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 sheet-like 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.

Biofilm formation in Staphylococcus implant infections. A review of molecular mechanisms and implications for biofilm-resistant materials.

Implant infections in orthopaedics, as well as in many other medical fields, are chiefly caused by staphylococci. The ability of growing within a biofilm enhances the chances of staphylococci to protect themselves from host defences, antibiotic therapies, and biocides. Advances in scientific knowledge on structural molecules (exopolysaccharide, proteins, teichoic acids, and the most recently described extracellular DNA), on the synthesis and genetics of staphylococcal biofilms, and on the complex network of signal factors that intervene in their control are here presented, also reporting on the emerging strategies to disrupt or inhibit them. The attitude of polymorphonuclear neutrophils and macrophages to infiltrate and phagocytise biofilms, as well as the ambiguous behaviour exhibited by these innate immune cells in biofilm-related implant infections, are here discussed. Research on anti-biofilm biomaterials is focused, reviewing materials loaded with antibacterial substances, or coated with anti-adhesive/anti-bacterial immobilized agents, or surfaced with nanostructures. Latter approaches appear promising, since they avoid the spread of antibacterial substances in the neighbouring tissues with the consequent risk of inducing bacterial resistance.

New trends in diagnosis and control strategies for implant infections.

In implant infections, a quick and reliable identification of the etiological agent is crucial to realizing efficacious therapies. Among molecular methods, automated ribotyping has proven to be an accurate and rapid technique. More recently, MALDI-TOF/MS and PCR-electrospray ionization (ESI)/MS have been applied successfully to microbiological diagnosis. In implant infections, biofilm is still the major problem for bacterial persistence and recalcitrance to antibiotic therapy. Among biofilm-disrupting agents, enzymes promise the greatest therapeutic possibilities. DNase I degrades biofilm extracellular DNA and has been shown to sensitize biofilm to various biocides and anionic detergents, while dispersin B acts on biofilm exopolysaccharide and, combined with antiseptic, gives a broad-spectrum antibiofilm and antimicrobial activity. The novel antimicrobial approach based on photodynamic treatment (PDT) applies, in combination with antibiotics, to the implant or medical devices reachable by optical fibers. Better progress could be gained by the development of infection-resistant biomaterials able to both inhibit bacterial adhesion and promote tissue integration. New knowledge regarding the fibronectin-mediated internalization of Staphylococcus aureus by osteoblasts, and on its role in the pathogenesis of implant-related osteomyelitis, paves the way for the development of vaccines against staphylococcal adhesins, to prevent both adhesion on biomaterials and bacterial invasion of bone cells.

New methods for the detection of orthopedic and other biofilm infections.

The detection and identification of bacteria present in natural and industrial ecosystems is now entirely based on molecular systems that detect microbial RNA or DNA. Culture methods were abandoned, in the 1980s, because direct observations showed that <1% of the bacteria in these systems grew on laboratory media. Culture methods comprise the backbone of the Food and Drug Administration-approved diagnostic systems used in hospital laboratories, with some molecular methods being approved for the detection of specific pathogens that are difficult to grow in vitro. In several medical specialties, the reaction to negative cultures in cases in which overt signs of infection clearly exist has produced a spreading skepticism concerning the sensitivity and accuracy of traditional culture methods. We summarize evidence from the field of orthopedic surgery, and from other medical specialties, that support the contention that culture techniques are especially insensitive and inaccurate in the detection of chronic biofilm infections. We examine the plethora of molecular techniques that could replace cultures in the diagnosis of bacterial diseases, and we identify the new Ibis technique that is based on base ratios (not base sequences), as the molecular system most likely to fulfill the requirements of routine diagnosis in orthopedic surgery.

Orthopaedic biofilm infections.

A recent paradigm shift in microbiology affects orthopaedic surgery and most other medical and dental disciplines because more than 65% of bacterial infections treated by clinicians in the developed world are now known to be caused by organisms growing in biofilms. These slime-enclosed communities of bacteria are inherently resistant to host defenses and to conventional antibacterial therapy, and these device-related and other chronic bacterial infections are unaffected by the vaccines and antibiotics that have virtually eliminated acute infections caused by planktonic (floating) bacteria. We examine the lessons that can be learned, within this biofilm paradigm, by the study of problems (e.g. non-culturability) shared by all biofilm infections and by the study of new therapeutic options aimed specifically at sessile bacteria in biofilms. Orthopaedic surgery has deduced some of the therapeutic strategies based on assiduous attention to patient outcomes, but much can still be learned by attention to modern research in related disciplines in medicine and dentistry. These perceptions will lead to practical improvements in the detection, management, and treatment of infections in orthopaedic surgery.

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.

A new procedure allowing the complete removal and prevention of hemodialysis biofilms.

Most currently used disinfectants for dialysis machines have a good bactericidal efficacy on biofilms but leave dead cells on the surface. This contributes to the regrowth of biofilm and the release of pyrogens. A new anti-biofilm procedure consisting of sequential treatment combining enzymes and detergents is able to detach adherent cells. The efficacy of this procedure was assessed both in vitro and in reality. For in vitro studies, a biofilm model was set up. Studies were also performed in reality in a clinically used dialysis machine. Biofilm removal was first monitored by image analysis. Then, the biomass was detached by scraping and quantified by plate counts and endotoxin level measurement. Treated samples were compared to untreated control samples. The procedure led to the complete detachment of the biomass, both in vitro and in the reality situation. The aim of this procedure is to replace or complete the usual disinfection methods for medical devices.