Recolonization after habitat restoration leads to decreased genetic variation in populations of a terrestrial orchid.

Colonization is crucial to habitat restoration projects that rely on the spontaneous regeneration of the original vegetation. However, as a previously declining plant species spreads again, the likelihood of founder effects increases through recurrent population founding and associated serial bottlenecks. We related Amplified Fragment Length Polymorphism markers genetic variation and fitness to colonization history for all extant populations of the outcrossing terrestrial orchid Dactylorhiza incarnata in an isolated coastal dune complex. Around 1970, D. incarnata suffered a severe bottleneck yet ultimately persisted and gradually spread throughout the spatially segregated dune slacks, aided by the restoration of an open vegetation. Genetic assignment demonstrated dispersal to vacant sites from few nearby extant populations and very limited inflow from outside the spatially isolated reserve. Results further indicated that recurrent founding from few local sources resulted in the loss of genetic diversity and promoted genetic divergence (F(ST) = 0.35) among populations, but did not influence population fitness. The few source populations initially available and the limited inflow of genes from outside the study reserve, as a consequence of habitat degradation and spatial isolation, may have magnified the genetic effects of recurrent population founding.

Biomaterial-centered infection: microbial adhesion versus tissue integration.

Biomaterials are being used with increasing frequency for tissue substitution. Complex devices such as total joint replacements and the total artificial heart represent combinations of polymers and metal alloys for system and organ replacement. The major barriers to the extended use of these devices are the possibility of bacterial adhesion to biomaterials, which causes biomaterial-centered infection, and the lack of successful tissue integration or compatibility with biomaterial surfaces. Interactions of biomaterials with bacteria and tissue cells are directed not only by specific receptors and outer membrane molecules on the cell surface, but also by the atomic geometry and electronic state of the biomaterial surface. An understanding of these mechanisms is important to all fields of medicine and is derived from and relevant to studies in microbiology, biochemistry, and physics. Modifications to biomaterial surfaces at an atomic level will allow the programming of cell-to-substratum events, thereby diminishing infection by enhancing tissue compatibility or integration, or by directly inhibiting bacterial adhesion.

Adherent bacterial colonization in the pathogenesis of osteomyelitis.

Direct scanning electron microscopy of material obtained during surgical debridement of osteomyelitic bone showed that the infecting bacteria grew in coherent microcolonies in an adherent biofilm so extensive it often obscured the infected bone surfaces. Transmission electron microscopy showed this biofilm to have a fibrous matrix, to contain some host cells, and to contain many bacteria around which matrix fibers were often concentrated. Many bacterial morphotypes were present in these biofilms, and each bacterium was surrounded by exopolysaccharide polymers, which are known to mediate formation of microcolonies and adhesion of bacteria to surfaces in natural ecosystems and in infections related biomaterials. The adherent mode of growth may reduce the susceptibility of these organisms to host clearance mechanisms and antibiotic therapy and thus may be a fundamental factor in acute and chronic osteomyelitis.

Scanning and transmission electron microscopy as tools for the study of phagocytosis of bacteria adherent to hard surfaces.

A technique for studying the phagocytosis of bacteria colonizing hard surfaces is described. Rabbit peritoneal macrophages were allowed to settle on the surface of high-molecular-weight polyethylene which had been previously colonized by Escherichia coli. To ascertain the presence of bacteria on the surface of the polyethylene and the degree of spreading of the attached macrophages, the preparations were observed by scanning electron microscopy. The ingestion of E. coli by the macrophages was studied by transmission electron microscopy on ultrathin sections of resin-embedded monolayers after their separation from the polyethylene surface. Numerous intracellular bacteria were located near the area of attachment of the macrophages to the substrata, suggesting that the phagocytosis of bacteria adherent to the surface of the plastic had taken place.

Particle-induced in vivo priming of alveolar macrophages for enhanced oxidative responses: a novel system of cellular immune augmentation.

A novel system for priming adult rabbit alveolar macrophages (AMs) in vivo for markedly enhanced oxidative responses is described. When adult rabbits were injected intravenously (i.v.) with 1- to 5-microns particles such as zymosan, latex particles, or heat-killed bacille Calmette-Guérin, AMs were primed in 1-3 days for greatly enhanced phorbol myristate acetate (PMA)- or opsonized zymosan (Op-zym)-elicited chemiluminescent (CL) responses. Intratracheal (i.t.) injection of zymosan particles also primed AMs for enhanced PMA- or Op-zym-elicited CL responses. AMs obtained from particle-injected rabbits showed up to 100-fold higher levels of PMA-elicited CL responses than AMs from normal rabbits. In contrast, Op-zym failed to prime normal AMs in vitro for enhanced CL responses. Whereas AMs could not be primed in vivo with an i.v. injection of particles of approximately 24 microns diameter. AMs could be primed if the particles were administered by the i.t. route. The priming appears to be independent of particle types. The priming effect was of short duration and declined after 5 to 7 days. The possibility that this system represents the primitive cellular immune response found in invertebrates is discussed. The potential use of this system as a means of immune augmentation prompts further investigation.

Altered oxidative responses and antibacterial activity of adult rabbit alveolar macrophages exposed to poly(methyl methacrylate).

The effect of poly(methyl methacrylate) (PMMA) on the oxidative responses and antibacterial activity of adult rabbit alveolar macrophages (AM) was studied. PMMA beads (ca. 0.3 micron diameter) elicited an acute respiratory burst within 6-8 min after the addition of the beads. In contrast. Teflon beads of comparable size (ca. 0.2 micron diameter) did not elicit an oxidative burst of AM. An oxidative response was elicited only by those PMMA samples that had affinity for AM adherence. Incubation of AM with PMMA beads reduced the subsequent phorbol myristate acetate (PMA)-elicited oxidative burst by more than 80%. The Staphylococcus epidermidis--RP12 killing capacity of AM was greatly increased when PMMA beads (ca. 0.3 micron) were added to the challenge dose of bacteria. Pre-incubation of freshly harvested AM with PMMA beads, which greatly reduced subsequent PMA-elicited chemiluminescent (CL) responses did not significantly affect the RP12 killing capacity of AM. Our data also suggest that killing of the RP12 strain of S. epidermidis does not involve reactive oxygen intermediates.

In vitro and in vivo comparative colonization of Staphylococcus aureus and Staphylococcus epidermidis on orthopaedic implant materials.

Clinically, Staphylococcus aureus appears to be the dominant organism associated with infected metal implants, whereas coagulase-negative staphylococcal strains are more frequently isolated from infected polymer implants. We reproduced this trend experimentally in vitro and in vivo. Discs of a titanium alloy, poly(methyl methacrylate) and ultra-high molecular weight polyethylene were exposed to a clinical isolate of Staphylococcus aureus or either of two strains of Staphylococcus epidermidis. Within 1 h Staphylococcus aureus was always the most rapid colonizer regardless of biomaterial. However, after 8 to 24 h, Staphylococcus aureus was present in higher numbers on metal and Staphylococcus epidermidis on polymers. Moreover, the exopolysaccharide produced by Staphylococcus epidermidis appeared to offer an effective protection against host defences in vivo.

Adhesive colonization of biomaterials and antibiotic resistance.

This study addresses the problem of antibiotic resistance in adhesive, biomaterial-centred infections. It is suggested that this anionic, extracapsular, polysaccharide slime produced by bacteria protects them from antibiotics and sequesters critical ions from the surface of biomaterials. Biofilm-enclosed bacteria on the surface of stainless steel substrata in a test chamber were challenged with incremental levels of tobramycin. In this setting, the minimum inhibitory concentration and minimum bactericidal level of tobramycin for Staphylococcus epidermis were well above normal.

Surgical biomaterials and differential colonization by Staphylococcus epidermidis.

The data presented in this communication demonstrate preferential colonization of certain biomaterials by Staphylococcus epidermidis. Using a laminar flow biomaterial colonization chamber and surgical-grade biomaterials (stainless steel, aluminium ceramic, methyl methacrylate and high-density polyethylene), the pattern of colonization was quantitated using plate count techniques and electron microscopy. Under comparable conditions, methyl methacrylate was colonized by S. epidermidis in greater numbers than the other biomaterials. Increased bacterial colonization and slime production on methyl methacrylate was time-dependent and 15 times higher than on stainless steel and aluminium and four times higher than on high-density polyethylene. The data reveal that certain biomaterials may promote infection by favouring colonization by potential pathogens. This variable should be explored extensively in an in vivo setting because of its implication in clinical infections.

Staphylococcal adhesion to collagen in intra-articular sepsis.

Ultrastructural studies of the cartilaginous articular surfaces of human and rabbit joints have shown that cartilage is the target substratum for adhesion by Staphylococcus aureus, leading to intra-articular sepsis. Transmission and scanning electron microscope studies demonstrated bacteria in intimate contact with acellular cartilage matrix surfaces, particularly with collagen fibres. Certain strains of Staphylococcus aureus used in these experiments reveal a high binding capacity to collagen that is derived from a cartilage matrix. These studies indicate that the pathogenesis of intra-articular sepsis is based on the ability of certain strains of staphylococci to bind preferentially to a cartilage matrix.

Site-specific adhesion of Staphylococcus epidermidis (RP12) in Ti-Al-V metal systems.

Staphylococcus epidermidis (RP12) adhesion patterns were studied on the following titanium (Ti)-aluminium (Al)-vanadium (V) metal systems: (i) microfabricated samples consisting of Ti, Al and V islands deposited onto Ti or V substrata, (ii) pure Ti, Al and V metals, and (iii) medical grade Ti6Al4-V alloy. All of these surfaces were covered with their respective oxides formed upon exposure of the metals to air. Quantitative analysis of the number of cells bound per unit area indicates that S. epidermidis (RP12) exhibits greatest adhesion to pure V surfaces. When exposed to surfaces having controlled spatial variations in chemical composition on the 10 microns scale (microfabricated samples), the bacteria preferentially populate V islands versus Ti or Al substrata. In the case of the biphasic Ti6Al4V alloy, the bacteria tend to adhere to V-rich, mixed phase regions and phase boundaries. These findings demonstrate that enhanced and preferential adhesion of S. epidermidis (RP12) occurs on V surfaces in Ti-Al-V metal systems and suggest that bacterial interactions are influenced by surface oxide composition.

Extended-wear lenses, biofilm, and bacterial adhesion.

While medical scientific knowledge pertaining to bacterial adhesion to biomaterials has become a rapidly growing field in most areas of medicine, its significance in ophthalmic infections has not been emphasized. Corneal bacterial ulceration in patients wearing extended-wear contact lenses has become a problem of epidemic proportions. The designation of the contact lens itself as a suitable substratum for bacterial colonization and as a source of subsequent inoculum to compromised epithelial cells are important factors in the pathophysiology of corneal ulcer formation. We demonstrate polysaccharide- (biofilm-)mediated adhesion to two ophthalmic pathogens (Pseudomonas aeruginosa and Staphylococcus epidermidis) to the surface of a typical extended-wear contact lens in vitro using cytochemistry and scanning and transmission electron microscopic techniques. This interaction between the biomaterial and bacterial organisms, which represents a favorable self-protective environment for propagation and inoculation, is a previously overlooked area of importance in the mechanism of corneal ulceration associated with hydrophilic (soft) contact lenses.

Bacterial colonization of percutaneous sutures.

The direct electron microscopic examination of 15 sutures and 15 staples removed from 10 healed surgical wounds showed, on the intradermal portions, consistent colonization by bacteria growing in adherent biofilms. This clearly demonstrable bacterial colonization of biomaterials within the wound tract had not resulted in infection or perceptible inflammation in any of the wounds. These bacterial cells were of several morphotypes, including gram-positive cocci, and all specimens yielded cultures of the autochthonous (native) skin bacterium, Staphylococcus epidermidis. The bacteria within the wound tracts were enveloped by extracellular material that appeared on scanning electron microscopy to be a condensed amorphous residue and on transmission electron microscopy to be a fibrous extracellular matrix. We suggest that this mode of growth, in which the colonizing bacteria are enveloped in a copious exopolysaccharide glycocalix, protects the bacteria from host defense factors and accounts for their persistence on the suture surfaces until they are removed with the sutures.

Musculoskeletal infection, microbial adhesion, and antibiotic resistance.

Osteomyelitis and intra-articular infection are septic diseases that present pathogenic features characteristic of molecular mechanisms involving adhesion to substrata. In this review, mechanisms of microbial adhesion to bone and cartilage as substrata are presented and related to host tissue response and to antibiotic treatment.

Bioimplant surfaces: binding of fibronectin and fibroblast adhesion.

The adhesion of baby hamster kidney 21C/13 fibroblasts to surfaces of passivated titanium, carbon fibers, bioactive glasses B5 and B6, fibronectin-precoated passivated titanium, and fibronectin-precoated B6 was quantified. The order of adhesive cell avidity for the uncoated surfaces was passivated titanium (greatest), B6 and carbon fibers (intermediate), and B5 (least). Precoating with fibronectin enhanced the adhesive characteristics of fibroblasts on passivated titanium and B6 by 74% and 118%, respectively.

Bacterial adherence to biomaterials and tissue. The significance of its role in clinical sepsis.

The direct examination of tissue and biomaterials from prosthesis-related infections of twenty-five patients showed that the causative bacteria grew in glycocalyx-enclosed biofilms that were adherent to surfaces of biomaterials and tissues in 76 per cent. This high rate of recovery of adherent biofilm-mediated growth suggests that the process occurs commonly in the presence of a foreign body or biomaterial-related infection. Because of the adherent mode of growth of the infecting organisms, accurate microbiological sampling was difficult. The analysis of joint fluids or of swabs of excised tissue and of prosthetic surfaces often yielded only one species from what was a polymicrobial population based on electron microscopic studies. We adapted direct quantitative sampling methods from environmental microbiology in order to recover a large number of species from these infections, but comparison of the organisms isolated by these techniques with the morphological types that were seen by electron microscopy indicated that in some instances all bacterial components of the biofilms were still not being recovered.

Spontaneous septic arthritis complicating rheumatoid arthritis.

Thirteen cases (in twelve patients) of septic arthritis complicating rheumatoid arthritis are reported. One ankle, one metacarpopophalangeal joint, one shoulder, and ten knees were involved. Staphylococcus aureus was cultured from twelve joints and Escherichia coli, from one. Treatment consisted of repeated needle aspirations in two patients, arthrotomy with Penrose drainage in six, and arthrotomy with through-and-through irrigation in four. Needle aspiration was the least effective therapy. The authors recommend as the treatment of choice: systemic antibiotic therapy and immediate arthrotomy followed by through-and-through irrigation with fluid containing the appropriate antibiotics.

Methylmethacrylate as an adjunct in internal fixation of pathological fractures. Experience with three hundred and seventy-five cases.

In 323 patients with 375 pathological fractures or impending fractures, local tumor resection and internal fixation supplemented by intramedullary methylmethacrylate proved highly successful. One hundred and thirty-nine patients had metastases from breast carcinoma; 142, metastases from other tumors; and forty-two, myeloma or lymphoma. The mean survival for the 210 patients who had undergone operation two years or more before final evaluation was 15.4 months. Ninety-four per cent of the patients who were ambulatory before fracture regained the ability to walk. Eighty-five per cent had excellent or good pain relief and in only five was pain relief rated poor. There were four failures of fixation and six functionally poor results. Twenty patients died within four weeks of operation, but the remaining patients benefited from the procedure in terms of pain relief, improved mobility, and ease of nursing care.

A novel spinal implant infection model in rabbits.

STUDY DESIGN: A new spinal implant model was designed to study device-centered infection with methicillin-resistant Staphylococcus aureus in multiple noncontiguous surgical sites in the lumbar spine region of a rabbit. OBJECTIVE: To develop a multiple-site spinal implant device-centered infection model in rabbits. SUMMARY OF BACKGROUND DATA: Results in many recent studies show that postoperative wound infection after spinal implant surgery and the increase in antibiotic-resistant bacteria are a concern. Anti-infection strategies must be tested in relevant animal models that will lead to appropriate clinical studies. METHODS: Eight anesthetized New Zealand White rabbits underwent completely isolated partial laminectomy and subsequent stainless steel Kirschner wire implantation directly into the transverse processes of vertebrae T13, L3, and L6. The middle sites (L3) were used as sterile control sites, and the outer sites (T13, L6) were challenged with different amounts of methicillin-resistant Staphylococcus aureus. Rabbits were killed after 7 days, and biopsies were performed to provide evidence for device-centered infection. Bacterial growth on the implant surfaces and in surrounding tissues and bone was assayed. RESULTS: Overall device-centered infection was established after 7 days in 100% of the sites challenged with 10(3) colony-forming units methicillin-resistant Staphylococcus aureus or higher. No infection was seen in any of the control sites located between infected vertebrae. Multiple blood and liver samples showed that the separate localized infections did not become systemic after 7 days. CONCLUSIONS: This new animal model demonstrates that multiple biomaterial implants can be evaluated in the same animal and provides a technique for investigating postoperative device-centered infection of the spine. Infection was demonstrated in noncontiguous lumbar sites of the spine, whereas adjacent control sites remained sterile. Because there was no cross contamination or systemic spread of the infection, multiple anti-infection strategies or implant materials can now be tested for efficacy in a single animal to combat dramatic and costly postoperative implant infections.

Musculoskeletal injuries in theatrical dance students.

Three hundred and fifty-two injuries in 185 theatrical dance students at the North Carolina School of the Arts were diagnosed and treated from September 1981 through May 1982, the most recent academic year. The total number of dancers enrolled in the school was 218; thus, 84.9% of the dance students were evaluated for an injury by a physician, with 87.8% of the injuries sustained during dance. Approximately one-fourth of the dance-related injuries involved the foot or ankle. The injuries were usually not severe, and proved amenable to treatment by conservative measures.