Death and transfiguration in static staphylococcus epidermidis cultures

The overwhelming majority of bacteria live in slime embedded microbial communities termed biofilms, which are typically adherent to a surface. However, when several Staphylococcus epidermidis strains were cultivated in static liquid cultures, macroscopic aggregates were seen floating within the broth and also sedimented at the test tube bottom. Light- and electron microscopy revealed that early-stage aggregates consisted of bacteria and extracellular matrix, organized in sheetlike structures. Perpendicular under the sheets hung a network of periodically arranged, bacteria-associated strands. During the extended cultivation, the strands of a subpopulation of aggregates developed into cross-connected wall-like structures, in which aligned bacteria formed the walls. The resulting architecture had a compartmentalized appearance. In late-stage cultures, the wall-associated bacteria disintegrated so that, henceforth, the walls were made of the coalescing remnants of lysed bacteria, while the compartment-like organization remained intact. At the same time, the majority of strand containing aggregates with associated culturable bacteria continued to exist. These observations indicate that some strains of Staphylococcus epidermidis are able to build highly sophisticated structures, in which a subpopulation undergoes cell lysis, presumably to provide continued access to nutrients in a nutrient-limited environment, whilst maintaining structural integrity

Microbial interactions and differential protein expression in Staphylococcus aureus –Candida albicans dual-species biofilms

The fungal species Candida albicans and the bacterial species Staphylococcus aureus are responsible for a majority of hospital-acquired infections and often coinfect critically ill patients as complicating polymicrobial biofilms. To investigate biofilm structure during polymicrobial growth, dual-species biofilms were imaged with confocal scanning laser microscopy. Analyses revealed a unique biofilm architecture where S. aureus commonly associated with the hyphal elements of C. albicans. This physical interaction may provide staphylococci with an invasion strategy because candidal hyphae can penetrate through epithelial layers. To further understand the molecular mechanisms possibly responsible for previously demonstrated amplified virulence during coinfection, protein expression studies were undertaken. Differential in-gel electrophoresis identified a total of 27 proteins to be significantly differentially produced by these organisms during coculture biofilm growth. Among the upregulated staphylococcal proteins was l-lactate dehydrogenase 1, which confers resistance to host-derived oxidative stressors. Among the downregulated proteins was the global transcriptional repressor of virulence factors, CodY. These findings demonstrate that the hyphae-mediated enhanced pathogenesis of S. aureus may not only be due to physical interactions but can also be attributed to the differential regulation of specific virulence factors induced during polymicrobial growth. Further characterization of the intricate interaction between these pathogens at the molecular level is warranted, as it may aid in the design of novel therapeutic strategies aimed at combating fungal–bacterial polymicrobial infection

The production of the alpha toxin of Clostridium perfringens in a chemically reproducible medium

Boyd, Logan and Tytell (1948) have shown that Clostridium perfringens BP6K will grow in a chemically reproducible medium. They recorded no lecithinase (∝toxin) production under the conditions of their experiment. This thesis reports conditions under which lecithinase is produced in the above medium. Time studies Involving hourly sampling proved to be the most satisfactory method of studying the production of the enzyme. The pH of each sample was determined, the bacterial density was recorded turbidimetrically, and the lecithinase was estimated by a modification of the egg yolk suspension method. Lecithinase was found to be produced during the logarithmic phase of growth, after which it rapidly diminished in quantity, disappearing at approximately twelve hours growth. The amount of the enzyme produced is relatively small compared with yields from complex organic media. However, the characteristics of the lecithinase appear identical with those of the lecithinase produced in complex media. Since the medium contains no lecithin, and the enzyme is produced during the logarithmic growth phase, it would appear that the enzyme is essentially constitutive and extracellular. Phase variation as indicated by colonial morphology has been shown to be of considerable importance in lecithinase production in the chemically reproducible medium. The observation of a peculiar colonial morphology designated as a "halo" colony is reported. This morphological type is seen to be closely related to high toxigenic activity in G.P.B.I. In the reproducible medium acid production during growth (pH 7.2 to 4.8) is closely related to enzyme destruction. Adjustment of the pH during growth does not greatly enhance lecithihase production but does markedly slow its destruction. The substitution of dextrin for glucose in the reproducible medium delays but does not reduce growth and lecithinase production. The destruction of the enzyme is slowed by the addition of proteinaceous material. The concentrations of cystine and of iron are also seen to affect the rate of destruction of lecithinase.Science, Faculty ofMicrobiology and Immunology, Department ofGraduat

A review of the scientific evidence for biofilms in wounds

Both chronic and acute dermal wounds are susceptible to infection due to sterile loss of the innate barrier function of the skin and dermal appendages, facilitating the development of microbial communities, referred to as biofilms, within the wound environment. Microbial biofilms are implicated in both the infection of wounds and failure of those wounds to heal. The aim of this review is to provide a summary of published papers detailing biofilms in wounds, the effect they have on infection and wound healing, and detailing methods employed for their detection. The studies highlighted within this paper provide evidence that biofilms reside within the chronic wound and represent an important mechanism underlying the observed, delayed healing and infection. The reasons for this include both protease activity and immunological suppression. Furthermore, a lack of responsiveness to an array of antimicrobial agents has been due to the biofilms’ ability to inherently resist antimicrobial agents. It is imperative that effective strategies are developed, tested prospectively, and employed in chronic wounds to support the healing process and to reduce infection rates. It is increasingly apparent that adoption of a biofilm-based management approach to wound care, utilizing the “antibiofilm tool box” of therapies, to kill and prevent reattachment of microorganisms in the biofilm is producing the most positive clinical outcomes and prevention of infection

Protease treatment affects both invasion ability and biofilm formation in Listeria monocytogenes

Listeria monocytogenes is a notably invasive bacterium associated with life-threatening food-borne disease in humans. Several surface proteins have been shown to be essential in the adhesion of L. monocytogenes, and in the subsequent invasion of phagocytes. Because the control of the invasion of host cells by Listeria could potentially hinder its spread in the infected host, we have examined the effects of a protease treatment on the ability of L. monocytogenes to form biofilms and to invade tissues. We have chosen serratiopeptidase (SPEP), an extracellular metalloprotease produced by Serratia marcescens that is already widely used as an anti-inflammatory agent, and has been shown to modulate adhesin expression and to induce antibiotic sensitivity in other bacteria. Treatment of L. monocytogenes with sublethal concentrations of SPEP reduced their ability to form biofilms and to invade host cells. Zymograms of the treated cells revealed that Ami4b autolysin, internalinB, and ActA were sharply reduced. These cell-surface proteins are known to function as ligands in the interaction between these bacteria and their host cells, and our data suggest that treatment with this natural enzyme may provide a useful tool in the prevention of the initial adhesion of L. monocytogenes to the human gut

Late stage structures.

<p>A. SEM picture, 14-day culture, MH strain: the thin compartment walls contain only few bacteria. B. Transmission electron microscopy (TEM) picture, 14-day culture, MH strain: the few bacteria (arrow 1) are a part of otherwise in ‘empty’ walls (arrow 2). C. TEM picture, 14-day culture, MH strain: an intact bacterium (arrow 1) and a partly disintegrated bacterium (arrow 2) are both integrated in the wall structure (arrow 3). D. TEM picture, 14-day culture, MH strain: the bare walls consist of fine, dispersed fibers (arrow 1) and intensely stained dots (arrow 2). E. SEM picture, 14-day culture, MH strain: empty compartment walls. F. SEM picture, 14-day culture, MH strain: bacteria depleted compartment walls. G. SEM picture: 14-day old cultures (MH strain) showed large compartmentalized areas. H. cLSM transmission mode, 14-day culture (MH strain): ‘empty’ compartment walls under hydrated conditions.</p

Specific staining, time-course and development model.

<p>A. cLSM picture, 14-day culture, strain #49134: the EUB338 probe labels the compartment structure (red), while the bacteria are double-stained by the EUB338 probe and Syto59 (yellow). B. cLSM picture, 14-day culture, MH strain: Concanavalin A stains the compartment structure, but not the bacteria (arrow 1). C. cLSM picture, 14-day culture, strain #49134: the Sypro protein stain (green) and the Syto59 nucleic acid stain label both the bacteria (yellow) but not the compartment structure. D. cLSM picture, 14-day culture, strain #49134: the lipophilic stain Nile Red (green) does not label compartment walls, only diffuse biofilm matrix. E. SEM picture, 28-day culture, MH strain: huge aggregates with intact bacteria and strands continued to be present in long-time cultures. F. SEM picture, MH strain, overnight grown colony on agar. G. The model depicts the main development stages and their timely appearances in the context with the CFU time-course (MH strain).</p

Compartmentalization.

<p>A. SEM picture, 10-day culture, MH strain: solid, parallel, wall-like structures. B. SEM picture, 14-day culture, MH strain: the top sheet (region 1) is situated on vertically stacked walls (region 2). C. cLSM transmission mode, 14-day culture, MH strain: a dense, horizontal top plate (region 1) is located on top of vertically stacked walls (region 2). D. SEM picture, 14-day culture, MH strain: compartmentalized structure of aligned walls and cross-walls without top sheet. E. cLSM transmission mode, 14-day culture, MH strain: aligned bacteria form compartment walls. F. SEM picture, 14-day culture, MH strain: matrix (arrow 1) embedded bacteria (arrow 2) form compartment walls. G. cLSM picture, 14-day culture, MH strain: LIVE/DEAD staining of compartment wall forming bacteria show varying stages of membrane integrity (arrows). H. SEM picture, 14-day culture, MH strain: compartment structure with abundant bacteria (region 1) adjacent to an area with bacteria-depleted compartments.</p