Susceptibility of European Escherichia coli clinical isolates from intra-abdominal infections, extended-spectrum ß-lactamase occurrence, resistance distribution, and molecular characterization of ertapenem-resistant isolates (SMART 2008-2009).

A total of 3160 clinical isolates of Escherichia coli from intra-abdominal infections were collected during 2008-2009 from 13 European countries. The frequency of extended-spectrum ß-lactamase (ESBL)-producing isolates in Europe was 11%. The most active antibiotics tested were typically imipenem, ertapenem, and amikacin, although the activity of all non-carbapenem antibiotics was lower when tested against ESBL-positive isolates than when tested against ESBL-negative isolates. Ertapenem exhibited 99.3% susceptibility with all isolates, and 96.8% susceptibility with ESBL-positive isolates. With application of the ertapenem CLSI clinical breakpoint for resistance (MIC ≥1 mg/L), only six isolates (0.2%) were ertapenem-resistant, and only three of these were available for molecular characterization. Of those three, only one was ESBL-positive (CTX-M-14), and two were carbapenemase-positive (OXA-48). All three were negative for, VIM, NDM and KPC carbapenemases. Although the level of ertapenem resistance in E. coli is very low, further monitoring of ertapenem susceptibility and molecular characterization of ertapenem-resistant isolates is needed.

Trending eight years of in vitro activity of ertapenem and comparators against Escherichia coli from intra-abdominal infections in North America--SMART 2002-2009.

During 2002 - 2009, 2,885 Escherichia coli intra-abdominal isolates were collected from North America in the Study for monitoring Antimicrobial Resistance trends (SmARt) surveillance program. the incidence of extendedspectrum beta-lactamase producing isolates ranged from 1.7% in 2005 to 7.2% in 2004 and 2006, and was 6.8% in 2009. Susceptibility trends showed that there were only minor fluctuations in susceptibility to ertapenem and imipenem with no significant decrease over time. By contrast, cefepime, cefotaxime, cefoxitin, ceftazidime, ceftriaxone, ciprofloxacin, and levofloxacin exhibited significantly higher minimum inhibitory concentrations against E. coli overall (p<0.05) and (except for cefoxitin) against extended-spectrum beta-lactamase producing isolates. Piperacillin-tazobactam also had significantly diminished activity against E. coli overall, but paradoxically showed significantly increased activity against extendedspectrum beta-lactamase producing isolates. Ertapenem and imipenem susceptibility of E. coli in North America remained consistently high during the period 2002 through 2009, and continuing updates from SMART will be helpful in detecting any changes that occur in the future.

Activity of tigecycline and comparators against recent clinical isolates of Finegoldia magna from Europe.

A total of 206 clinical isolates of Finegoldia magna were collected during the period 2007-2009 from six European countries. The majority of isolates were from body fluids (n = 83; 40.3%) or wounds (n = 82; 39.8%). All isolates were susceptible to tigecycline, meropenem, metronidazole and piperacillin / tazobactam, though susceptibility to penicillin (86.4-87.4%) and clindamycin (78.2-93.3%) were more variable.

Utility of 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenyl-amino)carbonyl]-2H-tetrazolium hydroxide (XTT) and minimum effective concentration assays in the determination of antifungal susceptibility of Aspergillus fumigatus to the lipopeptide class compounds.

The susceptibility of Aspergillus fumigatus to mulundocandin, an echinocandin-like compound, and other antifungal agents was assessed by the National Committee for Clinical Laboratory Standards (NCCLS) M38-P method, a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenyl-amino)carbonyl]-2H-tetrazolium hydroxide (XTT)-based colorimetric assay, and determination of morphologic alterations by microscopy. In contrast to the NCCLS M38-P method, which does not predict the activity in vivo, the XTT-based assay showed that A. fumigatus is susceptible to mulundocandin. Thus, the XTT-based assay might be useful for determination of the susceptibilities of molds to echinocandins. Further evaluation is warranted.

Effect of antifungal agents on the binding of Candida albicans to immobilized amino acids and bovine serum albumin.

In this study the effects of different antifungal agents on the binding of Candida albicans yeast cells to different supports were examined. Pre-treatment with amphotericin B or dithiothreitol (DTT) severely reduced the ability of C. albicans yeasts to bind to plastic, while the effects of pre-treatment with fluconazole, ketoconazole or flucytosine were less marked. Both DTT and amphotericin B reduced the binding of yeasts to bovine serum albumin (BSA) and amino acids at low concentrations, while the other antifungal agents were effective at concentrations several-fold higher than their MICs. These data suggest that DTT and amphotericin B affect the yeast cell wall components, and alter both hydrophobic interactions with plastic, and the more specific interactions with BSA and amino acids. By contrast, the effect of the azoles and flucytosine appears to be largely restricted to hydrophobic interactions.

Aspirochlorine: a highly selective and potent inhibitor of fungal protein synthesis.

Aspirochlorine, a compound belonging to the gliotoxin family of compounds, exhibits antifungal and antibacterial activity but its mechanism of action remains unknown. In this study we show that aspirochlorine inhibits the pathogenic fungus Candida albicans by acting on fungal protein synthesis. The compound selectively inhibits cell-free protein synthesis when using a C. albicans system, but does not inhibit this synthesis in vitro when tested with bacterial and mammalian systems. Moreover, in intact C. albicans cells, aspirochlorine inhibits protein synthesis but does not inhibit chitin, DNA or glucan synthesis though at high concentrations some inhibition of RNA synthesis is observed. By contrast, in intact Bacillus subtilis cells, aspirochlorine did not inhibit protein, DNA, or cell wall synthesis though it significantly inhibited RNA synthesis. Furthermore, using heterologous systems (mammalian ribosomes and C. albicans cytosolic factors) the data suggest that the inhibitory action of aspirochlorine is not exerted through a direct interaction with C. albicans EF-1 or EF-2.

Comparison of a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-t etrazolium hydroxide (XTT) colorimetric method with the standardized National Committee for Clinical Laboratory Standards method of testing clinical yeast isolates for susceptibility to antifungal agents.

MICs for clinical Candida and Cryptococcus isolates were determined by a method incorporating the colorimetric indicator 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl] -2H-tetrazolium hydroxide (XTT), and the results were compared with MICs obtained by the National Committee for Clinical Laboratory Standards approved standard method (M27-A). One hundred percent of all isolates demonstrated agreement within 2 dilutions between the MICs of amphotericin B, fluconazole, itraconazole, ketoconazole, and flucytosine obtained by the two methods. These data suggest that an XTT-based method could provide a useful means for the determination of antifungal susceptibility of yeasts.

Production of extracellular matrix by Candida albicans biofilms.

Growth of Candida albicans biofilms and production of extracellular matrix were monitored by dry weight, colorimetric and radioisotope assays, and by scanning electron microscopy. Under static incubation conditions synthesis of matrix material was minimal, but increased dramatically when developing biofilms were subjected to a liquid flow with the result that the cells were enveloped in extracellular polymer. These findings suggest that production of matrix material could contribute to the resistance of biofilm cells to antifungal agents in vivo.

Binding of Candida albicans to immobilized amino acids and bovine serum albumin.

In this study, we examined the binding of Candida albicans synchronized yeast-phase cells to plastic, immobilized amino acids and bovine serum albumin (BSA) and quantified the binding by using an XTT tetrazolium salt assay and absorbance determination. Our results show that C. albicans binds efficiently and specifically to several nonpolar aliphatic amino acids and positively charged amino acids and to BSA immobilized on tissue culture plastic but not to polar uncharged, negatively charged, or aromatic amino acids. Adhesion of yeasts to immobilized amino acids was not affected by preincubation of cells with BSA, whereas binding to immobilized BSA was affected by preincubation of yeasts with alanine, proline, and leucine but not by arginine or lysine. The ability to distinguish the chirality of these amino acids was also examined by using both the D and L amino acid configurations, and the results show that C. albicans yeasts recognize only the L configuration of these amino acids. The observations that C. albicans specifically binds to certain amino acids indicate that these amino acids may prove useful tools for studying the binding interactions of C. albicans yeasts with host proteins such as components of the extracellular matrix.

MSI-78 Magainin Pharmaceuticals.

Magainin is developing MSI-78, a 22-amino acid peptide, based on compounds discovered in frog skin, as a topical anti-infective. It has broad-spectrum activity, covering Gram-positive and -negative bacteria, anaerobic bacteria and Candida albicans. The compound also has potential for the treatment of impetigo and healing wounds with various infections. In July 1998, Magainin filed an NDA with the US FDA for the treatment of infections in diabetic foot ulcers [292671]. It expects to launch the drug during the second quarter of 1999 [275844]. A completed pivotal, 584 patient, phase III trial demonstrated statistical equivalence between MSI-78 and orally-administered ofloxacin, for the treatment of infection in diabetic foot ulcers. MSI-78 was comparable to ofloxacin with respect to the primary endpoint of clinical response of infection at day ten of treatment, and at subsequent time points through to day 28, and at follow-up [220339]. These data were confirmed by the company's second phase III trial for the same indication, for which successful results were announced in March 1997 [239274]. Additional data from this second trial, presented at the 37th Annual Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC), demonstrated that both drugs were comparable in terms of eradication rate of individual organisms and wound healing [264410]. Between 10% and 15% of wounds in patients treated with a combination of both drugs reached closure within four weeks. After six weeks, the closure rate increased to between 18% and 30%. This suggested that additional studies should be performed to evaluate the wound-healing effects further [275279]. The side-effects of both treatments were well-tolerated, although treatment with ofloxacin was associated with a significant excess of insomnia compared to MSI-78 [275844]. Further phase III trials are planned for treatment of surgical wounds, decubitus ulcers, venous stasis ulcers and infections associated with burns [173293]. The primary clinical endpoint is the cure of the infection and the secondary endpoint is the eradication of the organism. The first study has enrolled approximately 400 patients [195065]. The drug was also being developed for impetigo, but proved no better than placebo in phase III trials for the treatmentprimarily because 75% of controls showed clinical improvement as a result of better hygiene [293751]. Magainin is attempting to develop a recombinant process for commercial synthesis of MSI-78 to allow it to compete on price with conventional antibiotics [174944], [176153]. Magainin has a contract with Abbott for the manufacture of the drug [174944]. In February 1997, Magainin entered into a development, supply and distribution agreement in North America with SmithKline Beecham (SB) for Cytolex [234035]. Magainin has retained all rights to the drug outside of North America [275844], although it has also signed an agreement with Ambalal Sarabhai Enterprises (ASE) to commercialize MSI-78 (as Cytolex) in India [274544], [275556]. Analysts estimate the potential revenues of this compound, including off-label usage is between $200 and $250 million in the US and up to half as much again outside the US [191231].

Resistance of Candida albicans biofilms to antifungal agents in vitro.

Biofilms formed by Candida albicans on small discs of catheter material were resistant to the action of five clinically important antifungal agents as determined by [3H]leucine incorporation and tetrazolium reduction assays. Fluconazole showed the greatest activity, and amphotericin B showed the least activity against biofilm cells. These findings were confirmed by scanning electron microscopy of the biofilms.

Biofilm formation by Candida species on the surface of catheter materials in vitro.

A model system for studying Candida biofilms growing on the surface of small discs of catheter material is described. Biofilm formation was determined quantitatively by a colorimetric assay involving reduction of a tetrazolium salt or by [3H]leucine incorporation; both methods gave excellent correlation with biofilm dry weight (r = 0.997 and 0.945, respectively). Growth of Candida albicans biofilms in medium containing 500 mM galactose or 50 mM glucose reached a maximum after 48 h and then declined; however, the cell yield was lower in low-glucose medium. Comparison of biofilm formation by 15 different isolates of C. albicans failed to reveal any correlation with pathogenicity within this group, but there was some correlation with pathogenicity when different Candida species were tested. Isolates of C. parapsilosis (Glasgow), C. pseudotropicalis, and C. glabrata all gave significantly less biofilm growth (P < 0.001) than the more pathogenic C. albicans. Evaluation of various catheter materials showed that biofilm formation by C. albicans was slightly increased on latex or silicone elastomer (P < 0.05), compared with polyvinyl chloride, but substantially decreased on polyurethane or 100% silicone (P < 0.001). Scanning electron microscopy demonstrated that after 48 h, C. albicans biofilms consisted of a dense network of yeasts, germ tubes, pseudohyphae, and hyphae; extracellular polymeric material was visible on the surfaces of some of these morphological forms. Our model system is a simple and convenient method for studying Candida biofilms and could be used for testing the efficacy of antifungal agents against biofilm cells.