Detection by PCR and analysis of the distribution of a fibronectin-binding protein gene (fbn) among staphylococcal isolates.

The fibronectin-binding proteins of Staphylococcus aureus are considered to be important virulence factors for colonisation and infection. The polymerase chain reaction (PCR) was used to detect part of a gene equivalent to the fbnA gene of S. aureus in 120 isolates of staphylococci (S. aureus, S. epidermidis, S. haemolyticus, S. simulans, S. hominis, S. warneri, S. cohnii and S. lugdunensis). Primers specific for the binding domain region of the fbnA gene of S. aureus produced PCR products of the predicted sizes (93 and 207 bp). The identity of the PCR products was confirmed by digestion with DdeI and nucleic acid hybridisation. The fibronectin-binding activity of the staphylococci was determined with a particle agglutination assay (PAA). The fbn gene was found to be present by PCR in 107 of the 120 staphylococci tested, irrespective of their site of isolation, and expression of the gene was detected by PAA in 101 of the 120 strains.

A double mobility acetabular implant for primary hip arthroplasty in patients at high risk of dislocation.

INTRODUCTION: Dislocation following total hip replacement continues to be a problem for which no completely satisfactory solution has been found. Several methods have been proposed to reduce the incidence of hip dislocations with varying degrees of success, including elevated rim liners, constrained liners and large diameter bearings. We present our experience with the double mobility acetabular component in patients at high risk of instability. METHODS: This was a retrospective review of 65 primary total hip arthroplasties in 55 patients (15 men, 40 women), performed between October 2005 and November 2009. The majority (80%) of patients had at least two and 26% had at least three risk factors for instability. The mean age was 76 years (range: 44-92 years). The patients were followed up for a mean duration of 60 months (range: 36-85 months). RESULTS: Fourteen patients died and one was lost to follow-up, leaving fifty hips for final assessment. Until the final follow-up appointment, no patients had dislocation and none required revision surgery. The mean Oxford hip score improved from 45.0 to 26.5 (p<0.0001). The mean Merle d'Aubigné pain score improved from 1.4 to 4.9 (p<0.0001), the walking score from 2.3 to 3.1 (p<0.07) and the absolute hip function score from 5.4 to 10.8 (p<0.0001). There were no clinical or radiographic signs of loosening. CONCLUSIONS: The double mobility acetabular component was successful at preventing dislocation during early to medium-term follow-up. However, as data are still lacking with regard to polyethylene wear rates at the additional bearing surface, it would be prudent to restrict the use of this implant to selected patients at high risk of instability.

The double-mobility acetabular component in revision total hip replacement: the United Kingdom experience.

We present our experience with a double-mobility acetabular component in 155 consecutive revision total hip replacements in 149 patients undertaken between 2005 and 2009, with particular emphasis on the incidence of further dislocation. The mean age of the patients was 77 years (42 to 89) with 59 males and 90 females. In all, five patients died and seven were lost to follow-up. Indications for revision were aseptic loosening in 113 hips, recurrent instability in 29, peri-prosthetic fracture in 11 and sepsis in two. The mean follow-up was 42 months (18 to 68). Three hips (2%) in three patients dislocated within six weeks of surgery; one of these dislocated again after one year. All three were managed successfully with closed reduction. Two of the three dislocations occurred in patients who had undergone revision for recurrent dislocation. All three were found at revision to have abductor deficiency. There were no dislocations in those revised for either aseptic loosening or sepsis. These results demonstrate a good mid-term outcome for this component. In the 29 patients revised for instability, only two had a further dislocation, both of which were managed by closed reduction.

The effects of tetradecyl-4-ethyl-pyridinium chloride on the maximum specific growth rate biomass and hydrolytic enzyme production of Bacteroides gingivalis in continuous culture.

Bacteroides gingivalis was grown in continuous culture in the presence of tetradecyl-4-ethyl-pyridinium chloride (TDEPC). The maximum specific growth rate and biomass levels decreased with increasing concentrations of antimicrobial and complete inhibition of growth occurred when the TDEPC concentration reached 40 mg/l. Hydrolytic enzymes were detected in cells, vesicles and supernatant fractions of whole culture. Levels of alkaline phosphatase initially increased with increasing concentrations of TDEPC, but at higher concentrations (15-20 mg/l) of antimicrobial decreased significantly to less than 20% of the control value. The levels of N-acetyl-beta-glucosaminidase remained approximately constant at lower concentrations of TDEPC (0 10 mg/l) but then decreased significantly at higher concentrations. In contrast, levels of trypsin-like protease were reduced significantly at even low concentrations of TDEPC (5 10 mg/l) and decreased further as the TDEPC concentration increased. Therefore, TDEPC exerts significant physiological effects on B. gingivalis at concentrations below those considered to be lethal to the cell.

The effects of chlorhexidine on the maximum specific growth rate, biomass and hydrolytic enzyme production of Bacteroides gingivalis grown in continuous culture.

Bacteroides gingivalis was grown in continuous culture in the presence of chlorhexidine. Maximum specific growth rates and biomass levels initially increased but then decreased as the chlorhexidine level increased from 0 to 30 micrograms/ml. Total inhibition of growth occurred when the chlorhexidine concentration reached 60 micrograms/ml. The steady-state levels of cell-bound, extracellular vesicle and extracellular soluble enzymes, trypsin-like protease, alkaline phosphatase and N-acetyl-beta-glucosaminidase were measured. With increasing sub-lethal concentrations of chlorhexidine, levels of alkaline phosphatase increased noticeably in all three fractions of culture, whilst cell-bound and extracellular vesicle levels of N-acetyl-beta-glucosaminidase remained approximately constant. Extracellular soluble levels of alkaline phosphatase and N-acetyl-beta-glucosaminidase increased with increasing levels of chlorhexidine. The levels of trypsin-like protease decreased significantly in all fractions of the culture when cells were grown in the presence of chlorhexidine. Thus, chlorhexidine has a differential effect on the production of B. gingivalis hydrolytic enzymes.

Production of cell-bound and vesicle-associated trypsin-like protease, alkaline phosphatase and N-acetyl-beta-glucosaminidase by Bacteroides gingivalis strain W50.

Bacteroides gingivalis strain W50 was grown in batch and continuous culture on complex medium with haemin. In batch culture, cell-bound levels of trypsin-like protease (EC 3.4.21.4), alkaline phosphatase (EC 3.1.3.1) and N-acetyl-beta-glucosaminidase (EC 3.2.1.30) increased during the exponential phase of growth. These enzyme activities were also detected in extracellular vesicles and in extracellular soluble forms in the supernatant fluid, but in lower amounts per unit biomass compared to cell-bound levels. In continuous culture, at high relative growth rates (0.7-0.9 murel), the highest proportions of enzyme activities were cell-bound. In contrast, at low relative growth rates (0.1-0.2 murel), highest enzyme levels were detected in the extracellular vesicle fraction. Levels of extracellular soluble enzymes were always low compared to cell-bound or extracellular vesicle levels, but were highest at low relative growth rates. All three enzymes appeared to be relatively stable in their soluble forms. Vesicle production appeared to be associated with actively growing cells but was influenced by growth rate. The results are consistent with the hypothesis that cell-bound 'periplasmic' enzymes are encapsulated into vesicles which are subsequently released by the cells. Therefore, levels of total extracellular enzyme (extracellular vesicle plus extracellular soluble) may depend on the rate of vesicle formation superimposed on the rates of production of 'periplasmic' enzymes in the cell.

The distribution and properties of some hydrolytic enzymes from Porphyromonas gingivalis W50.

The production, distribution (as cell-bound, extracellular or associated with vesicles) and properties of four hydrolytic enzymes from Porphyromonas gingivalis were studied. During batch culture, enzymes appeared as cell bound until 72 h when extracellular trypsin-like protease and glycylprolyl dipeptidase were found. Alkaline phosphatase and N-acetyl-beta-glucosaminidase continued to be mainly cell-bound until 96 and 144 h post-inoculation, respectively. The pH optima for trypsin-like protease and dipeptidase activities were 7.5 and 8.0, respectively. Activity was most stable between pH 6.5 and 9.0 for the trypsin-like protease and pH 8.0 for dipeptidase. For phosphatase and N-acetyl-beta-glucosaminidase, double peaks of activity occurred at pH 8.0, 10.5 and 6.5, 7.5, respectively. Phosphatase was most stable at pH 7.0, whilst N-acetyl-beta-glucosaminidase was stable over a wide range of pH from 5.0-10.0. Molecular weight estimation by gel filtration gave 170 and 65 kD for trypsin-like protease, 200 and 24 kD for glycylprolyl dipeptidase, 125 and 24 kD for phosphatase, and 22 kD for N-acetyl-beta-glucosaminidase.

The influence of haemin levels on growth and enzyme production by Porphyromonas gingivalis in continuous culture.

Porphyromonas gingivalis was grown in continuous culture in brain heart infusion medium with haemin (5 microgm 1(-1) to 10 mg 1(-1)). Growth was haemin-limited up to 400 microgm 1(-1), and steady-state growth could not be obtained in the absence of haemin. It was confirmed that protoporphyrin IX could replace the haemin requirement. The results indicate that P. gingivalis has more than one uptake system for haemin, one of which is high affinity. The effect of haemin on hydrolytic enzyme production revealed a complex picture. Trypsin-like protease activity peaked at 200 microgm 1(-1) with a substantial and highly significant reduction at high (2 and 10 mg 1(-1)) concentrations of haemin. Alkaline phosphatase also peaked around 100 to 400 microgm 1(-1) while N-acetyl-beta-glucosaminidase was not greatly affected except that a greater proportion of enzyme was secreted into the medium at the lowest haemin concentration. In general, haemin-limitation enhanced export of the hydrolytic enzymes to the extracellular vesicle fraction. Protoporphyrin IX at 2 mg 1(-1) caused a highly significant reduction i the production of all three enzymes.