PCR-hybridization after sonication improves diagnosis of implant-related infection.

PURPOSE: We wanted to improve the diagnosis of implant-related infection using molecular biological techniques after sonication. METHODS: We studied 258 retrieved implant components (185 prosthetic implants and 73 osteosynthesis implants) from 126 patients. 47 patients had a clinical diagnosis of infection (108 components) and 79 patients did not (150 components). The fluids from sonication of retrieved implants were tested in culture and were also analyzed using a modified commercial PCR kit for detection of Gram-positive and Gram-negative bacteria (GenoType BC; Hain Lifescience) after extraction of the DNA. RESULTS: 38 of 47 patients with a clinical diagnosis of infection were also diagnosed as being infected using culture and/or PCR (35 by culture alone). Also, 24 patients of the 79 cases with no clinical diagnosis of infection were identified microbiologically as being infected (4 by culture, 16 by PCR, and 4 by both culture and PCR). Comparing culture and PCR, positive culture results were obtained in 28 of the 79 patients and positive PCR results were obtained in 35. There were 21 discordant results in patients who were originally clinically diagnosed as being infected and 28 discordant results in patients who had no clinical diagnosis of infection. INTERPRETATION: For prosthetic joint infections and relative to culture, molecular detection can increase (by one tenth) the number of patients diagnosed as having an infection. Positive results from patients who have no clinical diagnosis of infection must be interpreted carefully.

Importance of antibiotic penetration in the antimicrobial resistance of biofilm formed by non-pigmented rapidly growing mycobacteria against amikacin, ciprofloxacin and clarithromycin.

OBJECTIVES: To study the resistance of biofilms developed by non-pigmented rapidly growing mycobacteria (NPRGM) against amikacin, ciprofloxacin and clarithromycin in an in vitro model using clinical strains of different species. DESIGN: Antimicrobial susceptibilities of different clinical strains of Mycobacterium abscessus, Mycobacterium chelonae, Mycobacterium fortuitum, Mycobacterium peregrinum, Mycobacterium mucogenicum and Mycobacterium mageritense using conventional techniques were measured. Biofilm resistance was measured by using the sandwich technique developed by Anderl et al. using a concentration of antibiotic of 50mg/L. Penetration of antibiotics through biofilm was measured using the same technique with minimal modifications. RESULTS: NPRGM biofilms showed drug resistance (percentages of viable bacteria >1% of those of controls) against antibiotics that are commonly used for the treatment of infections caused by these organisms, although there are intraspecies differences between strains. We have detected differences in antibiotic penetration through biofilms with an important permeability barrier for ciprofloxacin. However, other mechanisms must be probably more important to explain the antimicrobial resistance of NPRGM biofilm. CONCLUSIONS: Biofilms formed by NPRGM are resistant to amikacin, ciprofloxacin and clarithromycin. As no resistance differences between the tested antibiotics have been observed, it is likely that biofilm permeability of antibiotics is of low importance for antimicrobial resistance of biofilms.

Biofilm development by clinical isolates of Staphylococcus spp. from retrieved orthopedic prostheses.

BACKGROUND: Biofilms are considered the key factor in the development of implant-related infections. However, only a few reports have dealt with the ability of organisms isolated from such infections to develop biofilms in vitro. METHODS: We evaluated different phenotypic techniques (2 microtiter plate assays and confocal laser scanning microscopy (CLSM) and genotypic techniques (detection of the ica operon) related to biofilm development by clinical isolates of Staphylococcus spp. RESULTS: All 26 strains tested (from 23 specimens) were biofilm producers. Stepanovic test detected biofilm formation in 85% of the strains, microtiter plate assay in 65%, and CLSM in 39%. The ica operon was detected in 73% of all strains (all 13 S. aureus strains and 6 of the 13 coagulase-negative Staphylococcus strains). 7 ica-negative strains were biofilm-positive by phenotypic methods. INTERPRETATION: The detection of ica genes could not be related to the phenotypic ability of the strains to develop a biofilm in vitro, so both studies (genetic and phenotypic) are required for a better evaluation of the biofilm-producing ability of clinical strains of Staphylococcus isolated from orthopedic infections.

Effect of different agents with potential antibiofilm activity on antimicrobial susceptibility of biofilms formed by Staphylococcus spp. isolated from implant-related infections.

An in vitro study aimed to evaluate the effect of N-acetyl cysteine (NAC) or sub-ICs of erythromycin on antimicrobial susceptibility of staphylococcal biofilms was performed. Staphylococcus aureus and Staphylococcus epidermidis strains were isolated from patients with prosthetic joint infections using a previously published sonication procedure. Conventional susceptibility studies were performed using microdilution according to the CLSI procedures. Biofilm susceptibility was performed using the Calgary methodology. The addition of NAC showed no effect with the S. aureus strains, and a strain-dependent effect with the S. epidermidis strains. No effect was detected with erythromycin for almost all tested strains.

In vitro susceptibility to antibiotics of staphylococci in biofilms isolated from orthopaedic infections.

Prosthetic joint infections (PJIs) are related to the formation of biofilms, mainly by Staphylococcus aureus and Staphylococcus epidermidis. Therapy is usually selected according conventional susceptibility testing, but these data may be insufficient to detect the true antibiotic susceptibility in the biofilm. In total, 32 clinical strains (17 S. aureus and 15 S. epidermidis) isolated from patients with PJIs as well as 2 collection strains (S. aureus 15981 and S. epidermidis ATCC 35984) were tested against nine antibiotics commonly used in the treatment of PJIs (rifampicin, vancomycin, tigecycline, clindamycin, trimethoprim/sulfamethoxazole, ciprofloxacin, cloxacillin, daptomycin and fosfomycin) using the Calgary Biofilm Device. None of the antibiotics proved to be totally effective against biofilms in both species, with minimum biofilm eradication concentrations (MBECs) highly above the minimum inhibitory concentrations for most of the antibiotics (>1024 mg/L). Rifampicin and tigecycline showed MBECs slightly lower, mainly against S. epidermidis biofilms, and only two strains of this staphylococcal species were susceptible to almost all of the antibiotics tested. These results show that the search for new compounds with antimicrobial and antibiofilm properties is mandatory as well as the development of other strategies that could lead to the prevention and treatment of PJIs. In addition, more studies are necessary to obtain a better understanding of the mechanisms involved in antimicrobial resistance of biofilms.

In vitro susceptibility of Staphylococcus aureus and Staphylococcus epidermidis isolated from prosthetic joint infections.

Prosthetic joint infections (PJI) are severe complications in Orthopedics, with Staphylococcus aureus and Staphylococcus epidermidis being the most commonly isolated pathogens. The variable antimicrobial susceptibility found in these microorganisms, along with the increasing number of methicillin-resistant strains, increases the difficulty of antibiotic selection and makes it necessary to perform individual susceptibility studies to select the optimal antibiotic treatment. The aim of this study was to evaluate the in vitro susceptibility pattern of 35 clinical strains isolated from PJI (17 S. aureus and 18 S. epidermidis) against rifampin, vancomycin, tygecicline, clindamycin, cotrimoxazole, cloxacillin, ciprofloxacin, daptomycin and fosfomycin. In vitro susceptibility assays were performed using the broth microdilution method and agar dilution for fosfomycin. MBC was also determined. Tygecicline and daptomycin showed the highest antimicrobial activity with low MIC(90) values, and no resistant strains were detected. On the other hand, ciprofloxacin and cloxacillin exhibited a poor antimicrobial effect with a high percentage of nonsusceptible strains in both species. Bactericidal activity rates revealed the bacteriostatic behavior of rifampin, tygecicline, cotrimoxazole, fosfomycin and clindamycin, whereas vancomycin and cloxacillin showed species- and strain-dependent behavior. Daptomycin and ciprofloxacin were observed to be efficient bactericidal agents against the tested strains. According to our data, rifampin, tigecycline, daptomycin and fosfomycin showed high in vitro activity against most staphylococcal strains isolated from the PJIs tested, although daptomycin seems to be the best alternative to vancomycin therapy.

Bacterial adherence to separated modular components in joint prosthesis: a clinical study.

Bacterial adherence on total joint replacement implants may lead to biofilm formation and implant-related osteoarticular infection. It is unclear if different biomaterials in the prosthetic components are more prone to facilitate this bacterial adherence, although ultrahigh molecular weight polyethylene (UHMWPE) component exchange in modular systems has been clinically utilized in the early management of these infections. To clarify if the amount of clinically adhered microorganisms was related to the material or the component, we investigated retrieved implants from infected joint replacements. Thirty-two patients were revised after confirmed implant-related infection through positive cultures. Eighty-seven total joint components (hip and knee) were obtained and separately sonicated following a previously published protocol. Cultures were quantified, and detected colony forming units (CFU) were adjusted according to the component surface and compared based on the component material and location. Variable adherence of bacteria to chrome cobalt alloys, UHMWPE, hydroxyapatite coated components, and titanium alloys. The commonest isolated organisms were Staphylococcus epidermidis (23 of 87 components) and Staphylococcus aureus (10 of 87). Twelve components did not show any microorganism adhered despite location in an infected joint, with positive cultures in other components. A mixed linear model adjusted for random effects (the random effect being the infected patient) obtained convergence for the CFU/mm(2) variable, but could not confirm a significantly higher adherence to a particular component or to a particular biomaterial. Therefore, the bacterial adherence primarily depends on the infective microorganism and the response of each individual patient, rather than materials or components.

Usefulness of SBA-15 mesoporous ceramics as a delivery system for vancomycin, rifampicin and linezolid: a preliminary report.

Bone infections are a challenge for modern medicine. The most common pathogen is Staphylococcus aureus, which usually develops a biofilm inside the infected bone. Local release of antibiotics within the infected tissue may diminish this problem because high concentrations of the antibiotic would be delivered to the required place. This study was carried out to evaluate silica-based mesoporous material SBA-15 as a delivery system for three antibiotics with activity against S. aureus, namely vancomycin, rifampicin and linezolid, alone or in combination. SBA-15 disks were loaded with antibiotics by adsorption using a 1000 mg/L solution. Measurements of biological activity were carried out by bioassay tests, and antibiotic release was monitored by high-performance liquid chromatography (HPLC). In all cases, the ceramic disks released most of the antibiotics at the initial stage of the experiments, with concentrations above the susceptibility breakpoints. The most active antibiotic was rifampicin, with an active concentration of 96.14 mg/L at 24 h, followed by linezolid (7.2 mg/L) and vancomycin (5.5 mg/L). In the HPLC measurements, the antibiotic that showed the best release was linezolid, followed by vancomycin; rifampicin alone could not be measured by HPLC with precision. Taking into account all these results, the antibiotic that remains most active after loading and release is vancomycin (77.46%), followed by linezolid (24%). The results presented here demonstrate the efficacy of SBA-15 bioceramics for local release of antibiotics, which could be of interest in the context of bone infection.