J. William Costerton

Antibiotic resistance of bacteria in biofilms

Bacteria that adhere to implanted medical devices or damaged tissue can encase themselves in a hydrated matrix of polysaccharide and protein, and form a slimy layer known as a biofilm. Antibiotic resistance of bacteria in the biofilm mode of growth contributes to the chronicity of infections such as those associated with implanted medical devices. The mechanisms of resistance in biofilms are different from the now familiar plasmids, transposons, and mutations that confer innate resistance to individual bacterial cells. In biofilms, resistance seems to depend on multicellular strategies. We summarise the features of biofilm infections, review emerging mechanisms of resistance, and discuss potential therapies.

Staphylococcus aureus biofilms: Properties, regulation, and roles in human disease

Increasing attention has been focused on understanding bacterial biofilms and this growth modality’s relation to human disease. In this review we explore the genetic regulation and molecular components involved in biofilm formation and maturation in the context of the Gram-positive cocci, Staphylococcus aureus. In addition, we discuss diseases and host immune responses, along with current therapies associated with S. aureus biofilm infections and prevention strategies.

Cystic fibrosis pathogenesis and the role of biofilms in persistent infection

Recent work has presented definitive evidence that the Pseudomonas cells that infect the lung in cystic fibrosis grow in the biofilm phenotype. These unequivocal data establish this chronic infection of compromised hosts as the archetypal biofilm infection, which is both refractory to antibiotic therapy and barely affected by host defenses.

Bacterial biofilms: a diagnostic and therapeutic challenge

Bacteria have traditionally been regarded as individual organisms growing in homogeneous planktonic populations. However, bacteria in natural environments usually form communities of surface-adherent organisms embedded in an extracellular matrix, called biofilms. Current antimicrobial strategies often fail to control bacteria in the biofilm mode of growth. Treatment failure is particularly frequent in association with intracorporeal or transcutaneous medical devices and compromised host immunity. The rising prevalence of these risk factors over the last decades has paralleled the increase in biofilm infections. This review discusses the shortcomings of current therapies against biofilms both in theory and with clinical examples. Biofilm characteristics are described with a focus on new diagnostic and therapeutic targets.

Identification of Staphylococcus aureus Proteins Recognized by the Antibody-Mediated Immune Response to a Biofilm Infection

ABSTRACT Staphylococcus aureus causes persistent, recurrent infections (e.g., osteomyelitis) by forming biofilms. To survey the antibody-mediated immune response and identify those proteins that are immunogenic in an S. aureus biofilm infection, the tibias of rabbits were infected with methicillin-resistant S. aureus to produce chronic osteomyelitis. Sera were collected prior to infection and at 14, 28, and 42 days postinfection. The sera were used to perform Western blot assays on total protein from biofilm grown in vitro and separated by two-dimensional gel electrophoresis. Those proteins recognized by host antibodies in the harvested sera were identified via matrix-assisted laser desorption ionization-time of flight analysis. Using protein from mechanically disrupted total and fractionated biofilm protein samples, we identified 26 and 22 immunogens, respectively. These included a cell surface-associated β-lactamase, lipoprotein, lipase, autolysin, and an ABC transporter lipoprotein. Studies were also performed using microarray analyses and confirmed the biofilm-specific up-regulation of most of these genes. Therefore, although the biofilm antigens are recognized by the immune system, the biofilm infection can persist. However, these proteins, when delivered as vaccines, may be important in directing the immune system toward an early and effective antibody-mediated response to prevent chronic S. aureus infections. Previous works have identified S. aureus proteins that are immunogenic during acute infections, such as sepsis. However, this is the first work to identify these immunogens during chronic S. aureus biofilm infections and to simultaneously show the global relationship between the antigens expressed during an in vivo infection and the corresponding in vitro transcriptomic and proteomic gene expression levels.

Identification of Microbial Biofilms in Osteonecrosis of the Jaws Secondary to Bisphosphonate Therapy

PURPOSE Biofilm theory has emerged to explain the etiology of the chronic infections that have come to constitute between 65% to 80% of the microbial diseases treated by physicians in the developed world. The purpose of this article is to report for the first time the observation of multispecies microbial biofilms on affected bone in patients with osteonecrosis of the jaws (ONJ) secondary to bisphosphonate therapy. PATIENTS AND METHODS A program has been established at the University of Southern California to monitor and evaluate patients with ONJ as a multidisciplinary collaboration between the School of Dentistry, Center for Biofilms, Center for Craniofacial Molecular Biology and the Keck School of Medicine. From this cohort, 4 patients with active ONJ who were scheduled for necessary treatment in the form of sequestrectomy gave informed consent for this study. Bone samples were evaluated using conventional histopathologic techniques and scanning electron microscopy, a technique applicable to biofilm characterization. RESULTS Bone specimens from affected sites in all patients showed large areas occluded with biofilms comprising mainly bacteria, and occasionally yeast, embedded in extracellular polymeric substance. The number of bacterial morphotypes in the biofilms ranged from 2 to 15, and they included species from the genus Fusobacterium, bacillus, actinomyces, staphylococcus, streptococcus, Selenomonas, and 3 different types of treponemes. The yeast identified was consistent with Candida species. Co-aggregation was observed between different species within the biofilms. CONCLUSION These findings have important clinical and therapeutic implications and may suggest a role for microbial biofilms in the disease process of ONJ.

Towards diagnostic guidelines for biofilm-associated infections.

Biofilms associated with the human body, particularly in typically sterile locations, are difficult to diagnose and treat effectively because of their recalcitrance to conventional antibiotic therapy and host immune responses. The study of biofilms in medicine today requires a translational approach, with examination of clinically relevant biofilms in the context of specific anatomic sites, host tissues, and diseases, focusing on what can be done to mitigate their pathologic consequences. This review, which grew out of a discussion session on clinical biofilms at the 5th ASM Biofilm Conference in Cancun, Mexico, is designed to give an overview of biofilm-associated infections (BAI) and to propose a platform for further discussion that includes clinicians, medical microbiologists, and biofilm researchers who are stakeholders in advancing the scientific pursuit of better diagnosis and treatment of BAI to mitigate their human and healthcare costs. It also highlights the need for better diagnostic markers, which exploit the difference between planktonic and biofilm cells.

Gene Expression in Pseudomonas aeruginosa: Evidence of Iron Override Effects on Quorum Sensing and Biofilm-Specific Gene Regulation

Prior studies established that the Pseudomonas aeruginosa oxidative stress response is influenced by iron availability, whereas more recent evidence demonstrated that it was also controlled by quorum sensing (QS) regulatory circuitry. In the present study, sodA (encoding manganese-cofactored superoxide dismutase [Mn-SOD]) and Mn-SOD were used as a reporter gene and endogenous reporter enzyme, respectively, to reexamine control mechanisms that govern the oxidative stress response and to better understand how QS and a nutrient stress response interact or overlap in this bacterium. In cells grown in Trypticase soy broth (TSB), Mn-SOD was found in wild-type stationary-phase planktonic cells but not in a lasI or lasR mutant. However, Mn-SOD activity was completely suppressed in the wild-type strain when TSB was supplemented with iron. Reporter gene studies indicated that sodA transcription could be variably induced in iron-starved cells of all three strains, depending on growth stage. Iron starvation induction of sodA was greatest in the wild-type strain and least in the lasR mutant and was maximal in stationary-phase cells. Reporter experiments in the wild-type strain showed increased lasI::lacZ transcription in response to iron limitation, whereas the expression level in the las mutants was minimal and iron starvation induction of lasI::lacZ did not occur. Studies comparing Mn-SOD activity in P. aeruginosa biofilms and planktonic cultures were also initiated. In wild-type biofilms, Mn-SOD was not detected until after 6 days, although in iron-limited wild-type biofilms Mn-SOD was detected within the initial 24 h of biofilm establishment and formation. Unlike planktonic bacteria, Mn-SOD was constitutive in the lasI and lasR mutant biofilms but could be suppressed if the growth medium was amended with 25 microM ferric chloride. This study demonstrated that (i) the nutritional status of the cell must be taken into account when one is evaluating QS-based gene expression; (ii) in the biofilm mode of growth, QS may also have negative regulatory functions; (iii) QS-based gene regulation models based on studies with planktonic cells must be modified in order to explain biofilm gene expression behavior; and (iv) gene expression in biofilms is dynamic.

Prevention of staphylococcal biofilm-associated infections by the quorum sensing inhibitor RIP

Staphylococcus aureus and Staphylococcus epidermidis associated with implantable medical devices, are often difficult to treat with conventional antimicrobials. Formation of a biofilm and subsequent production of toxins are two distinct mechanisms considered important in foreign body infections. Staphylococcal virulence is caused by a complex regulatory process, which involves cell-to-cell communication through the release and response to chemical signals in a process known as quorum sensing. We explored the possibility of preventing infections by interfering with biofilm formation and toxin production using the quorum sensing inhibitor ribonucleic-acid-III-inhibiting peptide. In our studies ribonucleic-acid-III-inhibiting peptide prevented graft-associated infections caused by all species of staphylococci tested so far, including methicillin resistant S. aureus and S. epidermidis. Ribonucleic-acid-III-inhibiting peptide also enhances the effects of antibiotics and cationic peptides in the clearance of normally recalcitrant biofilm infections. Ribonucleic-acid-III-inhibiting peptide is nontoxic, highly stable, and no resistant strains have been found so far, suggesting that ribonucleic-acid-III-inhibiting peptide may be used to coat medical devices or used systemically to prevent infections. When the target of ribonucleic-acid-III activating protein activity is disrupted, biofilm formation is reduced under flow and static conditions and genes important for toxin production or biofilm formation are down-regulated. These in vitro data help explain why ribonucleic-acid-III-inhibiting peptide seems to be effective in preventing staphylococcal infections.

Resolution of Staphylococcus aureus biofilm infection using vaccination and antibiotic treatment

ABSTRACT Staphylococcus aureus infections, particularly those from methicillin-resistant strains (i.e., MRSA), are reaching epidemic proportions, with no effective vaccine available. The vast number and transient expression of virulence factors in the infectious course of this pathogen have made the discovery of protective antigens particularly difficult. In addition, the divergent planktonic and biofilm modes of growth with their accompanying proteomic changes also demonstrate significant hindrances to vaccine development. In this study, a multicomponent vaccine was evaluated for its ability to clear a staphylococcal biofilm infection. Antigens (glucosaminidase, an ABC transporter lipoprotein, a conserved hypothetical protein, and a conserved lipoprotein) were chosen since they were found in previous studies to have upregulated and sustained expression in a biofilm, both in vitro and in vivo. Antibodies against these antigens were first used in microscopy studies to localize their expression in in vitro biofilms. Each of the four antigens showed heterogeneous production in various locations within the complex biofilm community in the biofilm. Based upon these studies, the four antigens were delivered simultaneously as a quadrivalent vaccine in order to compensate for this varied production. In addition, antibiotic treatment was also administered to clear the remaining nonattached planktonic cells since the vaccine antigens may have been biofilm specific. The results demonstrated that when vaccination was coupled with vancomycin treatment in a biofilm model of chronic osteomyelitis in rabbits, clinical and radiographic signs of infection significantly reduced by 67 and 82%, respectively, compared to infected animals that were either treated with vancomycin or left untreated. In contrast, vaccination alone resulted in a modest, and nonsignificant, decrease in clinical (34% reduction) and radiographic signs (9% reduction) of infection, compared to nonvaccinated animal groups untreated or treated with vancomycin. Lastly, MRSA biofilm infections were significantly cleared in 87.5% of vaccinated and antibiotic-treated animals, while antibiotics or vaccine alone could not significantly clear infection compared to controls (55.6, 22.2, and 33.3% clearance rates, respectively). This approach to vaccine development may lead to the generation of vaccines against other pathogenic biofilm bacteria.