Summary StatementA study to evaluate biofilm development on different coatings of UHMWPE was performed. We observed a species-specific effect, with S. aureus affected mainly by DLC-F and S. epiderm...

Summary StatementCombination of antibiotics with N-acetylcisteine and sub-MIC concentration of erythromycin was evaluated in two collection and 16 clinical strains of staphylococci isolated from PJI. The results were strain-dependent, so it evidences the necessity of perform individual studies of biofilm susceptibility.ObjectivesStaphylococci are the most common cause of prosthetic joint infections (PJI) (1), making the treatment of this disease difficult due to the increased resistance to antibiotics of biofilms. Combination between antibiotics and other compounds could be a good alternative. The aim of this study was to evaluate the effect of the combination of two compounds with nine antibiotics in biofilms formed by staphylococcal strains isolated from PJI.Methods16 clinical strains (8 S. aureus and 8 S. epidermidis) isolated from patients with PJI as well as 2 collection strains (S. aureus 15981 and S. epidermidis ATCC 35984) were tested against 9 antibiotics (rifampin, vancomycin, tigecycline, clind...

Because of their intrinsic properties, carbon nanotubes (CNTs) have been suggested for biomedical applications. We studied the anti-adherent performance of two ultra-high-molecular-weight polyethylene (UHMWPE) surfaces which contains 3% multi-wall CNT variant (MWCNT): Nanocyl/UHMWPE and Arc/UHMWPE. These surfaces were obtained by hot pressure forming after mechanical mixture. Additional nanoindentation studies were performed and hardness and stiffness were determined. Mechanical properties of the MWCNT/UHMWPE composites were also compared to raw UHMWPE and correlated with their anti-adherent performance. Comparing with UHMWPE, Nanocyl/UHMWPE was the least adherent surface. Bacterial adherence was also significantly reduced in Arc/UHMWPE for four strains. [GRAPHICS] .

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.

Artemisia absinthium is an aromatic and medicinal plant of ethnopharmacological interest and it has been widely studied. The use of A. absinthium based on the collection of wild populations can result in variable compositions of the extracts and essential oils (EOs). The aim of this paper is the identification of the active components of the vapour pressure (VP) EO from a selected and cultivated A. absinthium Spanish population (T2-11) against two parasitic protozoa with different metabolic pathways: Trypanosoma cruzi and Trichomonas vaginalis. VP showed activity on both parasites at the highest concentrations. The chromatographic fractionation of the VP T2-11 resulted in nine fractions (VLC1-9). The chemical composition of the fractions and the antiparasitic effects of fractions and their main compounds suggest that the activity of the VP is related with the presence of trans-caryophyllene and di-hydrochamazulene (main components of fractions VLC1 and VLC2 respectively). Additionally, the cytotoxicity of VP and fractions has been tested on several tumour and no tumour human cell lines. Fractions VLC1 and VLC2 were not cytotoxic against the nontumoural cell line HS5, suggesting selective antiparasitic activity for these two fractions. The VP and fractions inhibited the growth of human tumour cell lines in a dose-dependent manner.

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.

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.

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.

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.

Development of intrinsically antibacterial surfaces is of key importance in the context of prostheses used in orthopedic surgery. This work presents a thorough study of several plasma-based coatings that may be used with this functionality: diamond-like carbon (DLC), fluorine-doped DLC (F-DLC), and a high-fluorine-content-carbon-fluor polymer (CF(X)). The coatings were obtained by a radio-frequency plasma-assisted deposition on ultra high molecular weight polyethylene (UHMWPE) samples and physicochemical properties of the coated surfaces were correlated with their antibacterial performance against collection and clinical Staphylococcus aureus and Staphylococcus epidermidis strains. The fluorine content and the relative amount of C-C and C-F bonds were controlled by X-ray photoelectron spectroscopy, and hydrophobicity and surface tension by contact angle measurements. Surface roughness was studied by Atomic Force Microscopy. Additional nanoidentation studies were performed for DLC and F-DLC coatings. Unpaired t test and regression linear models evaluated the adherence of S. aureus and S. epidermidis on raw and coated UHMWPE samples. Comparing with UHMWPE, DLC/UHMWPE was the least adherent surface with independence of the bacterial species, finding significant reductions (p ≤ 0.001) for nine staphylococci strains. Bacterial adherence was also significantly reduced in F-DLC/ UHMWPE and CFx/UHMWPE for six strains.

The aim of this study was to evaluate the effects of human serum albumin (HSA), ibuprofen sodium (IBU) and N-acetyl-L-cysteine (NAC) against biofilm formation by seven biofilm-producing strains of Escherichia coli. Biofilm formation was studied using polystyrene microtitre plates in static conditions. The impact of the three compounds on bacterial growth and biofilm formation was tested by applying each compound in solution and as pre-treatment (coating) of polystyrene wells. When studied in solution, the minimum biofilm inhibitory concentrations of HSA, IBU and NAC were 8 mg/L (all strains), 2-125 mg/L (five strains) and 30-125 mg/L (five strains), respectively. Pre-treatment of polystyrene plates with HSA at 8 and 32,000 mg/L significantly reduced biofilm formation by all strains, whereas coating with 125 mg/L IBU and 1000 mg/L NAC did not. When HSA at 8 and 32,000 mg/L was combined with either 125 mg/L IBU or 1000 mg/L NAC in pre-treatment assays, more potent inhibition of biofilm was observed for some strains. Our results suggest that biofilm formation by E. coli may be prevented by coating medical devices with HSA alone or in combination with IBU or NAC. In addition, IBU and NAC could be useful in the treatment of urinary tract infections caused by E. coli due to their inhibitory effect on both bacterial growth and biofilm formation.

The in vitro activity of 22 antibiotics (including novobiocin) and beta-lactam/gentamicin combinations was assessed against 11 multidrug-resistant pneumococcal strains. Among orally administered drugs, only telithromycin, levofloxacin, and linezolid were active against all isolates, but their use is not indicated in pediatrics. Novobiocin could be a potential therapeutic alternative.

Streptococcus pneumoniae ability to produce biofilms may induce persistent infections and difficulties for eradication in vivo. We investigated the ability of 11 pneumococcal strains (serotypes 3, 6B, 9V, 19F, and 23F) to form biofilms on polystyrene plates at 16 and 24 h. The extent of biofilm was greater at 24 h in 10 strains, being the highest magnitude for serotype 3 strains. Human serum albumin at 25 000 mu g/mL and ibuprofen at 128 mu g/mL significantly reduced biofilm formation in 7 and 5 strains, respectively. Amoxicillin, erythromycin, and levofloxacin at supra-MIC concentrations were very active against planktonic cells of 3 selected strains but lower on biofilm-associated organisms in 2 strains and null against the third. Although N-acetyl-L-cysteine had very little activity against both planktonic and biofilm-forming organisms, when combined with the 3 antibiotics, a slightly enhanced activity against biofilm-embedded organisms in 1 strain and combined with amoxicillin in another one was observed. (C) 2010 Elsevier Inc. All rights reserved.

Development of intrinsically antibacterial surfaces is of key importance in the context of prostheses used in orthopaedic surgery. In this work we present a thorough study of several plasma based coatings that may be used with this functionality: diamond like carbon (DLC), fluorine doped DLC (F-DLC) and a high fluorine content carbon-fluor polymer (CFX). The study correlates the surface chemistry and hydrophobicity of the coating surfaces with their antibacterial performance. The coatings were deposited by RF-plasma assisted deposition at room temperature on ultra high molecular weight polyethylene (UHMWPE) samples. Fluorine content and relative amount of C-C and C-F bond types was monitored by X-ray photoelectron spectroscopy and hydrophobicity by water contact angle measurements. Adherence of Staphylococcus aureus and Staphylococcus epidermidis to non-coated and coated UHMWPE samples was evaluated. Comparisons of the adherence performance were evaluated using a paired t test (two materials) and a Kruskall Wallis test (all the materials). S. aureus was statistically significant (p< 0.001) less adherent to DLC and F -DLC surfaces than S. epidermidis. Both bacteria showed reduction of adherence on DLC/UHMWPE. For S. aureus, reduction of bacterial adherence on F-DLC/UHMWPE was statistically significant respect to all other materials.

Therapeutic alternatives are needed against infections caused by highly multidrug-resistant Streptococcus pneumoniae. Novobiocin, an old antibiotic, was tested in vitro and in a murine sepsis model against one amoxicillin-susceptible and three amoxicillin-resistant strains [minimum inhibitory concentrations (MICs) 8-64 mg/L]. Novobiocin MICs for all strains were 0.25-0.5 mg/L. In sepsis, novobiocin and amoxicillin were evaluated at 25, 50, 100 and 200 mg/kg given at 1, 5, 24 and 48 h post bacterial challenge. The most effective regimens in animals infected with the amoxicillin-susceptible strain were 200 mg/kg novobiocin and 25 mg/kg amoxicillin, achieving 100% survival and undetectable organisms in the peritoneum. Among mice infected with amoxicillin-resistant S. pneumoniae, 200 mg/kg novobiocin gave the highest protection (90-100% survivors), followed by 200mg/kg amoxicillin (60-100%), 100 mg/kg novobiocin (50-87.5%) and 50 mg/kg amoxicillin (14.3-25%). The killing effect of antibiotics in the peritoneum (mean Deltalog(10) colony-forming units/mL between treated and control mice) was as follows: 200 mg/kg novobiocin (-6.6)>200 mg/kg amoxicillin (-5.6)>100 mg/kg novobiocin (-3.7) > 50 mg/kg amoxicillin (-0.7). Total plasma and ultrafiltrate pharmacokinetics of novobiocin (200 mg/kg, single dose) in non-infected mice showed, respectively, half-lives of 151 min and 215 min, area under the concentration-time curves (AUCs) of 945.0 mgh/L and 136.6 mgh/L and maximal concentrations of 147 mg/L and 18 mg/L. Novobiocin may be a promising agent for therapy of highly beta-lactam-resistant pneumococcal infections.

The in vitro susceptibility of seven Escherichia coli biofilm-producing strains in their planktonic and biofilm-associated forms to amoxicillin, amoxicillin/clavulanic acid, cefotaxime, gentamicin, and ciprofloxacin was studied. Minimum inhibitory concentrations (MICs) were determined by the standard microdilution method and by the Alamar blue assay (providing the AB-MIC) at two levels of metabolic suppression and using standard and large inocula. Minimal biofilm inhibitory concentrations (AB-MBICs) on preformed biofilms on polystyrene plates were higher than the MICs and AB-MICs. Differences in magnitude depended on the antibiotic, strain, inoculum size, and the level of suppression of metabolism. Ciprofloxacin and gentamicin showed the greatest differences in the AB-MBIC as compared to AB-MIC. the possibility of antibiotic-resistant mutant selection within the biofilms was ruled out since bacteria recovered from the biofilm maintained the same MICs as before exposure to the antimicrobial agents. E. coli biofilms were much less sensitive than their planktonic counterparts.

Aimed to prevent Streptococcus pneumoniae biofilm infections, we studied the adherence of nine pneumococcal strains to polystyrene plates and on epithelial cells and the antiadhesive effect of albumin and xylitol. The adherence was variable among strains, but there was a good correlation between their adherent ability and binding to abiotic material and cells. Strains of serotypes 6B and 23F were the most adherent organisms, whereas serotype 3 strains were the least adherent. Human serum albumin (HSA) enhanced bacterial growth at low concentrations (0.5–2.5%) but inhibited it at 10%. Xylitol inhibited bacterial growth of all strains at concentrations ranging from 5 to 15%. Exposure to 0.5–5% HSA in solubilized form and to 5% HSA precoating of plates diminished adherence to polystyrene >80% for all strains, except for serotype 3 strains. Contrarily, 0.5 and 5% xylitol did not diminish significantly pneumococcal adherence to polystyrene plates or on epithelial cells. Our results suggest that 1) the potential application of HSA coatings on medical devices to inhibit pneumococcal adherence and 2) the possible beneficial effect of xylitol in preventing some pneumococcal infections could be because of its antimicrobial activity rather than to an antiadhesive effect.

The in vitro activity of penicillin, ampicillin, cefditoren, cefotaxime, erythromycin, clarithromycin, and levofloxacin against 763 clinical isolates of Streptococcus pyogenes was determined. Clinically significant isolates collected from November 2005 to December 2006 in the Czech Republic, Slovakia, Hungary, Poland, Romania, Estonia, Latvia, and Lithuania (the latter 3 analyzed as Baltic countries) were studied. No resistance to β-lactams and levofloxacin was found. The rate of erythromycin resistance in S. pyogenes varied among countries, being low (<10%) in Romania and Baltic countries, intermediate (10–20%) in Poland and Czech Republic, and high (>25%) in Hungary and Slovakia. The predominant (75.0%) erythromycin-resistant phenotype among S. pyogenes isolates was MLSB. The identification of the prevalence of erythromycin resistance mechanism could have impact on the choice of empiric antibiotic therapy for the clinicians in such countries.

OBJECTIVESTo study the ability of Corynebacterium urealyticum to produce biofilms and to compare the in vitro activity of antimicrobials against planktonic and biofilm-associated organisms.METHODSBiofilm formation on polystyrene plates by three C. urealyticum strains was studied in artificial urine under static conditions. The bactericidal activities of ciprofloxacin, moxifloxacin, vancomycin and erythromycin were studied against biofilm-associated organisms, and the results were compared with those obtained against planktonic organisms. Persister biofilm-associated organisms of each strain exposed to antibiotics were retested to determine the MIC of the same antibiotic.RESULTSThe three strains tested consistently produced biofilms. Planktonic organisms was susceptible to ciprofloxacin, moxifloxacin and vancomycin, and their MBC values were two to eight times higher than their corresponding MICs. Bactericidal effect on biofilm-associated organisms required very high antibiotic concentrations; the minimum biofilm bactericidal concentrations for ciprofloxacin, moxifloxacin and vancomycin ranged from 128 to > or =1024 times their respective MBCs for planktonic organisms. Erythromycin was not bactericidal against either planktonic or biofilm-associated organisms for the single susceptible strain tested. Persister biofilm-associated organisms exposed to erythromycin increased their MIC by a factor >8000, but no changes in susceptibility were observed with the other compounds.CONCLUSIONSThis work demonstrates that C. urealyticum produces biofilms on polystyrene plates and biofilm-associated organisms are much less susceptible to the bactericidal effect of the antibiotics; and the exposure of C. urealyticum to erythromycin may favour resistance selection. Overall, these results may explain the difficulties for bacterial eradication in chronic infections caused by C. urealyticum.

The ability of 15 Escherichia coli strains to form biofilms on polystirene plates was studied. The strains were serotyped, and their phenotypic expression of surface virulence factors (VFs), and antibiotic susceptibility was also determined. Moreover, 30 VFs-associated genes were analysed, including 15 adhesins (papC, papG and its three alleles, sfa/focDE, sfaS, focG, afa/draBC, iha, bmaE, gafD, nfaE, fimH, fimAvMT78, agn43, F9 fimbriae and type 3 fimbriae-encoding gene clusters), four toxins (hlyA, cnf1, sat and tsh), four siderophore (iron, fyuA, iutA and iucD), five proctetins/invasion-encoding genes (kpsM II, kpsMT III, K1 kps variant- neuC, traT and ibeA), and the pathogenicity island malX and cvaC. Morphological appearance and thickness of biofilms of two strong and three weak biofilm producers were also studied by confocal laser scanning microscopy (CLSM). Seven strains were classified as strong biofilm producers and the remaining eight strains were regarded as weak biofilm producers. Mannose-resistant haemagglutination was the only phenotypically expressed surface virulence factor more frequently found in the strong biofilm group. Five virulence-associated genes were more common (p<0.05) in strong biofilm producers: papC and papG alleles, sfa/focDE, focG, hlyA and cnf1. CLSM images showed irregular biofilms with projections at the top mainly in strong biofilm.