The vaginal microbiome: new information about genital tract flora using molecular based techniques.

Vaginal microbiome studies provide information that may change the way we define vaginal flora. Normal flora appears dominated by one or two species of Lactobacillus. Significant numbers of healthy women lack appreciable numbers of vaginal lactobacilli. Bacterial vaginosis (BV) is not a single entity, but instead consists of different bacterial communities or profiles of greater microbial diversity than is evident from cultivation-dependent studies. BV should be considered a syndrome of variable composition that results in different symptoms, phenotypical outcomes, and responses to different antibiotic regimens. This information may help to elucidate the link between BV and infection-related adverse outcomes of pregnancy.

Bacterial vaginosis: culture- and PCR-based characterizations of a complex polymicrobial disease's pathobiology.

Bacterial vaginosis (BV) is an enigmatic polymicrobial disease, and its evolution and pathobiology will not be solved by traditional culture-based methods. Characterization of the vaginal microbiota by polymerase chain reaction-based methods holds great promise. Molecular studies have identified species not detected by culture, but they also have missed some species identified by culture. These studies allow classification of both normal and BV patients based on distinct microbiologic profiles, which may prove important in accessing risk of BV, response to treatment, and risk of complications. More studies using new generations of primers and standardized methods are needed, and data must be analyzed after grouping patients according to microbiologic profiles.

Fungal endophthalmitis diagnosis by detection of Candida albicans DNA in intraocular fluid by use of a species-specific polymerase chain reaction assay.

Candida endophthalmitis is a serious infection secondary to hematogenous dissemination or direct inoculation of the organisms following trauma or eye surgery. The diagnosis is based on the characteristic findings in the infected eye and on culture of vitreous samples. Unfortunately, the yield of vitreous cultures is limited. The use of a Candida albicans species-specific polymerase chain reaction (PCR) assay in the diagnosis of Candida endophthalmitis is reported herein. Four patients with suspected fungal endophthalmitis underwent vitrectomy for diagnostic and therapeutic purposes. In 2 of the 4, vitreous cultures were negative. However, characteristic PCR products were generated in all 4 patient specimens, enabling the rapid diagnosis of Candida endophthalmitis in all 4. Clinical response was observed in all cases. These results demonstrate the utility of PCR-mediated detection of C. albicans in vitreous samples.

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.

High-frequency, in vitro reversible switching of Candida lusitaniae clinical isolates from amphotericin B susceptibility to resistance.

Recent studies have revealed an increase in the incidence of serious infections caused by non-albicans Candida species. Candida lusitaniae is of special interest because of its sporadic resistance to amphotericin B (AmB). The present in vitro study demonstrated that, unlike other Candida species, C. lusitaniae isolates frequently generated AmB-resistant lineages form previously susceptible colonies. Cells switching from a resistant colony to a susceptible phenotype were also detected after treatment with either UV light, heat shock, or exposure to whole blood, all of which increased the frequency of switching. In some C. lusitaniae lineages, after a cell switched to a resistant phenotype, the resistant phenotype was stable; in other lineages, colonies were composed primarily of AmB-susceptible cells. Although resistant and susceptible lineages were identical in many aspects, their cellular morphologies were dramatically different. Switching mechanisms that involve exposure to antifungals may have an impact on antifungal therapeutic strategies as well as on standardized susceptibility testing of clinical yeast specimens.

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.

Identification of Candida species by randomly amplified polymorphic DNA fingerprinting of colony lysates.

We have characterized a method that produces simple yet diagnostic fingerprints that are unique to isolates of Candida species. DNA from individual colonies can be amplified from crude single-colony lysates. Randomly amplified polymorphic DNA (RAPD) fingerprints generated from a single primer correctly identified the species of most (>98%) of the isolates identified with CHROMagar Candida plates as non-Candida albicans Candida species. RAPD fingerprints were much more informative than the plates, since they distinguished between all tested species and required less time. Most (91%) of these identifications agreed with those assigned by API 20C tests. In almost every incident of species identity mismatch, electrophoretic karyotyping showed that the RAPD fingerprint was correct. This underscores the improved objectivity and reliability of this method over those of conventional diagnostic tools. The identities of approximately 30% of C. albicans isolates identified in clinical laboratories by positive germ tube tests are not verified by either testing on CHROMagar Candida plates or RAPD fingerprinting. Data suggest that clinical isolates conventionally identified as C. albicans in clinical settings are heterogeneous, consisting of both misidentified and atypical yeasts. RAPD fingerprints obtained from primary culture plate colonies allows for rapid, highly accurate determinations of Candida species, hence permitting earlier selection of appropriate antifungal agents 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.

Evidence that a 1.6 kilobase region of Neurospora mtDNA was derived by insertion of part of the LaBelle mitochondrial plasmid.

The LaBelle mitochondrial plasmid hybridizes to a small region of the mtDNA of different Neurospora species. Here, we show that the region of homology encompasses 1385 bp of plasmid sequence and 1649 bp of mtDNA sequence. Several findings--that the region of homology is not found in the mtDNAs of other organisms, that it includes the C-terminus of the ORF encoding the plasmid DNA polymerase, and that the ORF sequence in the mtDNA is interrupted by insertions--suggest that the region was part of the plasmid that integrated into mtDNA prior to the divergence of Neurospora species. Since the LaBelle plasmid has been found in only one Neurospora strain, we infer that the plasmid was lost subsequently from most strains. The LaBelle plasmid is transcribed by the host Neurospora mitochondrial RNA polymerase and the major promoter is located upstream of the long ORF, within the region of homology to mtDNA. A promoter used for the transcription of the mitochondrial small rRNA is found at a corresponding position in Neurospora mtDNA and may have been acquired via integration of the plasmid sequence. Our results provide evidence that an autonomous infectious element may contribute to sequences that functionally constitute an organism's mtDNA.

Structural analysis of the Neurospora mitochondrial large rRNA intron and construction of a mini-intron that shows protein-dependent splicing.

The gene encoding the Neurospora mitochondrial large rRNA contains a single group I intron of 2.3 kilobases that is not self-splicing in vitro. We showed previously that the splicing of this intron in vivo and in vitro is dependent on the Neurospora cyt-18 protein, mitochondrial tyrosyl-tRNA synthetase. In the present work, we carried out further structural analysis of the intron and constructed mutant derivatives of it in order to identify features that are either required for splicing or prevent it from self-splicing. Previous studies showed that the intron contains a large hairpin structure near the 5' splice site. By mapping RNase III cleavage sites, we identified this hairpin structure as an extended P2 stem. We construct a mini-intron of 388 nucleotides by deleting the 426-amino acid intron open reading frame, most of the 5' intron hairpin, and all of L8. This mini-intron shows the same protein-dependent splicing as the full length intron, but is still not self-splicing. Further deletions, which remove all of P2 or all or part of P4, P6, P7, or P9, inactivate splicing, suggesting that an intact group I intron core structure is required. Strengthening the P1, P10, or P9.0 pairings did not enable the mini-intron to self-splice. Our findings indicate that the inability of the mitochondrial large rRNA intron to self-splice reflects deficiency of a structure or activity required for cleavage at the 5' splice site, either in the intron core itself or in the interaction between the core and the P1 stem.

Analysis of large deletions in the Mauriceville and Varkud mitochondrial plasmids of Neurospora.

The Mauriceville and Varkud mitochondrial plasmids are closely related, closed-circular DNAs (3.6 and 3.7 kb, respectively) that have characteristics of mtDNA introns and retroid elements. Both plasmids contain a 710 amino acid open reading frame (ORF) that encodes an 81 kDa protein having reverse transcriptase activity. Here, we analyzed two mutant plasmids, V5-36 and M3-24, that have undergone relatively large deletions (approximately 0.35 and 0.5 kb, respectively). Both deletions occur downstream of the long ORF in a non-coding region of the plasmids that contains a direct repeat of 160 bp and a cluster of five PstI-palindromes, a repetitive sequence element in Neurospora mtDNA. In V5-36, the deletion end points are at the bases of two hairpin structures that are centered around PstI-palindromes and flank the deleted region. In M3-24, the deletion junction contains an extra T-residue that is not encoded in the plasmid. In both plasmids, the deletion end points do not correspond to homologous or directly repeated sequences of more than one nucleotide, whose pairing could account for the deletion junction. The characteristics of the deletion end points can be accounted for either by illegitimate recombination, possibly following double strand breaks at cruciform structures, or by interruption of reverse transcription followed by reinitiation downstream. The finding that the deletions encompass the 160 bp direct repeat and all five PstI-palindromes indicates that neither are required for propagation of the plasmids and supports the hypothesis that PstI-palindromes are selfish DNA elements that inserted into a nonessential region of the plasmid.

Function of Neurospora mitochondrial tyrosyl-tRNA synthetase in RNA splicing requires an idiosyncratic domain not found in other synthetases.

Neurospora mitochondrial tyrosyl-tRNA synthetase (mt TyrRS), which is encoded by nuclear gene cyt-18, functions in splicing group I introns. Analysis of intragenic partial revertants of the cyt-18-2 mutant and in vitro mutants of the cyt-18 protein expressed in E. coli showed that splicing activity of the cyt-18 protein is dependent on a small N-terminal domain that has no homolog in bacterial or yeast mt TyrRSs. This N-terminal splicing domain apparently acts together with other regions of the protein to promote splicing. Our findings support the hypothesis that idiosyncratic sequences in aminoacyl-tRNA synthetase may function in processes other than aminoacylation. Furthermore, they suggest that splicing activity of the Neurospora mt TyrRs was acquired after the divergence of Neurospora and yeast, and they demonstrate one mechanism whereby splicing factors may evolve from cellular RNA binding proteins.

Identification of Neurospora mitochondrial promoters and analysis of synthesis of the mitochondrial small rRNA in wild-type and the promoter mutant [poky].

Using an in vitro transcription assay, we previously identified promoters in Neurospora mtDNA at the 5' ends of the genes encoding the mitochondrial (mt) small and large rRNAs and cob pre-mRNA. Here, we identified two additional promoters in mtDNA restriction fragment EcoRI-6, 3.8 and 5.5 kilobases upstream of the 5' end of the mt small rRNA. By comparing the two new promoters with the three identified previously, we derived a modified promoter consensus sequence (AT-rich)15-27TTAG(A/T)RR(G/T)(G/C)N(A/T). The mt small rRNA in Neurospora is transcribed from at least two promoters, a major promoter at the 5' end of the small rRNA and one or both of the newly identified promoters in EcoRI-6. The latter gives a series of putative pre-rRNAs that contain 5' end extensions of various sizes. The 5' ends of a number of these RNAs map at or near hairpin structures. The [poky] mutant, which is grossly deficient in the mt small rRNA, has a 4-base pair deletion in the major promoter at the 5' end of the mt small rRNA. The residual small rRNAs in [poky] appear to be synthesized via the upstream promoter(s), but are missing 37-44 nucleotides from their 5' ends, indicating either that pre-rRNAs are processed abnormally or that abnormal 5' RNA ends are unstable. The effect of the promoter mutation in [poky] on other transcripts suggests that the mt small rRNA is cotranscribed with downstream genes encoding tRNAs, coIII and ND6. Seven nonallelic nuclear suppressors of [poky] result in increased concentrations of the mt small rRNA and pre-rRNAs, but do not restore the ability to synthesize small rRNAs having the correct 5' ends. The suppressor mutations could act by increasing transcription, processing, or stability of the mt small rRNA or its precursors. The suppressors provide a genetic approach for identifying components that affect transcription and processing of the mt small rRNA.

Involvement of tyrosyl-tRNA synthetase in splicing of group I introns in Neurospora crassa mitochondria: biochemical and immunochemical analyses of splicing activity.

We reported previously that mitochondrial tyrosyl-tRNA synthetase, which is encoded by the nuclear gene cyt-18 in Neurospora crassa, functions in splicing several group I introns in N. crassa mitochondria (R. A. Akins and A. M. Lambowitz, Cell 50:331-345, 1987). Two mutants in the cyt-18 gene (cyt-18-1 and cyt-18-2) are defective in both mitochondrial protein synthesis and splicing, and an activity that splices the mitochondrial large rRNA intron copurifies with a component of mitochondrial tyrosyl-tRNA synthetase. Here, we used antibodies against different trpE-cyt-18 fusion proteins to identify the cyt-18 gene product as a basic protein having an apparent molecular mass of 67 kilodaltons (kDa). Both the cyt-18-1 and cyt-18-2 mutants contain relatively high amounts of inactive cyt-18 protein detected immunochemically. Biochemical experiments show that the 67-kDa cyt-18 protein copurifies with splicing and synthetase activity through a number of different column chromatographic procedures. Some fractions having splicing activity contain only one or two prominent polypeptide bands, and the cyt-18 protein is among the few, if not only, major bands in common between the different fractions that have splicing activity. Phosphocellulose columns resolve three different forms or complexes of the cyt-18 protein that have splicing or synthetase activity or both. Gel filtration experiments show that splicing activity has a relatively small molecular mass (peak at 150 kDa with activity trailing to lower molecular masses) and could correspond simply to dimers or monomers, or both, of the cyt-18 protein. Finally, antibodies against different segments of the cyt-18 protein inhibit splicing of the large rRNA intron in vitro. Our results indicate that both splicing and tyrosyl-tRNA synthetase activity are associated with the same 67-kDa protein encoded by the cyt-18 gene. This protein is a key constituent of splicing activity; it functions directly in splicing, and few, if any, additional components are required for splicing the large rRNA intron.

Characterization of mutant mitochondrial plasmids of Neurospora spp. that have incorporated tRNAs by reverse transcription.

The Mauriceville and Varkud mitochondrial plasmids of Neurospora spp. are closely related, closed-circular DNAs (3.6 and 3.7 kilobases, respectively) whose nucleotide sequences and genetic organization suggest relationships to mitochondrial introns and retroelements. We have characterized nine suppressive mutants of these plasmids that outcompete mitochondrial DNA and lead to impaired growth. All nine suppressive plasmids contain small insertions, corresponding to or including a mitochondrial tRNA (tRNATrp, tRNAGly, or tRNAVal) or a tRNA-like sequence. The insertions are located at the position corresponding to the 5' end of the major plasmid transcript or 24 nucleotides downstream near a cognate of the sequence at the major 5' RNA end. The structure of the suppressive plasmids suggests that the tRNAs were inserted via an RNA intermediate. The 3' end of the wild-type plasmid transcript can itself be folded into a secondary structure which has tRNA-like characteristics, similar to the tRNA-like structures at the 3' ends of plant viral RNAs. This structure may play a role in replication of the plasmids by reverse transcription. Major transcripts of the suppressive plasmids begin at the 5' end of the inserted mitochondrial tRNA sequence and are present in 25- to 100-fold-higher concentrations than are transcripts of wild-type plasmids. Mapping of 5' RNA ends within the inserted mtDNA sequences identifies a short consensus sequence (PuNPuAG) which is present at the 5' ends of a subset of mitochondrial tRNA genes. This sequence, together with sequences immediately upstream in the plasmids, forms a longer consensus sequence, which is similar to sequences at transcription initiation sites in Neurospora mitochondrial DNA. The suppressive behavior of the plasmids is likely to be directly related to the insertion of tRNAs leading to overproduction of plasmid transcripts.