Antibacterial effect of novel biodegradable and bioresorbable PLDA/Mg composites.

Polylactic acid/Mg composites have been recently proposed for biodegradable osteosynthesis devices because, with regards to the neat polymer, they combine an enhanced biocompatibility and bioactivity with better mechanical properties, particularly creep strength. A question still arises about their bacterial behavior. For this purpose, composites of poly-L-D-lactic acid (PLDA) loaded with 1 and 10 wt.% of Mg microparticles were evaluated using Staphylococcus epidermidis, with special emphasis on the study of bacterial adhesion and biofilm formation. During biofilm formation the bacteria viability of the composites decreased up to 65.3% with respect to PLDA. These antibacterial properties do not compromise the cytocompatibility of the material as the composites enhanced the viability of mesenchymal stem cells and their osteogenic commitment. These findings provide an important added value to the biodegradable and biocompatible PLDA/Mg composites for the manufacture of osteosynthesis devices.

In vivo bactericidal efficacy of the Ti6Al4V surface after ultraviolet C treatment.

BACKGROUND: Biomaterial-associated infections are one of the most important complications in orthopedic surgery. The main goal of this study was to demonstrate the in vivo bactericidal effect of ultraviolet (UV) irradiation on Ti6Al4V surfaces. MATERIALS AND METHODS: An experimental model of device-related infections was developed by direct inoculation of Staphylococcus aureus into the canal of both femurs of 34 rats. A UV-irradiated Ti6Al4V pin was press-fit into the canal by retrograde insertion in one femur and the control pin was inserted into the contralateral femur. To assess the efficacy of UV radiation, the mean colony counts after inoculation in the experimental subjects and the control group were compared at different times of sacrifice and at different inoculum doses. RESULTS: At 72 h, the mean colony counts after inoculation in experimental femurs were significantly lower than those of the control group, with a reduction percentage of 76 % (p = 0.041). A similar difference between control and experimental pins was observed at 24 h using an inoculum dose <104 colony-forming units (CFU), for which the reduction percentage was 70.48 % (p = 0.017). CONCLUSION: The irradiated surface of Ti6Al4V is able to reduce early bacterial colonization of Ti6AlV pins located in the medullar channel and in the surrounding femur. The reductions depend on the initial inoculums used to cause infection in the animals and the greatest effects are detected for inoculums <104 CFU. LEVEL OF EVIDENCE: Not applicable.

Eficacia bactericida in vivo del farnesol sobre implantes de Ti6Al4V / In vivo bactericidal efficacy of farnesol on Ti6Al4V implants

Objetivo. Evaluar in vivo la actividad bactericida antiestafilocócica del farnesol sobre superficies de Ti6Al4V. Material y métodos. Se desarrolló un modelo experimental de infecciones en biomateriales inoculando Staphylococcus aureus ATCC 29213 en los fémures de 15 ratas wistar. Seguidamente se insertó una aguja de Ti6Al4V impregnada con farnesol 30 mM en el fémur estudio y una aguja control en el fémur control. Para valorar la eficacia bactericida se compararon las medianas de unidades formadoras de colonias recuperadas después de la inoculación en el grupo estudio y en el grupo control, para diferentes tiempos de eutanasia y tamaño de inóculos. Resultados. La mediana expresada en Log10 de los recuentos de UFC obtenidos en agujas de titanio con farnesol fue de 4,26 y en agujas sin farnesol, controles, fue de 4,86. Esta diferencia, al aplicar la prueba de t de Student para muestras relacionadas, resultó ser estadísticamente significativa (p = 0,001). La reducción mediana obtenida en las agujas con farnesol respecto a las agujas control fue del 74%. Conclusiones. El tratamiento con farnesol de agujas de Ti6Al4V, a una concentración de 30 mM, parece disminuir la tasa de colonización por Staphylococcus aureus en dichas agujas (AU)

Objective. To evaluate the in vivo anti-staphylococcal bactericidal activity of farnesol on Ti6Al4V surfaces. Material and methods. An experimental model of infection in biomaterials was developed by inoculation of Staphylococcus aureus ATCC 29213 into the canal of both femurs of 15 Wistar rats. A Ti6Al4V pin impregnated with 30 mM of farnesol was inserted into study femur, and a Ti6Al4V control was inserted into the control femur. To evaluate the bactericidal efficacy, a comparison was made between the median of the colony forming units recovered after inoculation in the study group and the control group for different times of euthanasia and inoculum size. Results. The median expressed as Log10 CFU counts obtained with farnesol titanium pin was 4.26, and in control group, it was 4.86, which was statistically significant (P=.001) on applying the Student t test for related samples. The median reduction obtained in farnesol pins relative to the control was 74%. Conclusions. Treatment with farnesol 30 mM on Ti6Al4V pins appears to decrease the rate of colonisation by Staphylococcus aureus (AU)

In vivo bactericidal efficacy of farnesol on Ti6Al4V implants. / Eficacia bactericida in vivo del farnesol sobre implantes de Ti6Al4V.

OBJECTIVE: To evaluate the in vivo anti-staphylococcal bactericidal activity of farnesol on Ti6Al4V surfaces. MATERIAL AND METHODS: An experimental model of infection in biomaterials was developed by inoculation of Staphylococcus aureus ATCC 29213 into the canal of both femurs of 15 Wistar rats. A Ti6Al4V pin impregnated with 30mM of farnesol was inserted into study femur, and a Ti6Al4V control was inserted into the control femur. To evaluate the bactericidal efficacy, a comparison was made between the median of the colony forming units recovered after inoculation in the study group and the control group for different times of euthanasia and inoculum size. RESULTS: The median expressed as Log10 CFU counts obtained with farnesol titanium pin was 4.26, and in control group, it was 4.86, which was statistically significant (P=.001) on applying the Student t test for related samples. The median reduction obtained in farnesol pins relative to the control was 74%. CONCLUSIONS: Treatment with farnesol 30mM on Ti6Al4V pins appears to decrease the rate of colonisation by Staphylococcus aureus.

Determination of biofilm production by Candida tropicalis isolated from hospitalized patients and its relation to cellular surface hydrophobicity, plastic adherence and filamentation ability.

Candida tropicalis is an emerging virulent species. The aim of this study is to determine the biofilm-forming ability of 29 strains of C. tropicalis isolated from inpatients, and to examine its relation with other virulence factors such as cellular surface hydrophobicity (CSH), immediate (15 min, IA) and late (24 h, LA) plastic adherence and filamentation ability. The study was performed in parallel using two incubation temperatures - 37 and 22 °C - to determine the effect of growth temperature variations on these pathogenic attributes of C. tropicalis. Biofilm formation (BF) was measured by optical density (OD) and by XTT reduction (XTT); Slime index (SI), which includes growth as a correction factor in BF, was calculated in both methods. All strains were hydrophobic and adherent - at 15 min and 24 h - at both temperatures, with higher values for 22 °C; the adhered basal yeast layer appears to be necessary to achieve subsequent development of biofilm. Filamentation ability varied from 76.2% of strains at 37 °C to 26.6% at 22 °C. All C. tropicalis strains were biofilm producers, with similar results obtained using OD determination and XTT measurement to evaluation methods; SI is useful when good growth is not presented. BF at 37 °C was similar at 24 h and 96 h incubation; conversely, at 22 °C, the highest number of biofilm-producing strains was detected at 96 h. CSH is an important pathogenic factor which is involved in adherence, is influenced by the filamentation of yeast, and plays a critical role in BF.

A Case of Human Oral Myiasis by Lucilia sericata in a Hospitalized Patient in Extremadura, Spain.

Myiasis is the term used to describe infestations, both obligatory and accidental, in vertebrate animals and humans by dipteral larvae. The oral cavity is rarely affected by this infestation and the circumstances which can lead to oral myiasis include persistent mouth opening together with poor hygiene, or facial traumatism. We present a case of oral myiasis by larvae of Lucilia sericata, a species present in the Iberian Peninsula, in a hospitalized patient with surgical problems.

Effect of allicin on the production of polysaccharide intercellular adhesin in Staphylococcus epidermidis.

AIMS: Polysaccharide intercellular adhesin (PIA) is the main agglutination agent in the biofilm forming strain Staphylococcus epidermidis. To find an explanation for the observed inhibition of biofilm formation by allicin, we studied the effect of allicin on PIA production in samples treated with sub MIC doses of allicin and compared this with a control culture without allicin. METHODS AND RESULTS: Bacteria (Staph. epidermidis ATCC 35984) were grown in glass tubes, and PIA was extracted by vortex vibration using microbeads and NN dimethyl acetamide/LiCl as solvent. The extracts were filtered and passed through size exclusion columns. Chromatographic fractions were analysed with an excess of sodium metaperiodate and the excess was determined spectrophotometrically using 2,4,6-tripyridyl-s-triazine. CONCLUSION: The amount of exopolysaccharides in samples previously treated with allicin is significantly lower than in the control. This finding suggests a specific enzymatic inhibition in PIA synthesis. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides an insight into the mechanism of biofilm formation, and is a biochemical model for PIA inhibition by allicin. The analysis proposed may be useful in studies of production of exopolysaccharides responsible for adherence and agglutination of Staph. epidermidis. Prevention of biofilm formation by allicin opens up a new field of in vitro studies and permits us to envisage future clinical applications.

Aspartyl proteinase, phospholipase, hemolytic activities and biofilm production of Candida albicans isolated from bronchial aspirates of ICU patients.

A correlation between mucosal colonization by Candida albicans and the subsequent development of invasive respiratory infection has been previously described. The aim of this study was to evaluate different enzymatic activities in vitro and to determine the capacity to form biofilms by 17 C. albicans isolates from bronchial aspirates of mechanically ventilated patients hospitalized in intensive care units. All the C. albicans clinical isolates tested were biofilm producers and displayed detectable levels of proteinase and hemolytic activities, although phospholipase activity was not detected in one strain. The correlation noted among the virulence factors studied suggests that the presence of more than one concurrent factor could facilitate the spread of infection.

Enzymatic activities of Candida tropicalis isolated from hospitalized patients.

Secretion of hydrolytic enzymes is considered a virulence factor in Candida spp. Extracellular enzymatic activities in 29 clinical isolates of Candida tropicalis were analyzed by plate assays. C. tropicalis, similar to Candida albicans, showed elevated hemolytic and esterase activities. However, unlike C. albicans, low aspartyl protease and very low phospholipase activities were detected in C. tropicalis isolates.

Modification of adherence to plastic and to human buccal cells of Candida albicans and Candida dubliniensis by a subinhibitory concentration of itraconazole.

Exposure to subinhibitory concentrations of antifungal agents can influence the adherence of Candida spp. to the host cell. In this study the adherence of Candida albicans ATCC 10231 and Candida dubliniensis CECT 11455 to plastic and to human buccal epithelial cells was evaluated following pre-exposure to 0.5 x minimum inhibitory capacity (MIC) of itraconazole and compared with the corresponding cellular surface hydrophobicity. The yeasts were grown in Sabouraud broth or RPMI-1640 with itraconazole (0.5 x MIC) for 24-26 h at 37 degrees C and the drug was then removed. The adhesion capacity to plastic was studied by turbidimetry in a polystyrene microtiter plate. The adhesion of the yeast to buccal epithelial cells was determined using microscopy techniques. The cellular surface hydrophobicity levels were determined by the microbial adhesion hydrocarbons test. Pre-exposure to itraconazole decreased plastic adherence and cellular surface hydrophobicity in both species when grown in RPMI. When C. albicans was grown in Sabouraud broth, it was nonhydrophobic and did not adhere and therefore no change was detected with the antibiotic. Itraconazole increased adherence to buccal epithelial cells in both species and media studied, as compared to controls without antifungal agents. To study the effects of these antifungal agents on pathogenicity mechanisms, it will be necessary to standardize the methodology for evaluation to determine their in vivo therapeutic efficacy.

Exposure to therapeutic concentrations of ritonavir, but not saquinavir, reduces secreted aspartyl proteinase of Candida parapsilosis.

The effect of ritonavir and saquinavir, HIV proteinase inhibitors, on the secreted aspartyl proteinase (Sap) activity of Candida parapsilosis was studied. In a proteinase-inducing medium (yeast carbon base-bovine serum albumin), Sap activity in all clinical isolates of C. parapsilosis (n = 20) was observed at 37 degrees C but not at 22 degrees C. The presence of ritonavir at a concentration of 8 microg/ml produced an inhibition close to 50% albumin consumption and also delayed yeast growth; however, saquinavir did not have any effect on growth or on Sap activity. In Sabouraud broth, which does not induce Sap production, no effect was shown on yeast growth by either of the two HIV proteinase inhibitors studied.

Moxifloxacin and biofilm production by coagulase-negative staphylococci.

The in vitro activity of moxifloxacin against 41 strains of coagulase-negative staphylococci was determined. A relationship between the activity of moxifloxacin and biofilm formation was detected. Biofilm-producing strains were more resistant to moxifloxacin than biofilm-negative strains. Our global results obtained with six strains of Staphylococcus epidermidis showed that subinhibitory concentrations of moxifloxacin did not significantly modify biofilm formation. On the other hand, moxifloxacin concentrations of 2, 10, 50 and 100 x MIC produced a log decrease in viable count (included in a biofilm) of 0.20, 0.37, 1.10 and 1.69, respectively.

The measurement temperature: an important factor relating physicochemical and adhesive properties of yeast cells to biomaterials.

Flow chambers applied to the study of the initial adhesion process of Candida parapsilosis are rarely found in the literature. The ability of these microorganisms to proliferate and form biofilms in environments at temperatures around 22 or 37 degrees C is reflected in the contamination of laboratory instruments and material or in human implant infections, respectively. The initial interaction between yeasts and substrata is mediated by physicochemical forces, which in turn originate from the physicochemical surface properties of both interacting phases. In this context, this work aims to relate the initial rates of adhesion rates to glass and silicone of Candida parapsilosis, strains 294 and 289, grown at 22 and 37 degrees C with the theoretical predictions of the adhesion process, expressed by the interaction free energies and calculated through the physicochemical parameters, which are also measured at 22 and 37 degrees C. The results indicate that physicochemical parameters of yeasts are changed not only by the culture temperature but also by the measurement temperature; only when the measurement temperature is equal to the growth temperature a coherent relation between in vitro adhesion data and interaction free energies can be established. In this sense, the adhesion to glass is mediated by long-range forces or, what amounts to the same thing, by Lifshitz-van der Waals interaction free energy. On the other hand, the adhesion to silicone rubber seems to be moderated by acid-base interaction free energy, which involves the presence of short-range forces. Based on these results, it can be assumed that the substratum surface properties are directly related to the kind of force acting on the initial microbial adhesion process, while cell surface properties dictate the changes in the strength of the force between different samples.

In vitro activity of allicin against Staphylococcus epidermidis and influence of subinhibitory concentrations on biofilm formation.

AIMS: The aim of this study is to determine the in vitro activity of allicin against Staphylococcus epidermidis and to evaluate the influence of allicin on biofilm formation. METHODS AND RESULTS: In vitro activity of allicin (diallyl thiosulphinate) against 38 strains of S. epidermidis was investigated. The activity of allicin was similar against S. epidermidis methicillin susceptible and methicillin resistant strains [minimum inhibitory concentration (MIC)90=8 mg l(-1)]. In general, subinhibitory concentrations (sub-MIC) of allicin diminished biofilm formation in the five strains analysed. CONCLUSION: The results confirm the antibacterial effect of allicin. Sub-MICs of allicin also diminished the biofilm formations by S. epidermidis. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study shows that allicin is active in vitro against S. epidermidis and that sub-MICs of allicin may play a role in the prevention of adherence of this bacteria to medical devices.

Effect of ritonavir and saquinavir on Candida albicans growth rate and in vitro activity of aspartyl proteinases.

An in vitro study to evaluate the antifungal effect and activity of aspartyl proteinases of the HIV-proteinase inhibitors ritonavir and saquinavir was conducted. Ritonavir diminished the growth rate of Candida albicans as well as the activity of its secreted aspartyl proteinases (Saps) in a nitrogen-limited medium, yeast carbon base and bovine serum albumin (YCB-BSA). This inhibition occurred in a dose-dependent fashion; with 8 mg l(-1) of ritonavir a partial growth inhibition (44%) was produced. The growth rate of C. albicans in medium with saquinavir was similar to that seen in the control, and Sap activity was inhibited only at high concentrations. In conventional medium (RPMI-1640), which does not induce the production of yeast proteases, no inhibitory effect was detected with either HIV-protease inhibitor.

Adhesion of Enterococcus faecalis 1131 grown under subinhibitory concentrations of ampicillin and vancomycin to a hydrophilic and a hydrophobic substratum.

The effect of two subinhibitory antibiotic concentrations of ampicillin and vancomycin during growth on the adhesion of Enterococcus faecalis 1131 to glass and silicone rubber was studied in a parallel plate flow chamber. Initial deposition rates and numbers of adhering bacteria after 4 h were higher on hydrophilic glass than on hydrophobic silicone rubber, regardless of growth conditions. The presence of 1/4 minimal inhibitory concentration (MIC) of ampicillin during growth reduced enterococcal adhesion to both substrata, but growth in the presence of 1/4 MIC vancomycin did not affect the adhesion of E. faecalis. Moreover, enterococcal adhesion increased after growth in the presence of 1/8 MIC vancomycin. The increased adhesion after growth in the presence of subinhibitory concentrations of vancomycin may have strong implications for patients living with implanted biomaterials, as they may suffer adverse effects from use of this antibiotic, especially since bacteria once adhered are less sensitive to antibiotics.

Resistance to vancomycin, LY333328, ciprofloxacin and trovafloxacin of community-acquired and nosocomial strains of Enterococcus faecalis isolated in Badajoz (Spain) with and without high-level resistance to streptomycin and gentamicin.

In vitro resistance of community-acquired and nosocomial strains of Enterococcus faecalis isolated in Badajoz (Spain) were determined by a microdilution method. The isolates were identified with conventional MicroScan Pos Combo 4 I dehydrated panels. No resistance to glycopeptides was found, but LY333328 was 2-4 times more active than vancomycin. In the nosocomial strains, high-level resistance to streptomycin (HLRS) was 54.7%, and high-level resistance to gentamicin (HLRG) was 38.1%. Resistance to ciprofloxacin and trovafloxacin was 45.3 and 38.9%, respectively. In the community-acquired isolates, HLRS, HLRG, resistance to ciprofloxacin and resistance to trovafloxacin were 44.2, 17.3, 15.4 and 13.5%, respectively. Trovafloxacin was 2-4 times more active than ciprofloxacin against both groups of strains. An association between high-level resistance to aminoglycosides and resistance to fluoroquinolones was noted. The resistance to aminoglycosides did not influence the activity of vancomycin and LY333328.

[Pathogenicity of Enterococcus spp. Characteristics of 169 hospital isolates]. / Patogenicidad de Enterococcus spp. Características de 169 aislamientos hospitalarios.

BACKGROUND: To know the characteristics of Enterococcus spp. strains isolated in the hospital; to analyse the importance of this microorganism and its resistance to antimicrobial agents. PATIENTS AND METHODS: Retrospective analysis of the case histories of 169 patients with Enterococcus spp. isolates, selected at random at the Infanta Cristina Hospital in Badajoz. Investigation was carried out on: age, date of admission and discharge, clinical symptoms, risk factors, previous antibiotic treatment, clinical and microbiological evolution, recommended treatment and prescribed treatment. Identification of microorganisms and antibiogram with Pos Combo 41 microScam panels, read on Baxter WalkAway-40 equipment. RESULTS: The most frequently found species was Enterococcus faecalis. In 75 cases the isolations were polymicrobial. The enterococci were isolated mainly from urinary infections (27%), skin infections (20%), intrabdominal infections (14%) and infections from surgical wounds (14%). The existence of peripheral catheter was the main risk factor. All the strains of E. faecalis were susceptible to the glycopeptides. Two strains of Enterococcus faecium were not susceptible to vancomycin and one of these was not susceptible to teicoplanin. Mortality was from 21-27.5%. CONCLUSIONS: Enterococcus spp. is frequently isolated in nosocomial infections, although in approximately half the cases it is associated with other bacteria. For this reason, it is not always possible to determine its pathogenic contribution. The isolated strains, except two strains of E. faecium, are susceptible to vancomycin. A relation exists between high resistance to aminoglucosides and resistance to fluoroquinolones.

Phagocytosis and killing of slime-producing Staphylococcus epidermidis bypolymorphonuclear leukocytes. Effects of sparfloxacin.

The interaction of slime-producing Staphylococcus epidermidis (S+ strain) with human polymorphonuclear leukocytes (PMNs) was analyzed. The phagocytosis index (PI) and the killing index (KI) were evaluated, and the effect of subinhibitory concentrations (sub-MICs) of sparfloxacin in this interaction was determined. The study was carried out in parallel with a non-slime-producing strain (S- strain). The MIC values of sparfloxacin against both strains was 0. 06 mg/ml. In the S- strain the mean values of PI and KI were 82% and 31%, respectively, whereas in the S+ strain these values diminished to 49% and 8%, respectively (p <0.05) when the inocula were prepared in aerobiosis. In a constant flow atmosphere of 5% CO2, the results were similar to the previous ones in the S- strain (PI = 79% and KI = 27%), whereas in the S+ strain a increase in the PI (59%) and a similar value to the KI (9%) were observed. Significant increases (p <0.05) in the PI were noted when PMNs were preincubated with 1/4 and 1/16 MIC of sparfloxacin, and S. epidermidis S+ grown in aerobiosis were used. In the other assays the variations in PI and KI were not statistically significant (p >0.05). Our results confirm that slime protects against phagocytosis and killing by PMNs, although they also suggest that the S+ strains possess additional properties which make them resistant to the action of the PMNs.

Influence of N-acetylcysteine on the formation of biofilm by Staphylococcus epidermidis.

The influence of various concentrations (0.003-8 mg/mL) of N-acetylcysteine on the formation of biofilms by 15 strains of Staphylococcus epidermidis has been studied. A dose-related decrease in biofilm formation was observed, except with the lowest concentrations. The 'slime' index relative to the control was 63%, 55%, 46%, 34%, 26% and 26% in the presence of 0.25, 0.5, 1, 2, 4, and 8 mg/mL of N-acetylcysteine, respectively. These data are statistically significant. The inhibitory effect of 2 mg/mL of N-acetylcysteine on slime formation was also verified by electron microscopy.