John William Costerton

Biofilms in chronic wounds

Chronic wounds including diabetic foot ulcers, pressure ulcers, and venous leg ulcers are a worldwide health problem. It has been speculated that bacteria colonizing chronic wounds exist as highly persistent biofilm communities. This research examined chronic and acute wounds for biofilms and characterized microorganisms inhabiting these wounds. Chronic wound specimens were obtained from 77 subjects and acute wound specimens were obtained from 16 subjects. Culture data were collected using standard clinical techniques. Light and scanning electron microscopy techniques were used to analyze 50 of the chronic wound specimens and the 16 acute wound specimens. Molecular analyses were performed on the remaining 27 chronic wound specimens using denaturing gradient gel electrophoresis and sequence analysis. Of the 50 chronic wound specimens evaluated by microscopy, 30 were characterized as containing biofilm (60%), whereas only one of the 16 acute wound specimens was characterized as containing biofilm (6%). This was a statistically significant difference (p<0.001). Molecular analyses of chronic wound specimens revealed diverse polymicrobial communities and the presence of bacteria, including strictly anaerobic bacteria, not revealed by culture. Bacterial biofilm prevalence in specimens from chronic wounds relative to acute wounds observed in this study provides evidence that biofilms may be abundant in chronic wounds.

Human leukocytes adhere to, penetrate, and respond to Staphylococcus aureus biofilms

ABSTRACT Staphylococcus aureus is a common pathogen responsible for nosocomial and community infections. It readily colonizes indwelling catheters, forming microbiotic communities termed biofilms. S. aureus bacteria in biofilms are protected from killing by antibiotics and the bodys immune system. For years, one mechanism behind biofilm resistance to attack from the immune systems sentinel leukocytes has been conceptualized as a deficiency in the ability of the leukocytes to penetrate the biofilm. We demonstrate here that under conditions mimicking physiological shear, leukocytes attach, penetrate, and produce cytokines in response to maturing and fully matured S. aureus biofilm.

Growth and Detachment of Cell Clusters from Mature Mixed-Species Biofilms

ABSTRACT Detachment from biofilms is an important consideration in the dissemination of infection and the contamination of industrial systems but is the least-studied biofilm process. By using digital time-lapse microscopy and biofilm flow cells, we visualized localized growth and detachment of discrete cell clusters in mature mixed-species biofilms growing under steady conditions in turbulent flow in situ. The detaching biomass ranged from single cells to an aggregate with a diameter of approximately 500 μm. Direct evidence of local cell cluster detachment from the biofilms was supported by microscopic examination of filtered effluent. Single cells and small clusters detached more frequently, but larger aggregates contained a disproportionately high fraction of total detached biomass. These results have significance in the establishment of an infectious dose and public health risk assessment.

Influence of hydrodynamics and cell signaling on the structure and behavior of Pseudomonas aeruginosa biofilms

ABSTRACT Biofilms were grown from wild-type (WT) Pseudomonas aeruginosa PAO1 and the cell signaling lasI mutant PAO1-JP1 under laminar and turbulent flows to investigate the relative contributions of hydrodynamics and cell signaling for biofilm formation. Various biofilm morphological parameters were quantified using Image Structure Analyzer software. Multivariate analysis demonstrated that both cell signaling and hydrodynamics significantly (P < 0.000) influenced biofilm structure. In turbulent flow, both biofilms formed streamlined patches, which in some cases developed ripple-like wave structures which flowed downstream along the surface of the flow cell. In laminar flow, both biofilms formed monolayers interspersed with small circular microcolonies. Ripple-like structures also formed in four out of six WT biofilms, although their velocity was approximately 10 times less than that of those that formed in the turbulent flow cells. The movement of biofilm cell clusters over solid surfaces may have important clinical implications for the dissemination of biofilm subject to fluid shear, such as that found in catheters. The ability of the cell signaling mutant to form biofilms in high shear flow demonstrates that signaling mechanisms are not required for the formation of strongly adhered biofilms. Similarity between biofilm morphologies in WT and mutant biofilms suggests that the dilution of signal molecules by mass transfer effects in faster flowing systems mollifies the dramatic influence of signal molecules on biofilm structure reported in previous studies.

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.

Role of the Streptococcus gordonii SspB protein in the development of Porphyromonas gingivalis biofilms on streptococcal substrates

Porphyromonas gingivalis is an aggressive periodontal pathogen that persists in the mixed-species plaque biofilm on tooth surfaces. P. gingivalis cells attach to the plaque commensal Streptococcus gordonii and this coadhesion event leads to the development of P. gingivalis biofilms. Binding of these organisms is multimodal, involving both the P. gingivalis major fimbrial FimA protein and the species-specific interaction of the minor fimbrial Mfa1 protein with the streptococcal SspB protein. This study examined the contribution of the Mfa1-SspB interaction to P. gingivalis biofilm formation. P. gingivalis biofilms readily formed on substrata of S. gordonii DL1 but not on Streptococcus mutans cells which lack a coadhesion-mediating homologue of SspB. An insertional inactivation of the mfa1 gene in P. gingivalis resulted in a phenotype deficient in S. gordonii binding and unable to form biofilms. Furthermore, analysis using recombinant streptococci and enterococci showed that P. gingivalis biofilms formed on Enterococcus faecalis strains expressing SspB or translational fusions of SspB with SpaP (the non-adherent SspB homologue in S. mutans) containing the P. gingivalis adherence domain (SspB adherence region, BAR) of SspB. In contrast, an isogenic Ssp null mutant of S. gordonii DL1 was unable to support biofilm growth, even though this strain bound to P. gingivalis FimA at levels similar to wild-type S. gordonii DL1. Finally, site-specific mutation of two functional amino acid residues in BAR resulted in SspB polypeptides that did not promote the development of P. gingivalis biofilms. These results suggest that the induction of P. gingivalis biofilms on a streptococcal substrate requires functional SspB-minor fimbriae interactions.

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.

Detection of Staphylococcus aureus Biofilm on Tampons and Menses Components

Culturing has detected vaginal Staphylococcus aureus in 10%-20% of women. Because growth mode can affect virulence expression, this study examined S. aureus-biofilm occurrence in 44 paired-tampon and vaginal-wash-specimens from 18 prescreened women, using fluorescent in situ hybridization (FISH). All 44 specimens were also analyzed for S. aureus by standard culturing on mannitol salt agar, which produced positive results for 15 of the 44 specimens. FISH detected S. aureus cells in all 44 specimens, and S. aureus biofilm was observed in 37 of the 44 specimens. Independent confirmation of the presence of S. aureus in specimens from all 18 women was also obtained by amplification, via polymerase chain reaction, of an S. aureus-specific nuclease gene. The results of this study demonstrate that S. aureus biofilm can form on tampons and menses components in vivo. Additionally, the prevalence of vaginal S. aureus carriage may be more prevalent than what is currently demonstrated by standard culturing techniques.

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.