Risk factors and clinical outcomes of candidaemia in patients treated for Clostridium difficile infection.

The alterations occurring in the intestinal flora during Clostridium difficile infection (CDI) may promote the translocation of Candida to the blood and the development of candidaemia. The aim of our study was to analyse clinical findings of these patients to determine the risk factors associated with the development of candidaemia subsequent to CDI. We compared 35 patients with candidaemia subsequent to CDI with 105 patients with CDI. Patients with candidaemia showed more severe infections and higher mortality. The ribotype 027 strain and vancomycin treatment at ≥ 1000 mg/day were prevalent in patients developing candidaemia. CDI may predispose to the translocation of Candida.

Clinical experience of anidulafungin for the treatment of patients with documented candidemia.

Candida species are the most common causes of invasive fungal infections in humans, producing infections that range from mucocutaneous disorders to invasive disease that can involve any organ. Here we present our clinical experience with anidulafungin for the treatment of documented nosocomial candidaemia. From february 2009 through January 2010 all patients with documented candidemia treated with anidulafungin in three medical centers in italy were reviewed. Demographics, clinical and microbiological data, and outcome were collected for each patient. Twenty-four patients were included in the study. most patients had a central venous catheter (CVC) or a port-a-cath (100%), had a history of recent surgery (87.5%), or were receiving total parenteral nutrition (79%), broad spectrum antibiotics (83%), steroids or chemotherapy (45.8%). C. albicans (54%) was the most commonly isolated pathogen. CVC was the source of candidemia in 79% of cases. Six patients (25%) developed severe sepsis or septic shock, and five patients had unfavorable outcomes, with an overall mortality rate of 20%. No patients experienced side effects related to anidulafungin therapy. Anidulafungin was effective in the treatment of patients with documented candidemia arising from different sites, and no significant side effects were observed.

Antimicrobial susceptibility, biochemical and genetic profiles of Staphylococcus haemolyticus strains isolated from the bloodstream of patients hospitalized in critical care units.

Staphylococcus haemolyticus strains (n=20), responsible of blood stream infections, were consecutively isolated from patients hospitalized in two different wards at high risk of infection. Strains displayed high rate of resistance to oxacillin (90%). All strains but two with decreased susceptibility (MIC = 4 microg/mL), were sensitive to vancomycin. Ten strains were resistant to teicoplanin. Among the strains susceptible to glycopeptides, three displayed heteroresistance to vancomycin and seven to teicoplanin, when tested by Etest technique with 2 x McFarland inoculum. Biochemical reactions allowed to assign strains to eight biotypes, with 11 strains clustering under two main biotype A and biotype B. Pulsed-field-gel-electrophoresis (PFGE) identified 11 different PFGE-types. Seven strains grouping under the major PFGE-type 1 and three strains clustering in PFGE-type 2, closely correlated to biotype A and biotype B respectively. Seven teicoplanin-resistant isolates clustered in the PFGE-type 1, two in the PFGE-type 2 and one in PFGE-type 5. Therefore, teicoplanin-resistant strains were biochemically and genetically related and clonally distributed, despite different clones of S. haemolyticus circulated in the units during the study period.

Extracorporeal circulation does not induce intra-alveolar release of Endothelin 1, but only a modest overproduction in pulmonary circulation.

OBJECTIVE: To investigate whether ECC may produce regional liberation of inflammatory mediators capable of inducing vascular effects and organ damage. EXPERIMENTAL DESIGN: Comparative study [corrected]. SETTING: Cardiac surgery department in a University hospital. PATIENTS: Fifteen patients undergoing coronary artery bypass grafting (CABG, group A) and ten patients operated for infrarenal abdominal aortic aneurysm (controls, group B) have been studied. MEASURES: Levels of Interleukin 1beta (IL1), Tumor Necrosis Factor alpha (TNF), Interleukin 6 (IL6), and Endothelin 1 (ET1) were measured in pulmonary capillary, arterial, and venous blood and in bronchoalveolar lavages (BAL) before, during and after extracorporeal circulation (ECC) or surgical intervention. RESULTS: TNF-alpha (never >35 pg/ml) and IL1beta (range 20-300 pg/ml) values did not change over time for both groups. IL6 concentrations in all samples of group A increased between five and twenty fold, during and after ECC (from 3-5 pg/ml up to 240 pg/ml, p<0.001). This trend was similar in controls after surgical stress. Endothelin 1 was always undetectable in the BAL fluid, with a modest, but significant increase in pulmonary capillary blood of group A, after ECC, (from 11+/-4 pg/ml to 18+/-5 pg/ml, p<0.001). This increment correlated well with the PVR increase, but was transient and after 24 hours, ET1 values returned to baseline levels. Mean values of ET1 increased also in controls, but not significantly. CONCLUSIONS: ECC may induce ET1 liberation in pulmonary circulation with transient pulmonary vasoconstriction, but wihout intra-alveolar release, or lung damage. Augmented concentrations of IL6 probably express a response to surgical procedure rather than an effect exclusively related to ECC.

Serum-mediated enhancement of TNF-alpha release by human monocytes stimulated with the yeast form of Candida albicans.

The role of serum in the production of tumor necrosis factor-alpha (TNF-alpha) by human monocytes stimulated with yeast-form Candida albicans was studied. Pre-exposure of C. albicans to human pooled serum enhanced both TNF-alpha mRNA and cytokine secretion compared with C. albicans preincubated with medium only. Serum factors involved were >30 kDa, were efficiently inhibited by D-mannose, and recognized both Ca++-dependent and -independent pathways. Preincubation of yeasts with rabbit mannose-binding protein (MBP) resulted in dose-related enhancement of TNF-alpha secretion, through a Ca++-dependent pathway inhibited by D-mannose. TNF-alpha levels were similarly induced in C. albicans preincubated with vitronectin and with serum. Ca++ depletion did not affect cytokine release, while D-mannose supplementation displayed inhibition. The latter effect was abolished after Ca++ depletion. These data call for an involvement of both MBP and vitronectin in the serum-mediated enhancement of TNF-alpha release upon stimulation of monocytes with yeast forms of C. albicans.

Balance of proinflammatory and antiinflammatory cytokines in mice immunized with Escherichia coli and correlation with mortality after lethal challenge.

The balance of proinflammatory and antiinflammatory cytokines, their correlation with endotoxin levels and mortality rate after lethal challenge of Escherichia coli was investigated in mice immunized weekly for 8 weeks with formalin-killed E. coli either untreated or treated with 0.5x minimal inhibitory concentration of aztreonam. Control mice treated in parallel with saline, died within 24 h after challenge with 100x lethal dose (LD50) of viable E. coli O6:K-. Mice immunized with antibiotic-treated bacteria showed a significantly higher survival than mice immunized with untreated E. coli. Cytokines were not detected in the sera of control mice during the entire period of immunization. At 90 min after immunization, mice immunized with antibiotic-treated E. coli showed tumor necrosis factor-alpha (TNF-alpha) levels significantly lower and interleukin (IL)-6 levels significantly higher (P < 0.05) than mice immunized with untreated E. coli, while comparable levels of interferon-gamma (IFN-gamma were measured in both groups. TNF-alpha and IL-10 levels measured 90 min after lethal challenge correlated with the mortality rate observed in each group (r = 0.96 for TNF-alpha and 0.94 for IL-10). IL-6 levels correlated with survival (r = 0.95), while IFN-gamma serum levels did not differ in the two immunized groups, but were significantly higher than those measured in the control mice. IL-4 was detected only after challenge of mice immunized with antibiotic-treated bacteria. Comparable levels of circulating endotoxin were measured after lethal challenge in both control and immunized mice. These data showed that in the presence of a protective immune response the survival of immunized mice was correlated with an early alteration of cytokine expression pattern including enhanced secretion of IL-4, IL-6 and IFN-gamma, and reduced secretion of TNF-alpha and IL-10.

The release of tumor necrosis factor alpha (TNF-alpha) by interferon gamma (IFN-gamma) induced THP-1 cells stimulated with smooth lipopolysaccharide is inhibited by MAbs against HLA-DR and CD14 receptors on the effector cell.

Bacterial lipopolysaccharide (LPS) plays a central role in the pathogenesis of gram-negative sepsis and shock. The glycosylphoshatidyl inositol (GPI) anchored glycoprotein CD14 on mononuclear cells binds LPS, especially in the presence of an LPS binding serum protein, activating the production of pro-inflammatory cytokines, i.e. TNF-alpha. However, since GPI anchorage to the cell membrane lacks the intracellular signalling capacity, the existence of at least a second receptor has been postulated. In attempt to identify additional LPS receptors, we used the human myelomonocytic cell line THP-1. This undifferentiated cell line did not respond to LPS in terms of TNF-alpha release, but when induced with 250 U/ml of IFN-gamma for 48 h, the cells released TNF-alpha (174 +/- 58.6 U/ml. L929 cell bioassay) in response to 10 vg/ml of E. coli 0111 LPS, in the absence of serum. Blockade of either HLA-DR or CD14 receptors with specific MAbs did not reduce the amount of cytokine released. However, when both the receptors were sequentially blocked involved on the effector cells a remarkable inhibition of TNF-alpha release was observed (8.6 +/- 1.4). It seems therefore, that HLA-DR receptor may be with CD14 in triggering TNF-alpha release by IFN-gamma, induced THP-1 cells.

Differential effect of human and murine polyclonal and monoclonal antisera on TNF-alpha production by human monocytes.

The capacity of human and murine polyclonal and monoclonal antibodies to inhibit lipopolysaccharide (LPS)-induced tumor necrosis factor-alpha (TNF-alpha) release from human monocytes was investigated. Human pooled immunoglobulin G (IVIG), human IgM monoclonal antibody (HA-1A) directed against the lipid A moiety of LPS, and murine IgG monoclonal antibody (MT-1F) raised in mice against antibiotic-treated Escherichia coli O6:K- were either added simultaneously with LPS to monocytes or preincubated for 1 h at 37 degrees C before being added to monocytes. TNF-alpha content in the monocyte supernatants was then tested. Simultaneous addition of increasing concentrations of IVIG (from 0.3 to 2.5 mg/ml) and 10 micrograms/ml of LPS to monocytes induced an enhanced release of TNF-alpha by monocytes in a dose dependent fashion. Preincubation of IVIG with LPS abolished the additive effect, but did not inhibit LPS-induced TNF-alpha release by monocytes. The simultaneous addition of LPS and HA-1A to monocytes had no additive effect nor did it inhibit TNF-alpha release. On the other hand, inhibition of TNF-alpha release was observed when HA-1A was preincubated with LPS before being added to monocytes. In all instances MT-1F inhibited TNF-alpha release when the monocytes were stimulated with smooth type LPS, but not with LPS isolated from rough mutants.

Preincubation of Candida albicans strains with amphotericin B reduces tumor necrosis factor alpha and interleukin-6 release by human monocytes.

The release of tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6) by human monocytes stimulated with whole heat-killed Candida albicans CA3 (a clinical isolate) and CA2 (a germ tube-negative mutant) either treated or not treated with amphotericin B was investigated. The optimal release of the cytokines was observed at 24 h of incubation of the yeasts with the monocytes for both TNF-alpha and IL-6. The levels ranged from 10,500 to 19,000 U/ml for TNF-alpha and from 350 to 460 pg/ml for IL-6. Germ tube-negative mutant CA2 induced the release of TNF-alpha at levels significantly (P < 0.05) lower than those induced by clinical isolate CA3, while no major differences were observed between the two strains with regard to their capacity to induce the release of IL-6. In all instances, preincubation of the yeasts with a sublethal concentration of amphotericin significantly reduced cytokine production. These results suggest that drug-induced alterations of fungal outer structures may affect the interactions between the yeasts and the monocytes, resulting in a reduced level of secretion of cytokines.

Culture filtrates and whole heat-killed Candida albicans stimulate human monocytes to release interleukin-6.

The release of interleukin-6 (IL-6) by human monocytes upon stimulation with culture filtrates and heat-killed Candida albicans cells was studied. Two strains of C. albicans (a wild strain CA3 and an agerminative mutant CA2) were cultured overnight at 28 degrees C in complete medium, and 10(6) cells/ml were either filtered at different time points (6, 12, 18, 24, 30 hours) or heat-killed. C. albicans preparations were then added to monolayers of monocytes isolated from healthy donors and incubated at 37 degrees C in 5% CO2 atmosphere. Cell culture supernatants were collected at different time points (every 6 hrs for 30 hrs), and IL-6 content was then measured by immunometric assay. Monocytes stimulated with heat-killed C. albicans cells released IL-6 in the supernatants with values ranging from 59 to 460 pg/ml, that peaked at 24 hrs of incubation. Using heat-killed whole cells of C. albicans no major differences were observed between the two strains used in their capacity to induce IL-6. Culture filtrates also stimulated monocytes to release IL-6 and maximal cytokine levels were observed when the monocytes were triggered with filtrates from yeasts cultured for 24 hours. CA2 filtrate induced IL-6 levels to an extent significantly higher than did CA3 filtrate. These data add further evidence to the immunomodulatory properties possessed by structures of C. albicans.

Modification of Pseudomonas aeruginosa virulence factors by sub-inhibitory concentrations of antibiotics.

This study's objectives were to evaluate the effects of subminimum inhibitory concentrations (MICs) of tobramycin, gentamicin, netilmicin, streptomycin, ciprofloxacin, cefotaxime and piperacillin on proteinase production, alginate and siderophore synthesis by two strains of Pseudomonas aeruginosa. One of these strains, of recent clinical isolation, was mucoid. In fact it is well known that mucoid strains are more resistant than non-mucoid; there is, moreover, evidence that in cystic fibrotic lungs the non-mucoid P. aeruginosa are invariably replaced by mucoid variants. Our results show that subinhibitory concentrations of beta-lactams and quinolones significantly reduced the amount of alginate. Protease production was affected by all antibiotics tested.

Adherence of Pseudomonas aeruginosa elastase deficient mutant.

Pseudomonas aeruginosa produces several extracellular substances such as enzymes and toxins which seem to contribute to its pathogenicity. In particular, alkaline protease and elastase production seems to affect bacterial adherence. Aim of this study was to isolate an elastase deficient mutant of P. aeruginosa and to demonstrate a possible correlation between enzyme production and adherence to WEHI cells. Mutant strain showed a significant reduction of elastase and protease alkaline activity, as the decrease of absorbance values demonstrate. Furthermore the adherence to WEHI cells of mutant strain was strongly reduced with respect to the wild strain. Our results prove that proteolytic enzymes play an important role in adherence, probably modifying the cell surfaces and so enhancing adherence.

Role of alkaline protease and elastase in the adherence of Pseudomonas aeruginosa to WEHI cells.

Recent clinical isolates were tested for production of some extracellular factors such as alkaline protease and elastase. They were also assayed for adhesiveness to WEHI cells. It is well known that extracellular production of substances other than toxins is related to virulence and may increase adherence. The present investigation aimed to evaluate the role of extracellular proteins in adherence. Alkaline protease production was assayed using a test performed with casein as substrate while elastase activity was investigated with the elastin-congored method. Our results demonstrated that P. aeruginosa strains which are good alkaline protease and elastase producers adher better than those showing no or low protease and elastase activity.

The influence of antifungal drugs on adherence of Candida albicans to buccal epithelial cells.

The adherence of two strains of Candida albicans serotype A to human epithelial cells was measured after exposure to different concentrations of amphotericin B, 5-fluorocytosine, nystatin, miconazole and ketoconazole. Germ-tube formation after different exposure times to the antifungal drugs as a preliminary test was carried out. Pretreatment of blastospores with minimum inhibitory concentrations (MIC) and sub-MIC (1/2 and 1/4 of MIC values) for 3 and 72 h did not affect adherence for all drugs tested except amphotericin B. This antimycotic agent reduces significantly the adherence either after 3 or 72 h exposure time. The other antifungal drugs interfere with adherence only after 72 h and at the highest concentrations tested, above MIC values. The decrease in adherence by antifungal drugs suggests that some of these drugs would be useful in the prophylaxis of patients at high risk for candidosis.

Susceptibility of Candida species strains to five antifungal agents: comparison between agar dilution and disk diffusion tests.

The in vitro activity of five antifungal agents were compared against 180 Candida strains. The drugs were: two imidazoles (miconazole and ketoconazole), nystatin, 5-fluorocytosine and amphotericin B. Agar dilution and disk diffusion methods were used. Nystatin, miconazole and amphotericin B were the most active agents. 5-fluorocytosine had high activity except against C. albicans serotype B, of which a high percentage were resistant. Finally, a good correlation between the two methods was observed.

Comparison between adherence of C. albicans and Candida spp. to human epithelial cells.

A comparison was made of the adherence of different Candida species to human epithelial cells. Three strains each of C. albicans serotype A, serotype B, C. stellatoidea, C. tropicalis, C. krusei and C. glabrata recently clinical isolated were studied. The adherence assay, run in triplicate, was carried out using pooled buccal epithelial cells from healthy donors. The results indicate that both serotypes of C. albicans adhere to buccal epithelial cells in a significantly greater degree than the other species tested and there is no differences between C. albicans serotypes A and B. The rate of adherence of C. stellatoidea and C. tropicalis was similar to that of C. albicans serotypes A and B respectively. Among different strains of C. stellatoidea, C. tropicalis and C. glabrata, the adherence varied significantly and it is possible that there exist a relationship with different degree of pathogenicity of these particular strains.

[In vitro activity of norfloxacin against gram-positive and gram-negative organisms from recent clinical isolates]. / Attività in vitro della norfloxacina nei confronti di organismi gram positivi e gram negativi di recente isolamento clinico.

A total of 274 recently isolated Gram-positive e Gram-negative strains have been tested. The method used for the determination of the minimal inhibitory concentration of the antibiotics was by agar dilution and the minimal bactericidal concentrations were determined by "replica plating" system. All strains demonstrated a good sensitivity, above all, to Norfloxacin which inhibited Gram-positive bacteria at the concentration of 3.12 micrograms/ml and, concerning Gram-negative, at the concentration of 0.10 microgram/ml for K.E.S. group, 0.19 microgram/ml for Escherichia coli, 6.25 micrograms/ml for Proteus sp and 1.56 microgram/ml for Pseudomonas aeruginosa. Also with the other antibiotics we obtained good results especially with Nitrofurantoin concerning Enterococcus and Ceftazidime concerning all the strains. On the basis of these results we can conclude that Norfloxacin is one of the most active antibiotics among those used against strains implicated in urinary infections.

[Incidence of Pseudomonas sp., Staphylococcus sp. and Candida sp. in a high-risk population]. / Incidenza di Pseudomonas sp, Staphylococcus sp e Candida sp in un reparto ad alto rischio.

Since infections represent one of the greatest complications in immunocompromised hosts, our group, concerned with bacteriological monitoring, has applied its attention to microorganisms such as Pseudomonas sp, Staphylococcus sp and Candida sp. Pseudomonas sp strains have been identified using conventional techniques and the API system 20 NE. According to Varaldo's scheme, Staphylococci sp have been divided into six "lyogroup" and then, the typing of Candida sp has been checked by cultural and biochemical tests. The results obtained have demonstrated that the Pseudomonas aeruginosa species is prevalent not only in the urinary tract but also in the respiratory tract. As for the Staphylococci sp, the VI lyogroup showed the greatest percentage of strains and than, as regards Candida sp it has been possible to observe that, over a given period, the percentage of pluricontamination has been almost constant.

In vitro susceptibility tests with cefoperazone compared with cefotaxime, cefoxitin, cefuroxime, cephaloridine and gentamicin.

Minimum inhibitory concentrations (MICs), minimum bactericidal concentrations (MBCs) and sensitivity disc tests of cefoperazone, cefotaxime, cefoxitin, cefuroxime, cephaloridine and gentamicin were determined from 300 Gram-positive and -negative isolates from kidney transplant recipients. Cefoperazone inhibited 50% of Pseudomonas aeruginosa strains at a concentration of 6.25 micrograms/ml. Only gentamicin had similar activity but if we consider the sources of our strains and the nephrotoxicity of the compound, its use is not suitable. Moreover, cefoperazone was able to inhibit from 50% to 90% of the other strains at variable concentrations between 0.39 micrograms/ml and 3.12 micrograms/ml.