Bacterial infections are serious complications after orthopaedic implant surgery. Staphylococci, with Staphylococcus epidermidis as a leading species, are the prevalent and most important species involved in orthopaedic implant-related infections. The biofilm mode of growth of these bacteria on an implant surface protects the organisms from the host's immune system and from antibiotic therapy. Therapeutic agents that disintegrate the biofilm matrix would release planktonic cells into the environment and therefore allow antibiotics to eliminate the bacteria. An addition of a biofilm-degrading agent to a solution used for washing-draining procedures of infected orthopaedic implants would greatly improve the efficiency of the procedure and thus help to avoid the removal of the implant. We have previously shown that the extracellular staphylococcal matrix consists of a poly-N-acetylglucosamine (PNAG), extracellular teichoic acids (TAs) and protein components. In this study, we accessed the sensitivity of pre-formed biofilms of five clinical staphylococcal strains associated with orthopaedic prosthesis infections and with known compositions of the biofilm matrix to periodate, Pectinex Ultra SP, proteinase K, trypsin, pancreatin and dispersin B, an enzyme with a PNAG-hydrolysing activity. We also tested the effect of these agents on the purified carbohydrate components of staphylococcal biofilms, PNAG and TA. We found that the enzymatic detachment of staphylococcal biofilms depends on the nature of their constituents and varies between the clinical isolates. We suggest that a treatment with dispersin B followed by a protease (proteinase K or trypsin) could be capable to eradicate biofilms of a variety of staphylococcal strains on inert surfaces.

Staphylococcus aureus and coagulase-negative staphylococci, primarily Staphylococcus epidermidis, are recognized as a major cause of nosocomial infections associated with the use of implanted medical devices. It has been established that clinical isolates often produce a biofilm, which is involved in adherence to biomaterials and provides enhanced resistance of bacteria against host defenses and antibiotic treatments. It has been thought that the staphylococcal biofilm contains two polysaccharides, one responsible for primary cell adherence to biomaterials (polysaccharide/adhesin [PS/A]) and an antigen that mediates bacterial aggregation (polysaccharide intercellular adhesin [PIA]). In the present paper we present an improved procedure for preparation of PIA that conserves its labile substituents and avoids contamination with by-products. Based on structural analysis of the polysaccharide antigens and a thorough overview of the previously published data, we concluded that PIA from S. epidermidis is structurally identical to the recently described poly-␤-(136)-Nacetylglucosamine from PS/A-overproducing strain S. aureus MN8m. We also show that another carbohydratecontaining polymer, extracellular teichoic acid (EC TA), is an essential component of S. epidermidis RP62A biofilms. We demonstrate that the relative amounts of extracellular PIA and EC TA produced depend on the growth conditions. Moderate shaking or static culture in tryptic soy broth favors PIA production, while more EC TA is produced in brain heart infusion medium.

At least three different sets of symbiotic signals (here, they are compared to locks and keys) are exchanged between legumes and rhizobia during nodule development. Flavonoids, the first of these, emanate from the plant and interact with rhizobial NodD proteins that serve as both environmental sensors and activators of transcription. A second set of signals is synthesized when NodD-flavonoid complexes activate transcription from nod boxes. Most of the genes immediately downstream of these promoters are involved in the synthesis of lipooligosaccharidic Nod factors that provoke deformation of root hairs and allow rhizobia to enter the root through infection threads. Fine-tuning of transcription of nodulation (nod) genes is probably related to sequence variations in individual nod boxes (there are 19 on the symbiotic plasmid of the broad-hostrange Rhizobium sp. strain NGR234). Other rhizobial products seem to be necessary for continued infection thread development, and these represent a third set of signals. Among them are extracellular polysaccharides (EPS) and related compounds, as well as proteins exported by the type three secretion system (TTSS). In the latter case, flavonoids also activate protein secretion, suggesting that the same keys can unlock different doors.Symbioses are nearly ubiquitous, and many are also persistent (56). Mutualistic, nitrogen-fixing associations between members of the plant family Leguminosae, and the soil bacteria Azorhizobium, Bradyrhizobium, Mesorhizobium, and Rhizobium (collectively called rhizobia) contribute substantially to plant productivity (111). Legume-Rhizobium symbioses are marriages between two vastly different genomes. In rhizobia, these include a chromosome plus zero to many plasmids, totalling 6 to 9 Mbp (71). In contrast, genomes of legumes are much larger, some comprising more than 20 chromosomes with total DNA contents that range from about 450 to 4,500 Mbp per haploid genome (1C) (1). As examples, the model legumes Lotus japonicus and Medicago truncatula have six or eight chromosomes totalling 450 and 500 Mbp/1C (14), respectively. Common beans (Phaseolus vulgaris) and related species (e.g., Vigna unguiculata) (both genera belong to the tribe Phaseoleae) have 11 chromosomes each (24) with DNA contents of 637 (1) and 540 Mbp/1C (50). Furthermore, several widely cultivated legumes such as Arachis hypogaea, Glycine max, and Medicago sativa are effectively polyploid, although in G. max most loci segregate as if they were diploid. Legume genomes are thus at least 50 times larger than those of their microsymbionts. Given this disparity, it is difficult to imagine that theirs is a marriage of equals. Nevertheless, their respective contributions are probably not vastly different (see reference 48). LEGUME BOWERS ARE DECORATED WITH FLAVONOIDSWhich partner initiates contact? Since plants are nonmotile, it is tempting to think that courtship begins when bacteria advance into the legume rhizosphere. Yet chemotaxis and motility among rhizobia are clearly not essential for no...

Most field isolates of the swine pathogen Actinobacillus pleuropneumoniae form tenacious biofilms on abiotic surfaces in vitro. We purified matrix polysaccharides from biofilms produced by A. pleuropneumoniae field isolates IA1 and IA5 (serotypes 1 and 5, respectively), and determined their chemical structures by using NMR spectroscopy. Both strains produced matrix polysaccharides consisting of linear chains of N-acetyl-D-glucosamine (GlcNAc) residues in beta(1,6) linkage (poly-beta-1,6-GlcNAc or PGA). A small percentage of the GlcNAc residues in each polysaccharide were N-deacetylated. These structures were nearly identical to those of biofilm matrix polysaccharides produced by Escherichia coli, Staphylococcus aureus and Staphylococcus epidermidis. PCR analyses indicated that a gene encoding the PGA-specific glycoside transferase enzyme PgaC was present on the chromosome of 15 out of 15 A. pleuropneumoniae reference strains (serotypes 1-12) and 76 out of 77 A. pleuropneumoniae field isolates (serotypes 1, 5 and 7). A pgaC mutant of strain IA5 failed to form biofilms in vitro, as did wild-type strains IA1 and IA5 when grown in broth supplemented with the PGA-hydrolyzing enzyme dispersin B. Treatment of IA5 biofilms with dispersin B rendered them more sensitive to killing by ampicillin. Our findings suggest that PGA functions as a major biofilm adhesin in A. pleuropneumoniae. Biofilm formation may have relevance to the colonization and pathogenesis of A. pleuropneumoniae in pigs.

Legumes form tripartite symbiotic associations with nodule-inducing soil bacteria of the genera Rhizobium, Brudyrhizobium, or Azorkizobium (Caetano-Anollés and Gresshoff, 1991;Hirsch, 1992) and with VAM fungi (BonfanteFasolo, 1987;Koide and Schreiner, 1992). Both the rhizobial and fungal microsymbionts improve the mineral nutrition of the host plant in exchange for assimilates provided by the latter. The nitrogenase enzyme of rhizobia fixes atmospheric nitrogen in the nodules (Thorneley, 1992), and fungal hyphae facilitate the uptake of ions, mainly phosphate, in mycorrhizal roots (Smith and Gianinazzi-Pearson, 1988). In most cases investigated, especially when both nitrogen and phosphate are limiting factors, VAM fungi '

Clinical isolates of the periodontopathogen Aggregatibacter actinomycetemcomitans form matrixencased biofilms on abiotic surfaces in vitro. A major component of the A. actinomycetemcomitans biofilm matrix is PGA, a hexosamine-containing polysaccharide that mediates intercellular adhesion. In this report we describe studies on the purification, structure, genetics and function of A. actinomycetemcomitans PGA. We found that PGA was very tightly attached to A. actinomycetemcomitans biofilm cells and could be efficiently separated from the cells only by phenol extraction. A. actinomycetemcomitans PGA copurified with LPS on a gel filtration column. 1 H-NMR spectra of purified A. actinomycetemcomitans PGA were consistent with a structure containing a linear chain of N-acetyl-D-glucosamine residues in β(1,6) linkage. Genetic analyses indicated that all four genes of the pgaABCD locus were required for PGA production in A. actinomycetemcomitans. PGA mutant strains still formed biofilms in vitro. Unlike wild-type biofilms, however, PGA mutant biofilms were sensitive to detachment by DNase I and proteinase K. Treatment of A. actinomycetemcomitans biofilms with the PGA-hydrolyzing enzyme dispersin B made them 3 log units more sensitive to killing by the cationic detergent cetylpyridinium chloride. Our findings suggest that PGA, extracellular DNA and proteinaceous adhesins all contribute to the structural integrity of the A. actinomycetemcomitans biofilm matrix.

Extracellular DNA is an adhesive component of staphylococcal biofilms. The aim of this study was to evaluate the antibiofilm activity of recombinant human DNase I (rhDNase) against Staphylococcus aureus and Staphylococcus epidermidis. Using a 96-well microtiter plate crystal violet binding assay, we found that biofilm formation by S. aureus was efficiently inhibited by rhDNase at 1–4 μg l−1, and pre-formed S. aureus biofilms were efficiently detached in 2 min by rhDNase at 1 mg l−1. Pre-treatment of S. aureus biofilms for 10 min with 10 mg l−1 rhDNase increased their sensitivity to biocide killing by 4–5 log units. rhDNase at 10 mg l−1 significantly inhibited biofilm formation by S. epidermidis in medium supplemented with subminimal inhibitory concentrations of antibiotics. We also also found rhDNase significantly increased the survival of S. aureus-infected C. elegans nematodes treated with tobramycin compared to nematodes treated with tobramycin alone. We concluded that rhDNase exhibits potent antibiofilm and antimicrobial-sensitizing activities against S. aureus and S. epidermidis at clinically achievable concentrations. rhDNase, either alone or in combination with antimicrobial agents, may have applications in treating or preventing staphylococcal biofilm-related infections.

Staphylococcus aureus and coagulase-negative staphylococci, primarily Staphylococcus epidermidis, are recognized as a major cause of nosocomial infections associated with the use of implanted medical devices. The capacity of S. epidermidis to form biofilms, allowing it to evade host immune defence mechanisms and antibiotic therapy, is considered to be crucial in colonizing the surfaces of medical implants and dissemination of infection. It has previously been demonstrated that the biofilm of a model strain S. epidermidis RP62A comprises two carbohydrate-containing moieties, a polysaccharide having a structure of a linear poly-N-acetyl-(1-->6)-beta-D-glucosamine and teichoic acid. In the present paper we show that, unlike this model strain, certain clinical isolates of coagulase-negative staphylococci produce biofilms that do not contain detectable amounts of poly-N-acetyl-(1-->6)-beta-D-glucosamine. In contrast to that of S. epidermidis RP62A, these biofilms are not detached with metaperiodate, while proteinase K causes their partial dispersal.