In vitro activity (MIC and MFC) of voriconazole, amphotericin B, and itraconazole against 192 filamentous fungi: the GISIA-2 study.

We evaluated the in vitro activity of voriconazole, amphotericin B, and itraconazole against 192 clinical mould isolates recovered in twenty Italian microbiology laboratories. The vast majority of isolates belonged to the genus Aspergillus (94.2%) with A. fumigatus (58.3%) being the most frequently isolated species. Antifungal susceptibility testing was performed using the broth microdilution method defined by the CLSI M38-A standard, and results were compared to those obtained with Sensititre panels. Aspergillus flavus ATCC 204304 was employed as reference strain and results were within all expected ranges. Voriconazole's activity against the 192 mould isolates was comparable to that of amphotericin B and itraconazole: voriconazole MIC90 (CLSI 1 microg/ml, Sensititre 1 microg/ml), itraconazole MIC90 (CLSI 0.5 microg/ml, Sensititre 0.5 microg/ml), amphotericin B MIC90 (CLSI 1 microg/ml, Sensititre 1 microg/ml). In conclusion, these in vitro data highlight voriconazole's broad spectrum activity against filamentous fungi and support its use as a first line agent for the treatment of fungal diseases.

Synthesis and in vitro selective anti-Helicobacter pylori activity of pyrazoline derivatives.

In order to develop new anti-Helicobacter pylori agents, a series of N1-substituted 3,5-diphenyl pyrazolines P1-P13 was prepared and evaluated for their antibacterial activity. All synthesized compounds showed little or no activity against different species of Gram-positive and Gram-negative bacteria of clinical relevance and against various strains of pathogenic fungi. The same derivatives exhibited a significant degree of activity against a range of H. pylori strains, including those resistant to the reference compound metronidazole. Among the prepared compounds those with an N1-acetyl group and a 4-methoxy substituent in the 5-phenyl ring showed the best activity against H. pylori metronidazole resistant strains in the 1-4 microg/mL MIC range.

In vitro testing of Aspergillus fumigatus clinical isolates for susceptibility to voriconazole, amphotericin B and itraconazole: comparison of sensititre versus NCCLS M38-A using two different inocula.

Voriconazole, amphotericin B and itraconazole were tested in vitro against 18 strains of Aspergillus fumigatus isolated from cystic fibrosis patients. Susceptibility was tested with the broth microdilution method (M38-A protocol-NCCLS). Results of this reference method were compared with those of an experimental commercial microdilution broth method (Sensititre). Two different inocula, prepared from 2- and 7-day cultures, were used. Minimum inhibitory concentrations (MICs) of the reference method ranged from 0.25 to 2 microg/ml for voriconazole, 0.06 to 1 microg/ml for amphotericin B, 0.016 to >16 microg/ml for itraconazole. There were no significant differences in the MIC ranges or MIC90 values obtained with the two testing methods or with the two types of inocula. These findings confirm the good in vitro activity of voriconazole, itraconazole and amphotericin B against A. fumigatus. They also indicate that reliable susceptibility data can be generated more rapidly by commercial systems and use of 2-day cultures for inoculum preparation.

In vitro activity of voriconazole and other antifungal agents against clinical isolates of Candida glabrata and Candida krusei.

The antifungal susceptibility of 309 Candida glabrata and 63 Candida krusei clinical isolates was tested via the Sensititre YeastOne-3 system (Trek Diagnostic Systems, East Grinstead, UK) to compare the in vitro activity of voriconazole with that of five other antifungal agents (amphotericin B, fluconazole, itraconazole, ketoconazole, and flucytosine). Voriconazole was highly active (MIC90, 0.5 microg/ml) against isolates of both species, including those for which the MICs of itraconazole and fluconazole were high (MIC90s of itraconazole, 2 microg/ml for C. glabrata and 0.5 microg/ml for C. krusei; MIC90s of fluconazole, 32 microg/ml for C. glabrata and 64 microg/ml for C. krusei). Ketoconazole MIC90 values for both species were identical to those of voriconazole. The MIC90 of amphotericin B was similar for both species (0.125 microg/ml for C. glabrata and 0.25 microg/ml for C. krusei). As expected, flucytosine was only moderately active against C. krusei isolates (MIC90, 16 microg/ml) but was highly active against C. glabrata isolates (MIC90, 0.03 microg/ml). Potential cross-resistance within the azole class was noted for some strains of C. glabrata (5.5%) that presented high MIC values for all the azoles tested. In order to consider voriconazole a viable alternative to other triazoles for the treatment of infections caused by Candida species, susceptibility testing of all clinically significant isolates of C. glabrata and C. krusei is recommended because of the potential for azole cross-resistance. The Sensititre YeastOne-3 seems to be a suitable commercial tool for this purpose.

Anti-Helicobacter pylori agents endowed with H2-antagonist properties.

New anti-Helicobacter pylori (H. pylori) agents endowed with H2-antagonists properties were obtained by combining the lamtidine derived pharmacophoric group with the antibiotic calvatic acid. All the compounds were tested for their irreversible H2-antagonist properties and for their ability to inhibit 20 H. pylori strains, two of them metronidazole resistant. The most active derivative (compound 4) displayed antimicrobial activity similar to metronidazole.

Helicobacter pylori: ureA, cagA and vacA expression during conversion to the coccoid form.

As viability of coccoid forms of Helicobacter pylori can only be verified by demonstrating the integrity of the DNA and active protein synthesis, we analysed the expression of ureA, cagA, vacA genes after prolonged incubation in a liquid medium. Exponentially growing and ageing phase cultures were used. Our results showed that, although the coccoid forms had decreased DNA and RNA levels after 31 days, they were not degraded and still expressed the urease, cytotoxic island and vacuolating toxin genes. Coccoid forms are therefore viable and may act as a transmissible agent that plays a crucial role in disease relapses after antibiotic therapy.

Helicobacter pylori: cultivability and antibiotic susceptibility of coccoid forms.

Helicobacter pylori is an actively dividing helical bacterium that changes to coccoid morphology as the culture ages. It has been suggested that the coccoid forms may be involved in transmission of infection and in relapses following antimicrobial therapy. The aim of this investigation was to determine the survival and susceptibility of the coccoid forms to amoxycillin, erythromycin, gentamicin and metronidazole. Colony counts and microscopic examination were performed after 1-4 weeks of culture. At 2 and 4 weeks, identical cultures were treated with the antibiotics for 24 h. Our results showed that 4-week cultures of coccoid forms were cultivable after antibiotic treatment.

Inhibitory and bactericidal activity of rokitamycin against Helicobacter pylori and morphological alterations.

Rokitamycin is a macrolide antibiotic, recently entered into clinical use. Its in vitro activity and kill kinetics against Helicobater pylori have been evaluated at 1 x the minimum inhibitory concentration (MIC), 2 x MIC and 4 x MIC at 2, 4, 8, 24 hours and compared with those of clarithromycin, erythromycin and amoxicillin. Morphological changes in H. pylori induced by rokitamycin incubation at these MICs and times were also investigated by scanning electron microscopy. All the antibiotics tested had good inhibitory activity against H. pylori, a slow growing microorganism. The order of MIC activity was clarithromycin > amoxicillin > rokitamycin > erythromycin. Rokitamycin killed more rapidly than the other antibiotics, in fact H. pylori strains were totally killed at 8 h (2 x MIC) and 4 h (4 x MIC) and after only 2 h incubation all concentrations greatly decreased the CFU/ml. These effects were also confirmed by the rapid appearance of surface and morphological alterations (focal blebs, constrictions, rounded forms) in the normal structure of H. pylori observed by scanning electron microscopy. Clinical studies should be conducted to investigate the in vivo activity of rokitamycin, as an agent to be used in the combination therapies against H. pylori.

'In vitro' development of metronidazole, erythromycin, amoxicillin and gentamicin resistance in Helicobacter pylori.

Serial passage of 37 Helicobacter pylori clinical isolates on increasing concentrations of metronidazole rapidly produced five strains with MICs up to 512 fold higher than those for the original strains. For these five metronidazole-resistant strains the MICs of erythromycin, gentamicin and amoxicillin were unchanged. When they were submitted to the same technique for these last antimicrobial agents, only one strain developed high level resistance to erythromycin and gentamicin having MIC values respectively up to 32 and 64-fold increased. Finally, no amoxicillin-resistant Helicobacter pylori could be obtained.

Activity of amoxicillin, metronidazole, bismuth salicylate and six aminoglycosides against Helicobacter pylori.

The in vitro activity of metronidazole, amoxicillin, bismuth salicylate and some aminoglycosides, such as ribostamycin, gentamicin, amikacin, tobramycin, streptomycin and netilmicin was evaluated against 60 clinical isolates of Helicobacter pylori using the agar dilution technique. All 60 strains were susceptible to amoxicillin, with minimum concentrations able to inhibit 50% (MIC 50) and 90% (MIC 90) of strains equal to 0.031 microgram/ml and 0.25 microgram/ml, respectively. Of the aminoglycosides, ribostamycin, streptomycin and amikacin had a little lower activity (MIC 50 of 2 micrograms/ml, MIC 90 of 4-8 micrograms/ml) than gentamicin, tobramycin and netilmicin, with MIC 50s of 0.125 microgram/ml and MIC 90s of 0.25 microgram/ml. Metronidazole was effective against the majority of the strains, but we found ten resistant strains. Finally, bismuth salicylate showed only slight antibacterial activity.

Susceptibility of Helicobacter pylori and pharmacokinetic properties in mice of new 5-nitroimidazole derivatives devoid of mutagenic activity in the Ames test.

The minimum inhibitory concentrations of metronidazole and four new 5-nitroimidazole derivatives (EU 11100, EU 11102, EU 11103, EU 11104), obtained by the reaction of 1-methyl-5-nitroimidazolyl-2-carboxyaldehyde and terbutyl-phenol, were determined against 25 clinical isolates of Helicobacter pylori. Three of them (EU 11100, EU 11103, EU 11104) exhibited an antibacterial activity higher than that of metronidazole. The last one, the molecule EU 11102, was less active than metronidazole. In mice, after a single equimolar oral administration, the molecules EU 11100 and 11103 were poorly absorbed and poorly excreted in urine. The molecular EU 11104 was well adsorbed and its urinary recovery was slightly lower than that of metronidazole. The substance EU 11102 was not demonstrable in blood and urine. In the Salmonella/microsome mutagenicity test only the molecule EU 11100 showed an increase of mutation frequency in S. typhimurium TA 100.