James R. Nixon

Formation of Propionibacterium acnes biofilms on orthopaedic biomaterials and their susceptibility to antimicrobials

Failure to treat and eradicate prosthetic hip infection with systemic antibiotic regimens is usually due to the fact that the infection is associated with biofilm formation and that bacterial cells growing within a biofilm exhibit increased resistance to antimicrobial agents. In this in vitro study, we investigated the susceptibility of prosthetic hip Propionibacterium acnes and Staphylococcus spp. isolates growing within biofilms on polymethylmethacrylate (PMMA) bone cement to a range of antibiotics. All P. acnes isolates in the biofilm mode of growth demonstrated considerably greater resistance to cefamandole, ciprofloxacin and vancomycin. In contrast, only four of the eight P. acnes isolates demonstrated an increase in resistance to gentamicin. All ten Staphylococcus spp. isolates in the biofilm mode of growth exhibited large increases in resistance to gentamicin and cefamandole with eight of the ten isolates also exhibiting an increase in resistance to vancomycin. However, only three of the ten Staphylococcus spp. isolates exhibited an increase in resistance to ciprofloxacin. Biofilms were also formed on three different titanium alloys and on PMMA bone cement using P. acnes, Staphylococcus epidermidis and Staphylococcus aureus strains to determine if the underlying biomaterial surface had an effect on biofilm formation and the antimicrobial susceptibility of the bacteria growing within biofilms. Although differences in the rate at which the three strains adhered to the different biomaterials were apparent, no differences in biofilm antibiotic resistance between the biomaterials were observed. In the light of these results, it is important that the efficacy of other antibiotics against P. acnes and Staphylococcus spp. prosthetic hip isolates growing within biofilms on orthopaedic biomaterials be determined to ensure optimal treatment of orthopaedic implant infection.

Improved detection of infection in hip replacements

Our aim was to determine if the detection rate of infection of total hip replacements could be improved by examining the removed prostheses. Immediate transfer of prostheses to an anaerobic atmosphere, followed by mild ultrasonication to dislodge adherent bacteria, resulted in the culture of quantifiable numbers of bacteria, from 26 of the 120 implants examined. The same bacterial species were cultured by routine microbiological techniques from only five corresponding tissue samples. Tissue removed from 18 of the culture-positive implants was suitable for quantitative tissue pathology and inflammatory cells were present in all samples. Furthermore, inflammatory cells were present in 87% of tissue samples taken from patients whose implants were culture-negative. This suggests that these implants may have been infected by bacteria which were not isolated by the techniques of culture used. The increased detection of bacteria from prostheses by culture has improved postoperative antibiotic therapy and should reduce the need for further revision.

Detection of prosthetic joint biofilm infection using immunological and molecular techniques

Publisher Summary This chapter describes how the detection of bacteria by culture from revision hip prostheses can be improved by employing strict anaerobic techniques and by using mild ultrasonication to dislodge bacteria adhering to the surface of the retrieved implants. Nonculture techniques that can be used to further improve the detection of infection, including examination of the inflammatory response in implant-associated tissue and the detection of bacteria using immunological and polymerase chain reaction (PCR)-based molecular approaches, are also described. The use of strict anaerobic techniques and mild ultrasonication has resulted in bacteria being cultured from 26 of 120 (22%) retrieved implants examined. The use of nonculture techniques to improve the detection of prosthetic joint biofilm infection coupled with appropriate postoperative antibiotic therapy should improve the clinical outcome for patients undergoing revision hip surgery.

Recurrent hip arthroplasty dislocation: Good outcome after cup augmentation in 20 patients followed for 2 years

Recurrent posterior dislocation occurs after primary total hip arthroplasty at rates of up to 7%. Component revision is regarded as standard management, but this major surgery may be unsuitable for elderly patients. We have developed a congruent polyethylene acetabular posterior lip augmentation device (PLAD) with a stainless steel backing plate. This can be used when there is no gross malalignment, wear or loosening of the primary components. We retrospectively compared 20 patients who had revision surgery with twenty patients who had been treated with the PLAD for recurrent posterior dislocation after primary Charnley total hip arthroplasty. In the PLAD group, the mean operative time, intraoperative blood loss, time spent in the high-dependency unit (HDU), transfusion requirements and the duration of hospital stay were all less than that in the revision group. There was no difference in the Oxford Hip Score between the groups at latest review 2 years after surgery. None of the patients in either group had suffered another dislocation.

Improved antibiotic therapy for elimination and prevention of prosthetic hip infection

Total hip replacement is one of the most successful and cost-effective surgical operations ever introduced with over 50,000 hip replacements performed annually in the UK. Bacterial infection is a significant complication following this procedure, with 22% of revision operations in a recent study resulting from implant infection (Tunney et al 1998). Failure of the second implant post-revision, however, is due to infection in up to 40% of cases (Dupont 1986). It has been suggested that this higher rate of infection post-revision may be due to the fact that bacteria colonising the surface of implanted biomaterials grow predominantly within adherent biofilms which confer a level of resistance to antibiotics and may render current antibiotic therapy ineffective. In Musgrave Park Hospital, antibiotic treatment to reduce the risk of recurrent infection includes the use of gentamicin-impregnated bone cement for prosthesis fixation at revision surgery and the intravenous adminis t ra t ion of e i the r cefamandole or erythromycin peroperatively. We have previously shown resistance of planktonic Staphylococcus spp. isolated from orthopaedic implants to these three antibiotics (Tunney et a1 1997). The aim of the present study was, therefore, to determine if treatment of hip implant infection could be improved by the use of alternative antimicrobial agents or combinations of antimicrobial agents. A total o f 49 clinical isolates (30 Staphylococcus spp., 19 Propionibacterium acnes) recovered from retrieved prosthetic hip implants were tested. Planktonic minimum inhibitory concentrations (MIC), Planktonic minimum bactericidal concentrations (MBC) and sessile MBCs were determined as described previously (Tunney et a1 1997). Paired combinations of antibiotics were cross-titrated using the chequerboard technique to investigate possible synergy. The summation of fractional inhibitory concentrations (CFIC) was calculated to provide an indication of the combined effect of the antibiotics (Hallender et a1 1982). All isolates tested were sensitive to vancomycin and ciprofloxacin (Table 1). Resistance of several aerobic isolates to fusidic acid was however apparent. Ciprofloxacin was the most effective bactericidal agent followed by vancomycin and fusidic acid in decreasing order of efficacy. Synergy between cefamandole and gentamicin was observed for five of the eight isolates tested. In contrast, a combination of vancomycin and ciprofloxacin was synergistic for only one isolate and antagonistic for a further four isolates. On the basis of MBCs, sessile populations of a range of isolates adherent to bone cement exhibited greater resistance to gentamicin, cefamandole and vancomycin compared with planktonic bacterial suspensions. In contrast, sessile populations of seven of the eleven isolates tested showed no increase in resistance to ciprofloxacin. Table 1 . Susceptibility of isolates to antibiotics

Use of 16S ribosomal (rRNA) genes and immunofluorescence microscopy to detect prosthetic hip infection

Although aseptic mechanical loosening is reported to be the most common cause of prosthetic joint failure (Fitzgerald 1992), we have recently shown that the incidence of pros the t ic h ip infect ion is grea t ly underestimated (Tunney et a1 1998). In our study, the use of mild ultrasonication to dislodge bacteria growing within adherent biofilms on the surface of prostheses resulted in bacteria being cultured from 22% of retrieved prostheses (26/120 implants). Review of the notes from 18 of these 26 individuals revealed that infection prior to revision was suspected in only 6 cases and that in only 2 of these cases were bacteria cultured from the pre-operative aspirates or tissue removed at the time of surgery. Parallel examination of the inflammatory response in associated tissues showed that inflammatory cells indicative of infection were present in all of the patients from whom bacteria were cultured. Examination of associated tissue samples taken from culture-negative patients revealed a similar inflammatory response in 87% of patients. This implies that the detection of infection by culture may still be an underestimate of the real infection rate. The aim of the present study was, therefore, to determine if the detection of prosthetic hip infection could be improved by using 16s rRNA gene probes and immunolabelling in conjunction with fluorescence microscopy. Bacterial DNA was extracted from sonicates which had been stored at -70°C following ultrasonication of the retrieved implants. The 16s rRNA gene was detected by electrophoresis following polymerase chain reaction (PCR) using oligonucleotide primers specific for the eubacterial domain. In parallel, samples were screened by immuno-labelling and fluorescence microscopy (Patrick et a1 1995) using monoclonal antibody hybridoma secreting cell lines which are specific for Propionibacterium acnes surface antigens and polyclonal antisera specific for P. acnes, Staphylococcus aureus and S. epidermidis. Bacterial 16s rRNA genes were detected in all 26 culture-positive implants and in 64 of the 94 (68%) culture negative implants (Table I ) . The PCR results related well to the presence of inflammatory cells in associated tissue with 55 of the 73 tissue samples positive for the presence of inflammatory cells having corresponding implants which were PCR positive. Bacteria were detected in both culturepositive and culture-negative samples using immunolabelling and fluorescence microscopy.