Biomedical subjects

G Donelli

Publications and source records attributed to G Donelli.

At least 19 recordsLinked to original sources

PubMed2007

Synthesis, characterization, and in vitro activity of antibiotic releasing polyurethanes to prevent bacterial resistance.

Central venous catheters are a major cause of nosocomial bloodstream infections. Different attempts have been made to incorporate antimicrobial agents into catheters, particularly directed at the surface-coating of devices. To facilitate the antimicrobial adsorption, various cationic surfactants, which however showed several problems, have been used. On the other hand, impregnated catheters with only antimicrobials have demonstrated a short-term duration due to the difficulties to deliver the drug slowly. Thus, in order to obtain high antimicrobial-polymer affinity we synthesized or modified polyurethanes to introduce different functional groups. Polymers were loaded with two antibiotics, cefamandole nafate and rifampin (RIF), chosen for both their functional groups and their action spectrum. The in vitro release behavior showed that the elution of drugs depended on the matrix hydrophilicity and on the antibiotic-polymer and antibiotic-antibiotic interactions. To increase the amount of drug released, polyethylene glycol (PEG) used as a pore forming agent at different molecular weights was incorporated in the polymer bulk with antibiotics. As for the in vitro antimicrobial activity of matrices, assessed by Kirby-Bauer test, it was seen that antibiotics released from various formulations inhibited the bacterial growth and exerted a synergistic effect when both were present. In particular, PEG10000-containing polymer was active against the RIF-resistant S. aureus strain up to 23 days. These results suggest that the combined entrapping of antibiotics and pore formers in these novel polymer systems could be promising to prevent the bacterial colonization and to control the emergence of bacterial resistance.

Anti-Bacterial Agents↗

PubMed2006

Pore formers promoted release of an antifungal drug from functionalized polyurethanes to inhibit Candida colonization.

AIMS: As a preventive strategy to inhibit fungal biofilm formation on medical devices, we planned experiments based on polyurethane loading with fluconazole plus pore-former agents in order to obtain a promoted release of the antifungal drug. METHODS AND RESULTS: Different functional groups including carboxyl, hydroxyl, primary and tertiary amino groups, were introduced in polyurethanes. Fluconazole was adsorbed in higher amounts by the most hydrophilic polymers and its release was influenced by the degree of polymer swelling in water. The entrapping in the polymer of polyethylenglycol as a pore former significantly improved the fluconazole release while the entrapping of the higher molecular weight porogen albumin resulted in a controlled drug release and in an improved antifungal activity over time. CONCLUSIONS: Among the tested in vitro models, best results were achieved with an hydrophobic polymer impregnated with 25% (w/w) albumin and fluconazole which inhibited Candida albicans growth and biofilm formation on polymeric surfaces up to 8 days. SIGNIFICANCE AND IMPACT OF THE STUDY: The combined entrapping in polymers of pore formers and an antifungal drug and the consequent controlled release over time is a novel, promising approach in the development of medical devices refractory to fungal colonization.

Adsorption↗

PubMed2005

A 700 MHz 1H-NMR study reveals apoptosis-like behavior in human K562 erythroleukemic cells exposed to a 50 Hz sinusoidal magnetic field.

PURPOSE: To study cell damage and possible apoptosis in K562 human erythroleukemic cells exposed for 2 h to an extremely low frequency (ELF) 50 Hz sinusoidal magnetic field with a magnetic induction of either 1 or 5 mT using high resolution proton nuclear magnetic resonance (1H-NMR) spectroscopy. MATERIALS AND METHODS: One-dimensional 1H-NMR spectra were obtained on whole K562 cells and perchloric acid extracts of these cells. In addition, two-dimensional 1H-NMR spectra were also acquired. Cell damage was examined by lactate dehydrogenase release and changes in cell growth were monitored by growth curve analyses, bromodeoxyuridine incorporation and Ki67 antigen localization. Cell death (necrosis and apoptosis) were also studied by using the chromatin dye Hoechst 33258. RESULTS: The variations in numerous metabolites observed with 1H-NMR reveal apoptosis-like behavior in response of K562 cells to ELF fields. CONCLUSION: 1H-NMR can be extremely useful in studying the effects of ELF fields on cells. In particular, the variations in metabolites which suggest apoptosis-like behavior occur when the cells are not identifiable as apoptotic by more traditional techniques.

Apoptosis↗

PubMed2004

Detection of free and plankton-associated Helicobacter pylori in seawater.

AIMS: To detect both free and plankton-associated Helicobacter pylori in seawater samples collected on the Italian coast of the Adriatic Sea using a nested-PCR. METHODS AND RESULTS: Dissolved oxygen, pH, salinity and chlorophyll 'a' were the parameters recorded together with the characterization of zooplanktonic organisms. Plankton-associated H. pylori DNA was searched for in water samples filtered through 200 and 64 microm nylon nets whereas free bacteria were retained with the subsequent filtration through 0.22 microm pore-size membranes. Nested-PCR using primers for the glmM (ureC) gene was performed to reveal the presence of H. pylori. The DNA sequencing of amplified products confirmed the specificity of the assay. The sensitivity of the nested-PCR assay for H. pylori detection was 62 CFU per 100 ml in spiked water samples. Helicobacter pylori either free or bound to planktonic organisms was found in seven of 12 monthly samples. In particular, free bacteria were detected during the summer sampling and in November, December and March associated to planktonic cells. CONCLUSIONS: The presence of free and plankton-associated H. pylori in seawater suggests that it can be a significant reservoir and a potential route of transmission for the microorganism. SIGNIFICANCE AND IMPACT OF THE STUDY: Our study seems to provide a promising background to define new and effective strategies for surveillance of this human pathogen.

Animals↗

PubMed2004

Usnic acid, a natural antimicrobial agent able to inhibit bacterial biofilm formation on polymer surfaces.

In modern medicine, artificial devices are used for repair or replacement of damaged parts of the body, delivery of drugs, and monitoring the status of critically ill patients. However, artificial surfaces are often susceptible to colonization by bacteria and fungi. Once microorganisms have adhered to the surface, they can form biofilms, resulting in highly resistant local or systemic infections. At this time, the evidence suggests that (+)-usnic acid, a secondary lichen metabolite, possesses antimicrobial activity against a number of planktonic gram-positive bacteria, including Staphylococcus aureus, Enterococcus faecalis, and Enterococcus faecium. Since lichens are surface-attached communities that produce antibiotics, including usnic acid, to protect themselves from colonization by other bacteria, we hypothesized that the mode of action of usnic acid may be utilized in the control of medical biofilms. We loaded (+)-usnic acid into modified polyurethane and quantitatively assessed the capacity of (+)-usnic acid to control biofilm formation by either S. aureus or Pseudomonas aeruginosa under laminar flow conditions by using image analysis. (+)-Usnic acid-loaded polymers did not inhibit the initial attachment of S. aureus cells, but killing the attached cells resulted in the inhibition of biofilm. Interestingly, although P. aeruginosa biofilms did form on the surface of (+)-usnic acid-loaded polymer, the morphology of the biofilm was altered, possibly indicating that (+)-usnic acid interfered with signaling pathways.

Anti-Bacterial Agents↗

PubMed2004

Polyurethanes loaded with antibiotics: influence of polymer-antibiotic interactions on in vitro activity against Staphylococcus epidermidis.

Acidic or basic polyurethanes were loaded with antibiotics to develop materials to prevent medical device-related infections. A correlation between polymer-antibiotic interactions and amount of drug absorbed by polymers and released over time was found. Since the employed antibiotics, i.e. amoxicillin, cefamandole nafate, rifampin and vancomycin, possessed at least an acidic group in their structural formula, the introduction of basic tertiary amines in the polyurethane side-chain resulted in an increased polymer ability to adsorb antibiotics. However, a stronger ionic interaction between this polymer and the antibiotics caused a release of lower amount of drug over time. Antibiotics released from polymers inhibited Staphylococcus epidermidis growth on agar. Antibiotic-loaded polyurethanes kept in water for increasing times were still able to show inhibition zones of bacterial growth. The antibacterial activity lasted up to 3 hours for amoxicillin, 24 hours for vancomycin, 8 days for cefamandole nafate and 8 months for rifampin.

Amoxicillin↗

PubMed2004

Population dynamics in ageing Helicobacter pylori.

The aim of this work was to characterize population changes occurring in aged broth cultures of Helicobacter pylori. Experiments were performed using clinical strains cultured immediately after isolation and after multiple subcultures in solid medium. Morphological changes in the ageing bacteria during a 7-day broth culture were analysed by optical and electron microscopy. The expression of the virulence factor, CagA, together with the presence of the cell cycle regulator, cGMP, were also assessed. The transition from bacillary to coccoid forms was the main morphological change observed in freshly isolated bacteria, together with the increase in cGMP from 1 to 2.25 nmoles/mg of proteins within the first 7 days of broth culture. A similar trend of morphological and physiological changes was observed in cells after multiple subcultures in solid medium with a major presence of large cell clusters. The cagA gene product was always expressed in all experimental conditions evaluated. These data show a significant morphological and physiological diversity in fresh, ageing and aged cultures of H. pylori.

Antigens, Bacterial↗

PubMed2003

Retention of virulence in viable but non-culturable halophilic Vibrio spp.

The viable but non-culturable (VBNC) forms of two environmental strains of Vibrio alginolyticus 1 and Vibrio parahaemolyticus 66 and one strain of V. parahaemolyticus ATCC 43996 showing virulence characteristics (hemolysin production, adhesive and/or cytotoxic ability, in vivo enteropathogenicity) were obtained by culturing bacteria in a microcosm consisting of artificial sea water (ASW) and incubating at 5 degrees C with shaking. Every 2 days, culturability of the cells in the microcosm was monitored by spread plates on BHI agar and total count and the percentage of viable cells were determined by double staining with DAPI and CTC. When cell growth was not detectable (<0.1 CFU/ml), the population was considered non-culturable and, then, the VBNC forms were resuscitated in a murine model. For each strain, eight male Balb/C mice were intragastrically inoculated with 0.1 ml of concentrated ASW bacterial culture. Two mice from each group were sacrificed at 2, 4, 8, and 12 days after challenge for autopsy and re-isolation of the microorganisms from the intestinal tissue cultures. Isolation was obtained in 25% of the animals challenged with the VBNC V. alginolyticus strain, in 37.5% of those challenged with the VBNC V. parahaemolyticus strain of environmental origin and in 50% of the animals infected with VBNC V. parahaemolyticus ATCC 43996. The strains thus isolated were again subjected to biological assays to determine the retention of pathogenicity. The virulence characteristics that seemed to disappear after resuscitation in the mouse were subsequently reactivated by means of two consecutive passages of the strains in the rat ileal loop model. The results obtained indicate that VBNC forms of the strains examined can be resuscitated and retain their virulence properties.

Animals↗

PubMed2002

New polymer-antibiotic systems to inhibit bacterial biofilm formation: a suitable approach to prevent central venous catheter-associated infections.

Intravascular catheters are widely employed in medical practice. However, complications such as local or systemic infections are frequently related to their use. The significant increase in this type of nosocomial infection has prompted the search for new strategies to prevent them. This paper reports on an experimental model to prevent catheter-related infections based on the adsorption of a beta-lactam antibiotic (cefamandole nafate) on functionalized urethane polymers. The polyurethanes synthesized were used to coat a commercial central venous catheter. The influence of functional groups on the polymer-antibiotic interaction was analyzed and the kinetics of the antibiotic release from the catheters was dynamically studied. We were able to realize a polymer-antibiotic system able to inhibit bacterial growth up to 7 days. These promising results have encouraged us to extend this experimental model to other polymer-antibiotic systems in order to identify those allowing bacterial growth inhibition for longer times.

Anti-Bacterial Agents↗

PubMed2001

Searching the point of no return in Helicobacter pylori life: necrosis and/or programmed death?

AIMS: Ultrastructural and molecular studies to support the hypothesis of programmed cell death in Helicobacter pylori were conducted. METHODS AND RESULTS: Evidence of programmed death in H. pylori is provided through electron microscopic detection and cytochemical labelling of electrondense bodies (EDB), containing packaged DNA in coccoid cells, resembling micronuclei of apoptotic eukaryotic cells. This morphological evidence is also supported by DNA cleavage in homogeneous fragments of about 100 base pairs. Programmed cell death was observed in H. pylori cultures at 37 degrees C, with a maximum of 37.5% of EDB coccoid cells after 7 days. The non-permissive temperature of 4 degrees C anticipated this process, with 40% of EDB coccoid forms within 3 days, and it remained substantially unaffected during the observation time of 14 days. CONCLUSION: In these experiments, deprivation of nutrients and a non-permissive temperature acted as a powerful trigger for programmed cell death. SIGNIFICANCE AND IMPACT OF THE STUDY: Helicobacter pylori bacterial populations, under stressing stimuli, can respond with programmed cell suicide as a means of species preservation.

Apoptosis↗

PubMed2001

Efficacy of antiadhesive, antibiotic and antiseptic coatings in preventing catheter-related infections: review.

In recent years, central venous catheters (CVCs) are increasingly used in clinical practice. However, complications such as local or systemic infections are frequent for both temporary and indwelling vascular catheters. Annually, in the United States of America there are more than 200,000 cases of nosocomial bloodstream infections (BSIs), of which 90% are related to the use of an intravascular device. These infections are associated with increased morbidity and mortality, prolonged hospitalization and growing medical costs. Technological treatments of polymer surfaces including coating the catheter with antimicrobial substances may be promising tools for prevention of catheter-associated infections. A large number of surface-treated central venous catheters are now commercially available. In this paper the features and the clinical efficacy of different antimicrobial coatings are reviewed.

Anti-Bacterial Agents↗

PubMed2001

A multicenter study on central venous catheter-associated infections in Italy.

In a 1-year multicenter study the microbial colonization of 1154 central venous catheters (CVCs) was investigated. Catheters explanted either from immunocompromised or immunocompetent patients were collected and analyzed by five clinical microbiology laboratories located in Ancona, Aviano, Catania, Pavia and Rome, Italy. A further aim was to investigate, by scanning electron microscopy, the features of currently used catheters, both new and explanted from patients, analyzing their surface quality, the influence of the host protein biofilm on their microbial colonization, the modifications caused by their permanence in the body and the relationship between these factors and the occurrence of infections.

Anti-Bacterial Agents↗

PubMed2000

Enterotoxicity and cytotoxicity of Vibrio parahaemolyticus thermostable direct hemolysin in in vitro systems.

Vibrio parahaemolyticus is a marine bacterium known to be a common cause of seafood gastroenteritis worldwide. The thermostable direct hemolysin (TDH) has been proposed to be a major virulence factor of V. parahaemolyticus. TDH causes intestinal fluid secretion as well as cytotoxicity in a variety of cell types. In this study, we investigated the interplay between the hemolysin's enterotoxic and cytotoxic effects by using both human and rat cell monolayers. As revealed by microspectrofluorimetry, the toxin causes a dose-dependent increase in intracellular free calcium in both Caco-2 and IEC-6 cells. This effect was reversible only when low toxin concentrations were tested. The TDH-activated ion influx pathway is not selective for calcium but admits ions such sodium and manganese as well. Furthermore, in the same range of concentration, the hemolysin triggers a calcium-dependent chloride secretion. At high concentrations, TDH induces a dose-dependent but calcium-independent cell death as assessed by functional, biochemical, and morphological assays.

Animals↗

PubMed1999

Resolution of microsporidial sinusitis and keratoconjunctivitis by itraconazole treatment.

PURPOSE: To report successful treatment of ocular infection caused by the microsporidium Encephalitozoon cuniculi in a person with acquired immunodeficiency syndrome (AIDS) and nasal and paranasal sinus infection. METHOD: Case report. RESULTS: Microsporidial infection in a person with AIDS and with chronic sinusitis and keratoconjunctivitis was diagnosed by Weber modified trichrome stain and transmission electron microscopy. Symptoms completely resolved with itraconazole treatment (200 mg/day for 8 weeks) after albendazole therapy (400 mg/day for 6 weeks) was unsuccessful. CONCLUSION: Itraconazole can be recommended in ocular, nasal, and paranasal sinus infection caused by E. cuniculi parasites when treatment with albendazole fails.

AIDS-Related Opportunistic Infections↗

PubMed1999

Occurrence, diversity, and pathogenicity of halophilic Vibrio spp. and non-O1 Vibrio cholerae from estuarine waters along the Italian Adriatic coast.

The occurrence, diversity, and pathogenicity of Vibrio spp. were investigated in two estuaries along the Italian Adriatic coast. Vibrio alginolyticus was the predominant species, followed by Vibrio parahaemolyticus, non-O1 Vibrio cholerae, and Vibrio vulnificus. By using a biochemical fingerprinting method, all isolates were grouped into nine phenotypes with similarity levels of 75 to 97.5%. The production of toxins capable of causing cytoskeleton-dependent changes was detected in a large number of Vibrio strains. These findings indicate a significant presence of potentially pathogenic Vibrio strains along the Adriatic coast.

Animals↗

PubMed1999

Vibrio parahaemolyticus thermostable direct hemolysin modulates cytoskeletal organization and calcium homeostasis in intestinal cultured cells.

Vibrio parahaemolyticus is a marine bacterium known to be the leading cause of seafood gastroenteritis worldwide. A 46-kDa homodimer protein secreted by this microorganism, the thermostable direct hemolysin (TDH), is considered a major virulence factor involved in bacterial pathogenesis since a high percentage of strains of clinical origin are positive for TDH production. TDH is a pore-forming toxin, and its most extensively studied effect is the ability to cause hemolysis of erythrocytes from different mammalian species. Moreover, TDH induces in a variety of cells cytotoxic effects consisting mainly of cell degeneration which often leads to loss of viability. In this work, we examined the cellular changes induced by TDH in monolayers of IEC-6 cells (derived from the rat crypt small intestine), which represent a useful cell model for studying toxins from enteric bacteria. In experimental conditions allowing cell survival, TDH induces a rapid transient increase in intracellular calcium as well as a significant though reversible decreased rate of progression through the cell cycle. The morphological changes seem to be dependent on the organization of the microtubular network, which appears to be the preferential cytoskeletal element involved in the cellular response to the toxin.

Animals↗

PubMed1998

The effect of oxygen on the growth and cell morphology of Helicobacter pylori.

The in vitro effect of progressive oxygen decrease on the growth and morphology of Helicobacter pylori was studied. H. pylori ATCC 43,504 was used for the experiments. The strain inoculated in Brucella broth plus fetal calf serum was incubated under a controlled atmosphere with oxygen concentration from 5 to 0%. CFU ml-1 and bacterial morphology were detected at the time of spreading and at 24 h, 72 h, 7 days and 14 days. A detailed ultrastructural investigation of the bacterial cells, grown in different experimental conditions, was performed by scanning electron microscopy. Oxygen deprivation produced a rapid reduction of CFU ml-1. In particular, a significant reduction of viable bacteria was recorded at 72 h of incubation in the presence of 1% oxygen and anaerobiosis, and 0 CFU ml-1 was found after 7 days of incubation at the above mentioned oxygen concentrations. The coccoid phenotype was already prevalent after 24 h of incubation with a progressive tendency to aggregate in clusters. These clusters were progressively larger, depending on the reduction of oxygen concentration, since the aggregation phenomenon can be the expression of a hypothesized mechanism of protection among bacterial cells.

Aerobiosis↗

PubMed1998

Toxin-induced activation of Rho GTP-binding protein increases Bcl-2 expression and influences mitochondrial homeostasis.

It is now well established that apoptosis plays a pivotal role in several physiological and pathological situations. Consequently, the mechanisms controlling the cell fate are currently the subject of intense investigation. In this work, we report that an Escherichia coli protein toxin (Cytotoxic Necrotizing Factor 1, CNF1) which activates the Rho GTP-binding protein and prevent apoptosis in epithelial cells was able to: (i) influence the mitochondrial homeostasis and (ii) modulate the expression of proteins belonging to the Bcl-2 family. In particular, the content of the antiapoptotic products Bcl-2 and Bcl-XL resulted to be increased in treated cells, whereas the expression of the proapoptotic protein Bax remained unaltered. CNF1 induces cell spreading via activation of Rho and cell spreading has been reported to promote cell survival. Cytochalasin B, which provokes most of the morphological changes typical of CNF1, including cell spreading, but without the involvement of Rho, was unable to counteract apoptosis. Altogether our results suggest a link between the Rho GTP-binding protein and the regulation of the mitochondrial homeostasis via an effect on the antiapoptotic proteins of the Bcl-2 family.

Apoptosis↗