agent to kill these cells is easy to understand. On the other hand, if the antibiotic can be shown to penetrate throughout the biofilm, then one must turn to biological explanations for antibiotic tolerance.The objective of this study was to directly visualize the penetration of an antibiotic-sized tracer molecule into the interior of biofilm cell clusters, noninvasively and in real time. This was accomplished by using fluorescent dyes that were imaged by confocal scanning laser microscopy. Quantitative image analysis was performed to extract numerical values of the effective diffusion coefficient in the biofilm. MATERIALS AND METHODSBacteria and media. Staphylococcus epidermidis strain RP62A (ATCC 35984) was grown on tryptic soy broth (TSB) at 37°C. Full strength TSB was used to grow shake flask cultures that provided the inoculum for biofilm experiments. Biofilms were grown on 1/10 strength TSB.Biofilm reactor. Biofilms were grown in glass capillary tubes (Friedrich and Dimmock, Millville, N.J.) under continuous-flow conditions. Glass tubes were used rather than a more clinically relevant material because glass provided an optically clear substratum for microscopy. In addition, these capillary tubes had a square cross-section, which facilitated microscopic observation of the biofilm through the capillary walls. The nominal inside dimension of the tube was 0.9 mm, and it was approximately 10 cm long. Autoclaved 1/10 strength TSB was delivered to the capillary by gravity feed from a 5-liter carboy. The head difference from the feed carboy to the waste outlet was approximately 1.5 m. The flow rate of medium, which was monitored by counting drops passing through a flow break between the feed carboy and capillary, was between 120 and 180 ml h Ϫ1 . This flow rate corresponds to a Reynolds number of 37 to 56 based on the hydraulic radius of the clean tube. The medium carboy and the reactor itself were placed inside separate 37°C incubators stacked on top of each other. The reactor was inoculated by injecting 1 to 2 ml of overnight culture into a septum j...
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 Damkö hler 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.
Wound healing depends on controlling bacterial balance while maintaining the viability of the healing tissues. In vitro toxicity indexes provide helpful guidelines subsequent to in vivo evaluations and clinical applications. The study findings suggest that NeutroPhase, in contrast with many commercially available wound cleansers, has rapid bactericidal activity at concentrations that are safe for human cells.
Bacterial pathogens have specific virulence factors (e.g., toxins) that contribute significantly to the virulence and infectivity of microorganisms within the human hosts. Virulence factors are molecules expressed by pathogens that enable colonization, immunoevasion, and immunosuppression, obtaining nutrients from the host or gaining entry into host cells. They can cause pathogenesis by inhibiting or stimulating certain host functions. For example, in systemic Staphylococcus aureus infections, virulence factors such as toxic shock syndrome toxin 1 (TSST-1), staphylococcal enterotoxin A (SEA), and staphylococcal enterotoxin B (SEB) cause sepsis or toxic shock by uncontrolled stimulation of T lymphocytes and by triggering a cytokine storm. In vitro, these superantigens stimulate the proliferation of human peripheral blood mononuclear cells (PBMC) and the release of many cytokines. NVC-422 (N,N-dichloro-2,2-dimethyltaurine) is a broad-spectrum, fast-acting topical anti-infective agent against microbial pathogens, including antibiotic-resistant microbes. Using mass spectrometry, we demonstrate here that NVC-422 oxidizes methionine residues of TSST-1, SEA, SEB, and exfoliative toxin A (ETA). Exposure of virulence factors to 0.1% NVC-422 for 1 h prevented TSST-1-, SEA-, SEB-, and ETA-induced cell proliferation and cytokine release. Moreover, NVC-422 also delayed and reduced the protein A-and clumping factor-associated agglutination of S. aureus cultures. These results show that, in addition to its well-described direct microbicidal activity, NVC-422 can inactivate S. aureus virulence factors through rapid oxidation of methionines.
NVC-422 has potent, rapid in vitro virucidal activity against major causes of conjunctivitis. Its broad-spectrum virucidal activity combined with favorable safety profile validates NVC-422 as a potential new therapeutic agent against viral conjunctivitis.
Long-term use of indwelling urinary catheters can lead to urinary tract infections and loss of catheter patency due to encrustation and blockage. Encrustation of urinary catheters is due to formation of crystalline biofilms by urease-producing microorganisms such as Proteus mirabilis. An in vitro catheter biofilm model (CBM) was used to evaluate current methods for maintaining urinary catheter patency. We compared antimicrobial-coated urinary Foley catheters, with both available catheter irrigation solutions and investigational solutions containing NVC-422 (N,N-dichloro-2,2-dimethyltaurine; a novel broad-spectrum antimicrobial). Inoculation of the CBM reactor with 10(8) colony-forming units of P. mirabilis resulted in crystalline biofilm formation in catheters by 48 h and blockage of catheters within 5 days. Silver hydrogel or nitrofurazone-coated catheters did not extend the duration of catheter patency. Catheters irrigated daily with commercially available solutions such as 0.25 % acetic acid and isotonic saline blocked at the same rate as untreated catheters. Daily irrigations of catheters with 0.2 % NVC-422 in 10 mM acetate-buffered saline pH 4 or Renacidin maintained catheter patency throughout 10-day studies, but P. mirabilis colonization of the CBM remained. In contrast, 0.2 % NVC-422 in citrate buffer (6.6 % citric acid at pH 3.8) resulted in an irrigation solution that not only maintained catheter patency for 10 days but also completely eradicated the P. mirabilis biofilm within one treatment day. These data suggest that an irrigation solution containing the rapidly bactericidal antimicrobial NVC-422 in combination with citric acid to permeabilize crystalline biofilm may significantly enhance catheter patency versus other approved irrigation solutions and antimicrobial-coated catheters.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.