In vitro susceptibilities of Candida and Aspergillus species to Melaleuca alternafolia (tea tree) oil.

Candida species are an important cause of opportunistic infection in the oral cavity of immunocompromised patients, especially HIV infected patients. Melaleuca oil obtained commercially was investigated since it is known to have broad antifungal properties. The in-vitro susceptibilities of Aspergillus and susceptible and resistant Candida species were performed utilizing serial dilutions in microtiter plates with Sabouraud dextrose agar and the commercial preparation of Melaleuca. As a comparator, in vitro susceptibilities to amphotericin B and fluconazole were also determined using the broth microdilution technique. The results demonstrate that Melaleuca inhibited the Candida species. However, the growth of Aspergillus was not inhibited at the concentrations tested. Thus, preparations containing Melaleuca alternafolia may be a useful alternative for superficial candidal infections. In fact, it may be a useful alternative regimen for advanced HIV-positive patients with oropharyngeal candidiasis refractory to fluconazole. However, controlled clinical studies to evaluate its efficacy are still needed.

Amino acid variations of cytochrome P-450 lanosterol 14 alpha-demethylase (CYP51A1) from fluconazoleresistant clinical isolates of Candida albicans.

We studied six clinical isolates of Candida albicans. All six isolates showed high level resistance to fluconazole (minimum inhibitory concentrations 64 microg/ml) with varying degrees of cross-resistance to other azoles but not to amphotericin B. Neither higher dosage nor upregulation of the gene encoding the cytochrome P- 450 lanosterol 14 alpha-demethylase (CYP51A1 or P-450LDM) was responsible for fluconazole resistance. The resistant and the susceptible isolates accumulated similar amounts of azoles. To examine whether resistance to fluconazole in these clinical isolates of C. albicans is mediated by an altered target of azole action, we cloned the structural gene encoding P-450LDM from the fluconazole resistant isolates. The amino acid sequences of the P-450LDMs from the isolates were deduced from the gene sequences and compared to the P-450LDM sequence of the fluconazole-susceptible C. albicans B311. The enzymes from the clinical isolates showed 2 to 7 amino acid variations scattered across the molecules encompassing 10 different loci. One-half of the amino acid changes obtained were conserved substitutions (E116D, K143R, E266D, D278E, R287K) compared to the susceptible strain. Non-conserved substitutions were T128K, R267H, S405F, G450E and G464S, three of which are in and around the hemebinding region of the molecule. R287K is the only amino acid change that was found in all six clinical isolates. One or more of these mutational alterations may lead to the expression of an azole-resistant enzyme.

Stable phenotypic resistance of Candida species to amphotericin B conferred by preexposure to subinhibitory levels of azoles.

The fungicidal activity of amphotericin B (AmB) was quantitated for several Candida species. Candida albicans and C. tropicalis were consistently susceptible to AmB, with less than 1% survivors after 6 h of exposure to AmB. C. parapsilosis and variants of C. lusitaniae and C. guilliermondii were the most resistant, demonstrating 50 to 90% survivors in this time period and as high as 1% survival after a 24-h exposure time. All Candida species were killed (<1% survivors) after 24 h of exposure to AmB. In contrast, overnight exposure to either fluconazole or itraconazole resulted in pronounced increases in resistance to subsequent exposures to AmB. Most dramatically, C. albicans was able to grow in AmB cultures after azole preexposure. Several other Candida species did not grow in AmB but showed little or no reduction in viability after up to 24 h in AmB. Depending on the growth conditions, Candida cells preexposed to azoles may retain AmB resistance for days after the azoles have been removed. If this in vitro antagonism applies to the clinical setting, treatment of patients with certain antifungal combinations may not be beneficial. The ability of some Candida isolates to survive transient exposures to AmB was not reflected in the in vitro susceptibility changes as measured by standard MIC assays. This finding should be considered in studies attempting to correlate patient outcome with in vitro susceptibilities of clinical fungal isolates. Patients who fail to respond to AmB may be infected with isolates that are classified as susceptible by standard in vitro assays but that may be resistant to transient antifungal exposures which may be more relevant in the clinical setting.

In vitro interaction between amphotericin B and azoles in Candida albicans.

The use of azole prophylaxis as a measure to prevent invasive fungal infections in high-risk patients is increasing and is now the standard of care in many institutions. Previous studies disagree on whether preexposure of Candida albicans to azoles affects their subsequent susceptibility to amphotericin B (AmB). The present in vitro study indicates that azole exposure generates a subpopulation of cells that are not affected by subsequent exposure to AmB. These cells that are phenotypically resistant to AmB tolerated by most cells not exposed to azole. The percentage of cells that convert to phenotypic resistance to AmB varies with the concentration and the azole. Itraconazole is more effective than fluconazole in generating cells that are phenotypically resistant to AmB and that tolerate an otherwise lethal transient exposure to AmB. Until cells that are not exposed to fluconazole are simultaneously challenged with AmB, they are not protected to a significant extent from killing by AmB. Cells that are challenged with continuous exposure to AmB also acquire phenotypic resistance to AmB at increased frequencies by azole preexposure, but this requires that the azole be continuously present during incubation with AmB. In addition, Candida cells taken from mature colonies that are not actively growing are not susceptible to the short-term killing effects of AmB without azole preexposure. The adaptive responses of C. albicans to AmB and potentially other antifungal agents that may result from prior exposure to azoles in vitro or potentially in microenvironments in vivo that induce physiological changes may have major clinical implications.

Recombinant mitochondrial plasmids in Neurospora composed of Varkud and a new multimeric mitochondrial plasmid.

A mitochondrial plasmid, V5124, in Neurospora intermedia isolate 5124 has a deletion in its sequence relative to the highly similar Mauriceville and Varkud plasmids. These insertions in the latter plasmids are 28 bp in length and are positioned at sites that correspond to their major transcript 5' termini. The 28-bp sequence is nearly identical to a putative processing site upstream of the ND4L gene on the mitochondrial genome. The absence of this 28-bp sequence in V5124 apparently results in transcripts whose 5' termini correspond to an upstream consensus promoter sequence. Two variant forms of V5124 coexist with V5124 and have either of two similar 0.3-kb inserts positioned exactly as is the 28-bp insert in Varkud. These long inserts are chimeric, partly deriving from a newly discovered multimeric plasmid, MP. MP has significant similarity to a short region of the mitochondrial satellite plasmid VS. Another part of the 0.3-kb inserts in V5124 variants derives from the mitochondrial genome, within restriction fragment EcoRI-8. Neurospora mitochondria in many isolates can have several types of mitochondrial plasmids belonging to different homology groups. We propose that a common ancestral plasmid acquired insertions from either the mitochondrial genome or from other plasmids. The V5124 variants are the first instance of a chimeric mitochondrial plasmid in which distinct plasmids have recombined. This recombination proves that different plasmids coexist currently, or else did so at some point in their evolution, within a single mitochondrion.

Alternative methods of preparing whole-cell DNA from fungi for dot-blot, restriction analysis, and colony filter hybridization.

There is a large and increasing number of methods for preparing whole-cell DNA from fungi. Modifications have evolved for two reasons. This first is to simplify the protocol as much as possible to allow processing of large sample numbers, in some cases for very specific uses, e.g., dot-blots. The second is to increase the quality of the DNA. Most preparations are contaminated with varying amounts of polysaccharides and unknown wall contaminants that can inhibit subsequent restriction or ligation. The extent of contamination varies with the species, the individual isolate, and at least in Neurospora, with the method or extent of growth. This paper offers three new methods. The first is a simplified procedure for isolating denatured DNAs from filamentous fungi for dot-blot analysis. The second is a rapid method for isolating DNAs from large numbers of small- to medium-scale cultures of filamentous fungi. These preparations are sufficiently pure for a variety of enzymatic reactions. The third is a nonenzymatic method for yeast colony filter hybridization that is simple, inexpensive, and efficient and results in uniform signals for a variety of species.

Suppressor mutants of Neurospora crassa that tolerate allelic differences at single or at multiple heterokaryon incompatibility loci.

Allelic differences at any one of at least 11 heterokaryon incompatibility (het) loci in Neurospora crassa trigger an incompatibility response: localized cell death at sites of hyphal anastomosis. We have isolated spontaneous and insertional suppressor mutants that are heterokaryon-compatible in spite of allelic differences at one or at several het loci. Some intra- and extragenic mutants tolerated allelic differences only at single het loci. Multi-tolerant spontaneous mutants were isolated by selecting simultaneously for tolerance of differences at het-c, -d and -e, or at each of these plus mating-type. Some suppressor mutants were specific for only one allele at the affected het locus; others suppressed both alleles. Insertional mutations were isolated from banks of transformants, each having a plasmid integrated into a random position in the chromosome. One mutant tolerated allelic differences at het-d. A homologous cosmid from a Neurospora genomic bank complemented the mutant phenotype. A second insertional inactivation mutant was tolerant of het-c differences. Inactivation of the wild-type locus corresponding to the integration site was accomplished by repeat-induced point mutation (RIP). The RIP progeny, like the original mutant, were tolerant of differences at het-c. It may be possible to use such suppressor mutants as universal donors of hypovirulence in pathogenic fungi.

Distribution of seven homology groups of mitochondrial plasmids in Neurospora: evidence for widespread mobility between species in nature.

A survey of mitochondrial DNAs from over 225 Neurospora and related fungal isolates from around the world uncovered three new homology groups of mitochondrial plasmids, two divergent subgroups of the Fiji plasmid family, and extended previous data about plasmid distribution patterns. Newly-discovered circular plasmids, Java and MB1, and the linear Moorea plasmids, were found in relatively-few isolates. A large proportion of isolates (51%) were found to have these or previously-discovered plasmids in the Varkud, kalilo, LaBelle, or Fiji families. Plasmids in most families were found in isolates worldwide and distributed nearly randomly with respect to species. As many as three types of plasmids were found in single isolates, and plasmids typically were found alone or in pairs in a random, independent pattern. The regional clustering of some plasmids was independent of species, providing a strong argument that horizontal transfer of plasmids occurs frequently in nature. Some plasmid families were much more diverse than others. The Fiji plasmids are a superfamily composed of distinct subgroups defined by degrees of cross-hybridization. Between some subgroups there were large regions of non-homology.