A transformation scheme for Cryptococcus neoformans to yield high-frequency, integrative events was developed. Adenine auxotrophs from a clinical isolate of C. neoformans serotype A were complemented by the cryptococcal phosphoribosylaminoimidazole carboxylase gene (ade2) with a biolistic DNA delivery system.Comparison of two DNA delivery systems (electroporation versus a biolistic system) showed notable differences. The biolistic system did not require linear vectors and transformed each auxotrophic strain at similar fr-equencies. Examination of randomly selected transformants by biolistics showed that 15 to 40%O were stable, depending on the recipient auxotroph, with integrative events identified in all stable transformants by DNA analysis. Although the ade2 cDNA copy transformed at a low frequency, DNA analysis found homologous recombination in each of these transformants. DNA analysis of stable transformants receiving genomic ade2 revealed ectopic integration in a majority of cases, but approximately a quarter of the transformants showed homologous recombination with vector integration or gene replacement. This system has the potential for targeted gene disruption, and its efficiency will also allow for screening of DNA libraries within C. neoformans. Further molecular strategies to study the pathobiology of this pathogenic yeast are now possible with this transformation system.Cryptococcus neoformans is a pathogenic yeast with an unexplained predilection for infecting the central nervous system. There has been a significant increase in the number of human infections with this yeast over the last decade as the immunocompromised population has enlarged because of AIDS. Although epidemiological studies suggest that the increase in cryptococcal infection is due to more-susceptible hosts rather than increased virulence, the pathobiology of the organism needs further investigation. Several phenotypic factors associated with virulence have been identified for C. neoformans: the polysaccharide capsule (10,16,20), melanin production (19,20,26), and growth at 370C (20,26 Recent studies of C neofonnans have established basic molecular techniques for performing site-directed mutagenesis (9, 31). A C. neoformans uraS auxotrophic strain (serotype D) was recently reported to be transformed at a low efficiency by complementation with a cloned uraS gene delivered by electroporation. However, the number of integrative events was low and generally nonhomologous (9, 31). A total of 80 to 90% of the transformants were unstable and apparently contained extrachromosomal DNA, consisting of sequences of rearranged uraS-vector DNA with the addition of cryptococcal genomic DNA (8,9,31

Itraconazole and fluconazole, two new triazoles, were examined for their antifungal activity in rabbits. Fluconazole easily crossed the blood-cerebrospinal fluid barrier, and active drug was eliminated in high concentrations in the urine. On the other hand, itraconazole did not cross the blood-cerebrospinal fluid barrier in measurable amounts, and urine concentrations were variable. Despite differences in pharmacokinetics at the site of infection, both agents were equally effective ill treating cryptococcal meningitis and candida pyelonephritis in animals. By using a ketoconazole-resistant strain of Candida albicans, we showed that there was cross-resistance in vivo between these two new triazole compounds.For over 25 years, amphotericin B has provided the standard treatment for most systemic mycotic infections (13,14). However, this drug is less than ideal in many respects, especially with regard to toxicity. In the search for alternative therapies, the azole compounds represent an important development. Imidazoles, such as clotrimazole and miconazole, have proven very effective for treating superficial dermatophyte and yeast infections. An intravenous preparation of miconazole had some success against disseminated mycoses (4, 11), but lack of clinical experience has limited its use for systemic disease. Ketoconazole is the most recently licensed addition to the meager stockpile of systemic antifungal drugs. It has proven to be effective against both superficial dermatophyte and yeast infections (9, 10), and some disseminated mycoses such as paracoccidioidomycosis, blastomycosis, histoplasmosis, and coccidioidomycosis (5,19,21).In the quest for new antifungal agents with lower toxicity, broader spectrum, and better pharmacokinetic profiles, the progressive development of azole compounds has produced the triazoles. These include itraconazole and fluconazole.In this study, we compared the pharmacokinetics, in vitro activity, and in vivo efficacy of these two agents in two animal model infections: cryptococcal meningitis in cortisone-treated rabbits and candida pyelonephritis in rabbits. In the first model, we evaluated the effect of these agents on a central nervous system infection in a compromised host. In the second model, we examined the treatment of the most common pathogenic yeast in the urinary tract (6).

We studied the penetration of three azole compounds, ketoconazole, itraconazole, and fluconazole, into the ocular tissues and fluids of rabbits in the presence and absence of ocular inflammation. Drug concentrations were compared with those found in serum and cerebrospinal fluid. The rank order of penetration into eye tissue was fluconazole > ketoconazole > itraconazole. Fluconazole penetrated freely into both inflamed and uninflamed eyes. The presence of inflammation improved penetration of all three compounds into ocular fluids and tissues. Penetration of these azoles into the anterior chamber of uninflamed eyes and into the cerebrospinal fluid was similar. All three azole compounds reduced the number of yeasts found in the eye in hematogenous Candida albicans endophthalmitis in rabbits when therapy was initiated within 24 h of inoculation. However, only ketoconazole significantly reduced yeast counts in the eye when therapy was postponed for 7 days.The incidence of ocular fungal infections has increased (10, 11, 26) due to a number of factors: increased prevalence of immunosuppression associated with organ transplantation and malignancies; prolonged recovery from complex surgical procedures; and increasing use of antibiotics, immunosuppressive agents, intravenous catheters, and hyperalimentation fluids. Endogenous Candida endophthalmitis (ECE) also occurs in heroin addicts

A B S T R A C T The ability of antibiotics to prevent Streptococcus sangutis endocarditis was tested in rabbits. Only vancomycin or a combination of penicillin G plus streptomycin always prevented infection when administered as a single dose. A loading dose of 30 mg/kg of phenoxymethyl penicillin (penicillin V) followed by additional 7.5 mg/kg doses for 48 h proved to be the only successful prophylactic program that could be given orally to man. Cefazolin alone or with streptomycin in multiple doses was also an effective alternative to penicillin or penicillin derivatives. Erythromycin uniformly failed to protect animals from bacterial endocarditis but showed greater prophylactic efficacy when a low inoculum of streptococci was used.