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Revertible hydrogen uptake-deficient mutants of Rhizobium japonicum.

We have developed mutants of Rhizobium japonicum which are deficient in H2 uptake capacity (Hup-) and which spontaneously revert to the parent type at a frequency consistent with that of a single-point mutation (ca. 1.0 x 10(-09)). The mutagenesis by nitrous acid and the selection of the Hup- phenotype by using penicillin and chemolithotrophy as enrichment for chemolithotrophy-deficient strains are described. Two mutants retain low but reproducible levels of ribulose bisphosphate-dependent CO2 fixation when grown on a low-carbon medium under an atmosphere of 1% O2, 4% H2, 5% CO2, and 90% N2. Neither O2 nor the artificial electron acceptors phenazine methosulfate or methylene blue supported detectable H2 uptake by the free-living Hup- mutants or by their bacteroids. Plant growth experiments under bacteriologically controlled conditions were conducted to assess the mutants' performance as inocula for soybean plants. Plants inoculated with Hup- strains had lower dry weights and contained less total N than did plants inoculated with the parent Hup+ strain. Use of either the Hup- mutants or the Hup+ parent strain as inocula, however, did not significantly affect the acetylene-reducing activity or the fresh weight of nodules. These results, obtained with apparently isogenic lines of H2 uptake-deficient R. japonicum, provide strong support for a beneficial role of the H2 uptake phenotype in legume symbiosis.

Carbon Dioxide↗

Importance of preabsorptive insulin release on oral glucose tolerance: studies in pancreatic islet transplanted rats.

The role of preabsorptive (cephalic phase) insulin release in oral glucose tolerance was investigated using diabetic rats treated by intraportal transplantation of isogenic islets. This early neurally mediated phase of insulin release has been shown to be absent in such rats. When the body weight of transplanted rats was normalised, glucose tolerance tests (GTTs) were performed in the unstressed state using permanent cardiac catheters. Transplanted rats had a normalised intravenous GTT, whereas, as we have shown previously, their oral GTT remained clearly pathological. During both tests peripheral insulin levels were decreased compared with controls. While during intravenous GTT the onset of insulin release occurred as early in transplanted rats as in controls, during oral GTT there was a clear delay, probably because of the absence of the cephalic phase. Re-establishment of normal preabsorptive insulin levels was attempted by a small intravenous insulin injection during this period. This resulted in a transient increase in peripheral insulin levels, which, at two minutes after glucose ingestion, gave values similar to those found in controls at that time. This small insulin injection caused a marked improvement of the oral GTT which was most evident after exogenous insulin had disappeared from the blood. While the injection did not affect the 60 minute incremental insulin area, the glucose area was decreased by 50%, to a value not significantly different from that of control rats. The cephalic phase of insulin release appears, therefore, to be one important factor in the control of glycaemia during food intake. Its absence plays a major role in the pathological oral glucose tolerance of diabetic rats treated by intraportal islet transplantation.

Animals↗

Cytoplasmic control of premature activation of a secreted protease zymogen: deletion of staphostatin B (SspC) in Staphylococcus aureus 8325-4 yields a profound pleiotropic phenotype.

The cytoplasmic protein SspC of Staphylococcus aureus, referred to as staphostatin B, is a very specific, tightly binding inhibitor of the secreted protease staphopain B (SspB). SspC is hypothesized to protect intracellular proteins against proteolytic damage by prematurely folded and activated staphopain B (M. Rzychon, A. Sabat, K. Kosowska, J. Potempa, and A. Dubin, Mol. Microbiol. 49:1051-1066, 2003). Here we provide evidence that elimination of intracellular staphopain B activity is indeed the function of SspC. An isogenic sspC mutant of S. aureus 8325-4 exhibits a wide range of striking pleiotropic alterations in phenotype, which distinguish it from the parent. These changes include a defect in growth, a less structured peptidoglycan layer within the cell envelope, severely decreased autolytic activity, resistance to lysis by S. aureus phages, extensively diminished sensitivity to lysis by lysostaphin, the ability to form a biofilm, and a total lack of extracellular proteins secreted into the growth media. The same phenotype was also engineered by introduction of sspB into an 8325-4 sspBC mutant. In contrast, sspC inactivation in the SH1000 strain did not yield any significant changes in the mutant phenotype, apparently due to strongly reduced expression of sspB in the sigma B-positive background. The exact pathway by which these diverse aberrations are exerted in 8325-4 is unknown, but it is apparent that a very small amount of staphopain B (less than 20 ng per 200 microg of cell proteins) is sufficient to bring about these widespread changes. It is proposed that the effects observed are modulated through the proteolytic degradation of several cytoplasmic proteins within cells lacking the inhibitor. Seemingly, some of these proteins may play a role in protein secretion; hence, their proteolytic inactivation by SspB has pleiotropic effects on the SspC-deficient mutant.

Bacterial Proteins↗

Morphology of stable muscle grafts of rats: effects of gender and muscle type.

Our purpose was to quantify morphological characteristics of extensor digitorum longus (EDL) and soleus (SOL) muscle grafts in female (N = 8) and male (N = 8) rats. Muscles were grafted orthotopically, with the nerve remaining intact, and were studied 56 days later. The mass of EDL and SOL grafts and control muscles of females was 60% to 65% of male values; this difference was directly related to gender differences in body mass. The fiber composition of EDL and SOL grafts did not differ from control, and no gender effects were noted. The mean fiber area (MFA) of control EDL and SOL muscles of females averaged 65% of male values. The MFA of grafts did not differ due to gender, and averaged 60% of control value for SOL and 70% for EDL grafts. We conclude there are no substantial differences in the regenerative capacity of EDL and SOL muscles grafted with the nerve intact.

Animals↗

Isolated islet transplantation in experimental diabetes.

Pancreatic islets have been isolated from the exocrine pancreas of inbred rats by the collagenase digestion method. Transplantation of isolated islets into the portal venous system of streptozotocin diabetic recipients resulted in complete abrogation of the diabetic state as measured by non-fasting serum glucose level, 24 h urinary output, rate of weight gain and glucose tolerance test. Transplantation to other sites resulted in less than optimal survival and function of islets. Allogeneic islets, transplanted across weak histocompatibility barriers, can survive and function for prolonged periods of time when transplanted recipients are immunosuppressed with antilymphocyte serum (ALS). Recipients of allogeneic islets, after a period of immunosuppression with ALS, become permanently tolerant to the allografted islets and to subsequent skin grafts from similar allogeneic donors. Allografted islets are able to prevent the occurrence of diabetic renal and ophthalmic changes that occur in control diabetic animals which had not undergone transplantation.

Animals↗

Cultivar-specific elicitation of barley defense reactions by the phytotoxic peptide NIP1 from Rhynchosporium secalis.

Resistance of barley to the phytopathogenic fungus, Rhynhosporium secalis race US238.1, was found to be controlled by resistance gene Rrs1, which segregated in a manner characteristics for a codominant gene. PRHv-1, a thaumatin-like pathogenesis-related protein, was shown to be encoded by a gene family on chromosome 1. As part of the barley defense response, significant accumulation of PRHv-1 and peroxidase transcripts was induced early during pathogenesis in two Rrs1 cultivars but not or to a lower level in a near-isogenic, susceptible rrs1 cultivar or a cultivar lacking known resistance genes. R. secalis secretes a small group of necrosis-inducing peptides. One of these, NIP1, which was detected in culture filtrates only of fungal race US238.1, was found to elicit the accumulation of PRHv-1 and peroxidase mRNAs in Rrs1 cultivars with a time course similar to that upon fungal infection. Therefore, NIP1 is a candidate for the product of fungal avirulence gene avrRrs1, which, together with barley resistance gene Rrs1, determines incompatibility of the interaction.

Amino Acid Sequence↗

Binding of Pseudomonas aeruginosa AlgZ to sites upstream of the algZ promoter leads to repression of transcription.

Mucoid variants of the opportunistic pathogen Pseudomonas aeruginosa produce the exopolysaccharide alginate and colonize the respiratory tracts of cystic fibrosis patients. The genes encoding the alginate biosynthetic enzymes are clustered in a single operon, which is under tight transcriptional control. One essential activator of the alginate operon is AlgZ, a proposed ribbon-helix-helix DNA binding protein that shares 30% amino acid identity with the Mnt repressor of Salmonella enterica serovar Typhimurium bacteriophage P22. In the current study, we examined the role of AlgZ as an autoregulator. Using single-copy algZ-lacZ transcription fusions, an increase in algZ transcription was observed in an algZ mutant compared to the isogenic wild-type strain, suggesting that AlgZ may have an additional role as a repressor. To identify the AlgZ binding site, overlapping regions upstream of algZ were incubated with AlgZ and analyzed by electrophoretic mobility shift assays. Specific binding activity was localized to a region spanning from 66 to 185 base pairs upstream of the algZ transcriptional start site. Two AlgZ binding sites were defined using copper-phenanthroline footprinting and deletion analyses, with one site centered at 93 base pairs and the other centered at 161 base pairs upstream of the algZ promoter. Deletion of both binding sites resulted in the loss of AlgZ binding. These results indicate that AlgZ represses algZ transcription, and this activity is mediated by multiple AlgZ-DNA interactions.

Bacterial Proteins↗

Regulation of branchial Na(+)/K(+)-ATPase in common carp Cyprinus carpio L. acclimated to different temperatures.

Isogenic carp Cyprinus carpio L. were acclimated to water temperatures of 15, 22 and 29 degrees C for at least 8 weeks. The acclimations consistently resulted in slightly, but significantly, different plasma osmolality, sodium, potassium and chloride concentrations between the groups studied. Plasma total and ionic calcium levels were unaffected, indicating successful adaptation. The apparent changes in set point for plasma ion levels are explained by altered sodium pump activity and hormonal control of branchial permeability to water and ions. It appears that in 15 degrees C-acclimated fish, a lower apparent Na(+)/K(+)-ATPase activity is compensated by strongly enhanced Na(+)/K(+)-ATPase expression (determined biochemically and immunohistochemically). In 29 degrees C-acclimated fish, the higher ambient temperature activates the enzyme. Arrhenius plots for branchial Na(+)/K(+)-ATPase preparations of the three groups of fish suggest the occurrence of different enzyme isoforms or protein (in)stability as explanations for differences in apparent enzyme activities, rather than temperature-dependent changes in membrane fluidity. As for hormonal control over permeability, prolactin mRNA expression (and anticipated production and release) is lower in fish kept at 29 degrees C, suggesting that control over branchial permeability to water and ions needs to be downregulated at higher temperatures. In so doing, enhanced sodium pump activity is balanced by a controlled passive ion loss to fine-tune plasma sodium levels. Basal plasma cortisol levels did not correlate positively with Na(+)/K(+)-ATPase expression, but doubling plasma cortisol levels in control fish by administering exogenous cortisol (for 7 days, using implanted minipumps and thus stress-free) enhanced Na(+)/K(+)-ATPase expression. This effect must be the result of a glucocorticoid action of the steroid: in fish, mineralocorticoid receptors have higher affinity for cortisol than glucocorticoid receptors. At a lower ambient temperature, branchial Na(+)/K(+)-ATPase expression is upregulated to counteract the temperature-inhibited activity of the sodium pump, perhaps via a mineralocorticoid receptor.

Acclimatization↗

Low-phytate barley cultivars improve the utilization of phosphorus, calcium, nitrogen, energy, and dry matter in diets fed to young swine.

A 28-d experiment was conducted using 45 crossbred barrows with an average initial BW of 9.5 kg and age of 35 d to evaluate low-phytate barley (LPB) mutants (M) M422, M635, and M955, which were hulled, near-isogenic progeny of the normal barley (NB) Harrington and had 47, 66, and 80% less phytic acid, respectively, than NB. A hull-less LPB, M422-H, which was not near-isogenic to the other cultivars, was also evaluated. Apparent nutrient balance, bone measurements, and growth performance were the response criteria evaluated. The barrows were fed the diets to appetite in meal form in individual metabolism crates. Barley and soybean meal were the only sources of phytic acid. Dietary protein supplementation and ME/kg were equalized in all diets. The treatments were diets containing NB, M422, M635, or M422-H without or with added inorganic P (iP), or M955 without added iP. Diets with added iP contained 0.30% available P (aP), the same concentration of aP provided by the diet containing M955 without added iP. There were linear increases (P < or = 0.02) in ADG, G:F, metacarpal and radius bone strength, and fat-free dry weight, and in the absorption and retention (g/d and % of intake) of P and Ca with increasing dietary concentration of aP from the near-isogenic cultivars NB, M422, M635, or M955 without added iP. There were linear decreases in the grams (P < or = 0.02) and percentages (P < 0.001) of P and Ca excreted per day with increasing dietary concentration of aP without added iP. There were no responses for N or energy balance. Growth performance and bone response criteria did not differ for barrows fed the diet containing M955 or the near-isogenic diets containing NB, M422, or M635 with added iP. However, barrows fed the diet containing M955 had greater (P < or = 0.02) percentages of P, N, and energy absorption and retention, Ca absorption, and DM digestibility and had less (P < or = 0.02, g/d and %) excretion of P, N, energy, and Ca (g) per day than barrows fed the diets containing the near-isogenic NB or LPB cultivars with added iP. When dietary aP was equalized with iP, the excretion of P in feces plus urine (g/d) was reduced by 20.2, 27.9, and 44.6%, respectively, in barrows fed the diets containing M422 + iP, M635 + iP, or M955 compared with barrows fed the diet containing NB + iP. Energy utilization did not differ for barrows fed the diets containing hulled or hull-less LPB when ME/kg was equalized with lard. In conclusion, the apparent utilization of P and Ca, the bone strength and fat-free dry weight, and growth performance increased with increasing dietary concentration of aP provided by LPB, in association with linear decreases in P and Ca excretion. Barrows fed the diet containing M955 also had greater utilization and less excretion of P, Ca, N, energy, and DM than barrows fed the diets containing the near-isogenic NB or LPB cultivars with added iP to equalize aP at 0.30%.

Animal Feed↗

[The effect of Listeria factor Ei, BCG vaccine and a combination of both on the growth of experimental syngeneic tumors in inbred mice].

The investigation assessed, whether a nonspecific single and repeated immunization induced by Listeria monocytogenes Ei factor, BCG vaccine and their combination influences the growth of transplanted syngenic methylcholanthrene tumour depending on tumor graft cellularity. The cellularity of 10(4) and 10(5) were investigated. Tumour growth was examined 10 and 17 days after the inoculation. It has been found that single Ei factor administration did not influence the tumour growth. Single BCG vaccine favourably influenced the tumour growth, but in progressing time and cellularity the effect was failing. In combination of BCG vaccine and Listeria Ei factor the antitumour effect appeared to be most profound. In progressing time after the tumour grafting and graft cellularity the effect was disappearing. It was probably due to the fact that immunotherapy fails to control tumour mass with high cellularity. The antitumour effect potentiation by combination of BCG vaccine with Ei factor suggests a way to a possible combined antitumour immunotherapy.

Animals↗

Control of beef spoilage by a sulfide-producing Lactobacillus sake strain with bacteriocinogenic Leuconostoc gelidum UAL187 during anaerobic storage at 2 degrees C.

Chill-stored, vacuum-packaged beef inoculated with sulfide-producing Lactobacillus sake 1218 developed a distinct sulfide odor within 3 weeks of storage at 2 degrees C, at which time the bacteria had reached maximum numbers of 10(6) CFU cm(-2). Coinoculation of the meat with the wild-type, bacteriocinogenic (Bac+) strain of Leuconostoc gelidum UAL187 delayed the spoilage by L. sake 1218 for up to 8 weeks of storage. Coinoculation of meat samples with an isogenic, slowly growing Bac+ variant, UAL187-22, or with the Bac- variant UAL187-13 did not delay the onset of spoilage by L. sake 1218. The study showed that spoilage of chill-stored, vacuum-packaged beef by a susceptible target organism could be dramatically delayed by the Bac+ wild-type strain of L. gelidum UAL187. Inoculation with L. sake 1218 can be used as a model system to determine the efficacy of biopreservation of vacuum-packaged meats.

Anaerobiosis↗

Identification of AFLP markers linked to fertility restorer genes for tournefortii cytoplasmic male-sterility system in Brassica napus.

The tournefortii cytoplasmic male-sterility system is being used as a method of pollination control to develop hybrids in Brassica napus. Genetic analyses have indicated that two dominant genes, one major ( Rft1) and another minor ( Rft2), were required to achieve complete fertility restoration. Though the major gene ( Rft1) can cause complete fertility restoration on its own, its expression was significantly enhanced in the presence of the minor gene ( Rft2). In the absence of Rft1, Rft2 caused only partial fertility restoration. We used a pair of near-isogenic lines (NILs), differing for the presence/absence of Rf genes, to identify AFLP markers linked to fertility restorer genes. A total of 64 EcoRI/ MseI primer combinations were surveyed which produced 3,225 bands, of which 19 (0.006%) were polymorphic between parental NILs. Primer combinations which led to the identification of polymorphic bands present in fertile parental NILs were used for assaying a mapping population of 70 F(2) plants for determining the segregation pattern of markers. Initial screening resulted in the identification of five AFLP markers. The recombination analyses of these AFLP markers revealed that at least two (EACC/MCTT(105), EAAG/MCTC(80)) were present in the same linkage group along with the Rf loci. Marker EACC/MCTT(105) was separated from the major gene ( Rft1) by a distance of 18.1 cM, while it was 33.2 cM away from the minor fertility restorer gene ( Rft2). Another marker EAAG/MCTC(80) was also located adjacent to Rft1 at a distance of 18.1 cM, but on other side. Identification of flanking markers (EACC/MCTT(105), EAAG/MCTC(80)) for the major fertility restorer gene ( Rft1) provides a crucial component for marker-assisted selection and map-based cloning of the restorer genes, and can hence be used to construct elite restorer genotypes.

Brassica↗

Regulatory and functional co-operation of flagella and type 1 pili in adhesive and invasive abilities of AIEC strain LF82 isolated from a patient with Crohn's disease.

Genetic determinants that co-operate with type 1 pili to mediate invasion were sought for in adherent-invasive Escherichia coli strain LF82 isolated from a patient with Crohn's disease. Two mutants selected for their impaired ability to invade epithelial cells carried insertions of a TnphoA transposon within genes of the flagellar regulon. An isogenic mutant LF82-DeltafliC deleted for the flagellin-encoding gene did not adhere, did not invade and, surprisingly, expressed only a few type 1 pili. Type 1 pili downregulation resulted from a preferential switch towards the off-position of the invertible DNA element located upstream of the fim operon. This was also correlated with a decrease in the flagellar regulator flhDC mRNA levels, suggesting that the transcriptional regulator FlhD2C2 could control type 1 pili expression directly or indirectly. Transformation with a cloned fim operon allowed bypass of the type 1 pili downexpression in the LF82-DeltafliC mutant. Thus, we showed that flagella play a direct role in the adhesion process via active motility. In addition to downregulating type 1 pili expression, flagella also play an undefined role in strain LF82 invasion, which is not restricted to motility or flagellar structure, but could be related to co-ordinate expression of invasive determinants.

Bacterial Adhesion↗

EilA, a HilA-like regulator in enteroaggregative Escherichia coli.

Enteroaggregative Escherichia coli (EAEC) is increasingly recognized as a diarrhoeal pathogen in developing and industrialized countries. Most EAEC virulence factors thus far described are encoded on virulence plasmid pAA, yet recent completion of the EAEC genome has suggested the presence of additional factors encoded on chromosomal islands. Previous reports have recognized the presence of a type III secretion system (T3SS), designated ETT2, at the glyU locus of prototype EAEC strain 042, along with possible T3SS effectors at the selC locus. The selC locus was also noted to harbour homologues of Salmonella enterica regulator HilA and of invasin from Yersinia spp., yet previous publications suggested that these loci may be silent. Here, we show that the genes of the selC locus are present inconsistently among a collection of well-characterized EAEC strains. Notably, however, there was perfect correlation between the presence of hilA-homologue eilA and predicted Yersinia invasin homologue gene eaeX. We hypothesized that if expressed, the putative gene product EilA would contribute to EAEC virulence in part by activation of the T3SS and its effectors. An eilA mutant was constructed in EAEC strain 042, and complementation was achieved by cloning the eilA gene under control of an arabinose-dependent promoter. In this system, we observed expression of at least seven genes to be affected by expression of eilA, either directly or indirectly: selC locus genes eipB, eipC, eipD, eicA and eaeX (renamed here air), as well as glyU ETT2 genes eivF and eivA. Notably, the eilA mutant was shown to be less adherent to epithelial cells in culture and to form less abundant biofilms than the isogenic parent. These effects were recapitulated in the air mutant, suggesting that the predicted outer membrane protein product of the air gene is involved as an accessory adhesin and aggregin of EAEC, coexpressed with the T3SS. Our data suggest that the T3SS of EAEC and presumed effectors located on different chromosomal islands may be coordinately activated by EilA, which also activates the genetically linked high molecular weight bacterial surface protein Air. Contributions of this new putative virulence-related regulon in EAEC may include adherence, aggregation, and as yet uncharacterized roles for the T3SS.

Bacterial Adhesion↗

Lung tissue mechanics in the early stages of induced paracoccidioidomycosis in rats.

Pulmonary dysfunction represents the most important cause of death in patients with paracoccidioidomycosis (PBM). In order to investigate the functional changes of the lungs in the early stages of PBM, a model of benign disease was developed by intratracheal challenge of 12-week old isogenic Wistar rats with 1 x 10(6) yeast forms of Paracoccidioides brasiliensis. Animals were studied 30 and 60 days after infection, when fully developed granulomas were demonstrable in the lungs. Measurements of airway resistance, lung elastance and tissue hysteresis were made during sinusoidal deformations (100 breaths/min, tidal volume = 2 ml) with direct measurement of alveolar pressure using the alveolar capsule technique. Infection caused a significant increase in hysteresis (infected: 1.69, N = 13; control: 1.13, N = 12, P = 0.024, ANOVA), with no alterations in airway resistance or lung elastance. Histopathological analysis revealed the presence of fully developed granulomas located in the axial compartment of the lung interstitial space. These results suggest that alterations of tissue mechanics represent an early event in experimental PBM.

Animals↗

Functional aspects of small-bowel transplantation in rats.

Although clinical small-bowel transplantation is still severely hindered by rejection of the graft, prolonged graft survival can be achieved by using cyclosporin A in several experimental models of small-bowel transplantation. In an immunologically quiescent phase after transplantation, the important question arises whether a small-bowel allograft has enough functional capacity to maintain a normal nutritional status. We investigated the functional capacity of orthotopic small-bowel transplants and evaluated the ability of the total small-bowel transplant to absorb orally given cyclosporin in the early postoperative period and the effect of this oral cyclosporin treatment on allograft survival as compared with intramuscular administration. Between 3 and 7 months postoperatively, recipients of syngeneic and allogeneic total small-bowel transplants and syngeneic jejunal segmental grafts had significantly decreased serum triglyceride levels. Total serum protein and albumin concentrations, serum cholesterol values, fecal fat excretion, and percentage of split fatty acids were normal. One year after transplantation the weight in the groups transplanted with a total small-bowel graft was not different from age-matched untreated controls. Animals grafted with a segmental graft, however, showed a significantly impaired growth and had not reached a normal weight 1 year after transplantation. Growth was also significantly impaired after near-total small-bowel resection. These animals had to be killed because of their poor condition. Cyclosporin absorption after small-bowel transplantation equalled that in normal controls. Graft survival after intramuscular treatment, however, was significantly better than after oral treatment.

Absorption↗

Intestine-like remodeling of adult mouse glandular stomach by implanting of fetal intestinal mesenchyme.

A morphogenetic response of adult glandular stomach grown in contact with implanted fetal intestinal mesenchyme has been demonstrated. Mesenchymal tissues from intestines of 14- to 16-day-old BALB/c mouse fetuses were introduced beneath the epithelial layer of glandular stomach in 2-month-old mice and allowed to develop. Three to 4 weeks later, remodeling of the epithelial architecture had occurred; the characteristic glandular pit structure of normal stomach had been replaced by immature villi and crypts composed of mucus-secreting columnar cells more characteristic of intestinal tissues. Chief and parietal cells had disappeared, but neither goblet nor Paneth cells were observed. Such intestine-like morphogenesis was not induced by similarly implanted mesenchymal controls from fetal glandular stomach, forestomach, and salivary gland. A possible role of the mesenchymal stroma in the pathogenesis of intestinal metaplasia in stomach is discussed.

Aging↗

Novel animal model for studying the molecular mechanisms of bacterial adhesion to bone-implanted metallic devices: role of fibronectin in Staphylococcus aureus adhesion.

Infection around metallic implants is a rare but severe complication of orthopaedic surgery. A novel animal model mimicking conditions of internal fixation devices was developed to evaluate the role of host proteins adsorbed on metallic devices in promoting adhesion and colonization of the material surfaces by Staphylococcus aureus. Small plates made of pure titanium were either fixed (three screws per plate) onto the iliac bones of guinea pigs or implanted into their subcutaneous space as controls. Five to 6 weeks after surgery, the plates and screws were removed from the previously killed animals, carefully rinsed in buffer, and tested in an in vitro assay of S. aureus adhesion to metallic surfaces. To evaluate the role of fibronectin in staphylococcal adhesion to explanted plates and screws, a mutant of S. aureus that is specifically defictive in fibronectin adhesion due to decreased expression of the fibronectin adhesin was compared with its isogenic parental strain. A significant reduction in adhesion of the fibronectin adhesin-defective mutant compared with the parental strain occurred on both the subcutaneously implanted and bone-implanted metallic plates. The results of this specific biological assay suggest that fibronectin is present on bone-implanted metallic devices and promotes attachment of S. aureus to their surfaces. This novel experimental model should help, to characterize several parameters of bacterial adhesion to metallic orthopaedic devices and to develop novel anti-adhesive strategies for preventing such infections.

Animals↗