Cryphonectria naterciae: a new species in the Cryphonectria-Endothia complex and diagnostic molecular markers based on microsatellite-primed PCR

In a recent study intended to assess the distribution of Cryphonectria parasitica in Portugal, 22 morphologically atypical orange isolates were collected in the Midwestern regions. Eleven isolates were recovered from Castanea sativa, in areas severely affected by chestnut blight and eleven isolates from Quercus suber in areas with cork oak decline. These isolates were compared with known C. parasitica and Cryphonectria radicalis isolates using an integrated approach comprising morphological and molecular methods. Morphologically the atypical isolates were more similar to C. radicalis than to C. parasitica. Phylogenetic analyses based on internal transcribed spacer (ITS) and β-tubulin sequence data grouped the isolates in a well-supported clade separate from C. radicalis. Combining morphological, cultural, and molecular data Cryphonectria naterciae is newly described in the Cryphonectria-Endothia complex. Microsatellite-primed PCR fingerprinting with (GACA)(4) primer discriminated between C. naterciae, C. radicalis, and C. parasitica.     

Molecular epidemiology of clinical and environmental isolates of the Cryptococcus neoformans species complex reveals a high genetic diversity and the presence of the molecular type VGII mating type a in Colombia

The aim of this study was to investigate the epidemiological relationships of clinical and environmental isolates of the Cryptococcus neoformans species complex in Colombia. The current study reflects data from 1987 to 2004. In Colombia serotypes A and B are most frequently recovered from patients and the environment. Of the 178 clinical isolates studied, 91.1% were of serotype A, 8.4% serotype B and 0.5% serotype C. Of the 247 environmental isolates, 44.2% were of serotype A, 42.6% serotype B and 13.2% serotype C. No serotype D isolates were isolated. Serotype AD has not been recovered in Colombia. PCR fingerprinting with the primers M13, (GACA)4 and (GTG)5 and URA5 gene restriction fragment length polymorphism analysis grouped the majority of clinical serotype A and environmental serotype B isolates into the molecular types VNI (98.1%) and VGII (100%), respectively. Mating type alpha was determined in 99.3% of serotype A isolates, but 96.6% of serotype B isolates were of mating type a. Similar profiles between clinical and environmental isolates suggest that the patients may have acquired the infection from the environment. The data presented form part of the Colombian contribution to the ongoing global survey of the C. neoformans species complex.     

Penicillium marneffei: types and drug susceptibility

The PCR fingerprints of 30 Penicillium marneffei isolates from Chiang Rai in northern Thailand and Bangkok in central Thailand were studied through use of single-nucleotide primers (GACA)₄ and the phage M13 core sequence. Discrimination of fingerprint patterns was based on differences in the number of major bands. The P. marneffei isolates were divided into four types, i.e., A, B, C, and D. Type A was found in two isolates from Chiang Rai (6.7%). Types B and C respectively were found in two (6.7%) and one (3.3%) isolates from Bangkok. The predominate type D (83.3%) was found in isolates obtained from Chiang Rai and Bangkok. The PCR fingerprinting method was found to be useful for the epidemiological study of P. marneffei, a dimorphic opportunistic fungus and an emerging pathogen in the HIV pandemic. In vitro drug susceptibility testing by broth macrodilution to four antifungal agents against the yeast form ofP. marneffei was performed. The MIC ranges for amphotericin B, fluconazole, itraconazole, and ketoconazole were 0.125–0.5, 4.0–8.0, <0.032, and <0.125 μg/ml respectively. This revised version was published online in June 2006 with corrections to the Cover Date.     

Candida dubliniensis identification in Brazilian yeast stock collection

We investigated the presence of Candida dubliniensis among isolates previously identified as Candida albicans and maintained in a yeast stock collection from 1994 to 2000. All isolates were serotyped and further evaluated for antifungal susceptibility profile. After doing a screening test for C. dubliniensis isolates based on the capability of colonies to grow at 42 C, its final identification was obtained by randomly amplified polymorphic DNA (RAPD) analysis using three different primers. A total of 46 out of 548 screened isolates did not exhibit growth at 42 C and were further genotyped by RAPD. Eleven isolates were identified as C. dubliniensis with RAPD analysis. Regarding serotypes, 81.5% of C. albicans and all C. dubliniensis isolates belonged to serotype A. Of note, 9 out of 11 C. dubliniensis isolates were obtained from patients with acquired immunodeficiency syndrome (Aids) and all of them were susceptible to azoles and amphotericin B. We found 17 (3%) C. albicans isolates that were dose-dependent susceptibility or resistant to azoles. In conclusion, we found a low rate of C. dubliniensis isolates among stock cultures of yeasts previously identified as C. albicans. Most of these isolates were recovered from oral samples of Aids patients and exhibited high susceptibility to amphotericin B and azoles. C. albicans serotype A susceptible to all antifungal drugs is the major phenotype found in our stock culture.     

Leishmania donovani: intraspecific polymorphisms of Sudanese isolates revealed by PCR-based analyses and DNA sequencing

Four polymerase chain reaction (PCR)-based approaches were used to analyze diversity within 23 Sudanese isolates of Leishmania donovani. Methods compared were fingerprinting with single nonspecific primers, restriction analysis of the amplified ribosomal internal transcribed spacer (ITS) locus, single-stranded conformation polymorphism (SSCP), and sequencing of the ITS region. When PCR fingerprinting and restriction analysis of ITS were applied, highly similar fragment patterns were observed for all strains of L. donovani studied. The ITS1 locus gave five different SSCP profiles among the 23 Sudanese isolates, whereas the ITS2 locus was highly conserved with the exception of 1 isolate. Strains of L. donovani derived from other geographical areas were found to have different ITS2 patterns. SSCP analysis correlated well with results of DNA sequencing and confirmed that SSCP was able to detect genetic diversity at the level of a single nucleotide. SSCP had advantages over the other methods employed for investigation of sequence variation within the species L. donovani. There was no correlation between the form of clinical manifestation of the disease and the PCR fingerprinting, ITS-RFLP, or ITS-SSCP characteristics.     

Distribution of Candida species in different clinical sources in Delhi, India, and proteinase and phospholipase activity of Candida albicans isolates

Eighty-five isolates of Candida recovered from three hundred and fifty diverse clinical sources, viz. respiratory tract (sputum, bronchial washing,bronchoalveolar lavage, tracheal aspirate), blood, urine, high vaginal swab, skin and plastic devices, were studied in detail for their morphological and biochemical characters. Seven species of Candida were identified, viz., C. albicans (45.8%), C. tropicalis (24.7%), C. parapsilosis (10.5%), C. krusei (7.0%), C. kefyr (7.0%), C. guilliermondii (3.5%), and C. glabrata (1.1%). C. albicans was the predominant species isolated from all clinical specimens, except blood from which C. krusei was most frequently (38.4%) recovered. Out of 39 isolates of C. albicans, 26 (66.6%) and 19 (48.7%) exhibited strong proteinase and phospholipase activity respectively. There was a higher prevalence of proteinase producing strains amongst the vaginal and skin isolates than that in urinary and respiratory isolates. Also a greater number of phospholipase producing strains was observed in the vaginal and urinary isolates than that in the respiratory and skin isolates.     

Multicenter Brazilian study of oral Candida species isolated from AIDS patients

Oropharyngeal candidiasis continues to be considered the most common opportunistic disease in Aids patients. This study was designed to investigate species distribution, serotype and antifungal susceptibility profile among Candida spp. isolated from the oral cavity of Aids patients recruited from six Brazilian university centers. Oral swabs from 130 Aids patients were plated onto CHROMagar Candida medium and 142 isolates were recovered. Yeast isolates were identified by classical methods and serotyped using the Candida Check or target system-Iatron. Antifungal susceptibility testing was performed according to the NCCLS microbroth assay. C. albicans was the most frequently isolated species (91%), and 70% of the isolates belonged to serotype A. We detected 12 episodes of co-infection (9%), including co-infection with both serotypes of C. albicans. Non-albicans species were isolated from 12 episodes, 50% of them exhibited DDS or resistance to azoles. Otherwise, only 8 out 130 isolates of C. albicans exhibited DDS or resistance to azoles. Brazilian Aids patients are infected mainly by C. albicans serotype A, most of them susceptible to all antifungal drugs.     

The discovery of enfumafungin, a novel antifungal compound produced by an endophytic Hormonema species biological activity and taxonomy of the producing organisms

In a screening of natural products with antifungal activity derived from endophytic fungi, we detected a potent activity in a culture belonging to the form-genus Hormonema, isolated from leaves of Juniperus communis. The compound is a new triterpene glycoside, showing an antifungal activity highly potent in vitro against Candida and Aspergillus and with moderate efficacy in an in vivo mouse model of disseminated candidiasis. The agent is especially interesting since its antifungal spectrum and its effect on morphology of Aspergillus fumigatus is comparable to that of the glucan synthase inhibitor pneumocandin B,,, the natural precursor of the clinical candidate MK-0991 (caspofungin acetate). An additional search for other Hormonema isolates producing improved titers or derivatives resulted in the isolation of two more strains recovered from the same plant host showing identical activity. The producing isolates were compared with other non-producing Hormonema strains by DNA fingerprinting and sequencing of the rDNA internal transcribed spacers. Comparison of rDNA sequences with other fungal species suggests that the producing fungus could be an undetermined Kabatina species. Kabatina is a coelomycetous genus whose members are known to produce Hormonema-like states in culture.     

Diversity of antifungal and plant-associated Serratia plymuthica strains

A total of 21 plant-associated Serratia plymuthica strains were characterized phenotypically by their nutritional patterns, susceptibility to antibiotics, antifungal and haemolytic properties, and genotypically by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rDNA, PCR fingerprints using BOX primers (BOX-PCR) and pulsed-field gel electrophoresis (PFGE) after digestion with SpeI. All of the investigated strains demonstrated antifungal activity in vitro against fungal pathogens while only six strains produced the antifungal antibiotic prodigiosin. Haemolytic activity and antibiotic resistance patterns were investigated to assess the risk associated with the use of isolates in plant protection. The strains were haemolytic at human-relevant temperatures. The level of resistance to antibiotics was low. This work shows that BOX-PCR and PFGE are useful fingerprinting methods to characterize Ser. plymuthica strains, although the discriminatory effect between the two methods differed. Computer-assisted analysis of phenotypic and genotypic features demonstrated relationships between the origin of isolation, the production of prodigiosin and the molecular fingerprint.     

Comparative analyses of classical phenotypic method and ribosomal RNA gene sequencing for identification of medically relevant Candida species

As the distribution of Candida species and their susceptibility to antifungal agents have changed, a new means of accurately and rapidly identifying these species is necessary for the successful early resolution of infection and the subsequent reduction of morbidity and mortality. The current work aimed to evaluate ribosomal RNA gene sequencing for the identification of medically relevant Candida species in comparison with a standard phenotypic method. Eighteen reference strains (RSs), 69 phenotypically identified isolates and 20 inconclusively identified isolates were examined. Internal transcribed spaces (ITSs) and D1/D2 of the 26S ribosomal RNA gene regions were used as targets for sequencing. Additionally, the sequences of the ITS regions were used to establish evolutionary relationships. The sequencing of the ITS regions was successful for 88% (94/107) of the RS and isolates, whereas 100% of the remaining 12% (13/107) of the samples were successfully analysed by sequencing the D1/D2 region. Similarly, genotypic analysis identified all of the RS and isolates, including the 20 isolates that were not phenotypically identified. Phenotypic analysis, however, misidentified 10% (7/69) of the isolates. Phylogenetic analysis allowed the confirmation of the relationships between evolutionarily close species. Currently, the use of genotypic methods is necessary for the correct identification of Candida species.     

Fluconazole-resistant pathogens Candida inconspicua and C. norvegensis: DNA sequence diversity of the rRNA intergenic spacer region, antifungal drug susceptibility, and extracellular enzyme production

The opportunistic fungal pathogens Candida inconspicua and C. norvegensis are very rarely isolated from patients and are resistant to fluconazole. We collected 38 strains of the two microorganisms isolated from Europe and Japan, and compared the polymorphism of the rRNA intergenic spacer (IGS) and internal transcribed spacer (ITS) regions, antifungal drug susceptibility, and extracellular enzyme production as a potential virulence factor. While the IGS sequences of C. norvegensis were not very divergent (more than 96.7% sequence similarity among the strains), those of C. inconspicua showed remarkable diversity, and were divided into four genotypes with three subtypes. In the ITS region, no variation was found in either species. Since the sequence similarity of the two species is approximately 70% at the ITS region, they are closely related phylogenetically. Fluconazole resistance was reconfirmed for the two microorganisms but they were susceptible to micafungin and amphotericin B. No strain of either species secreted aspartyl proteinase or phospholipase B. These results provide basal information for accurate identification, which is of benefit to global molecular epidemiological studies and facilitates our understanding of the medical mycological characteristics of C. inconspicua and C. norvegensis.     

Genetic Diversity, Aggressiveness and Metalaxyl Sensitivity of Pythium spinosum Infecting Cucumber in Oman

A total of 24 isolates of Pythium spinosum from cucumber obtained from five regions in Oman were characterized for genetic diversity using amplified fragment length polymorphism (AFLP) fingerprinting and three isolates from the Netherlands, South Africa and Japan were included for comparison. Isolates from Oman were also characterized for aggressiveness on cucumber seedlings and sensitivity to metalaxyl. Identity of all isolates was confirmed using sequences of the internal transcribed spacer (ITS) region of the ribosomal DNA (rDNA), which showed more than 99% nucleotide similarity among all isolates. Using six primer‐pair combinations, AFLP fingerprinting resolved 295 AFLP markers of which 193 were polymorphic among isolates from other countries and only six were polymorphic among isolates of P. spinosum from Oman. Seven different AFLP phenotypes of P. spinosum were recovered in Oman; two of them were found to contain over 79% of isolates and one was recovered from all regions in Oman. Phenotypes from Oman showed very high (≥99%) levels of genetic similarity to each other compared to moderate (mean =53%) levels of genetic similarity with phenotypes from other countries. In addition, all isolates from Oman were found to be highly sensitive to metalaxyl and all were aggressive on cucumber seedlings at 25°C. The high genetic similarity among phenotypes of P. spinosum in Oman as well as recovering two major clones across regions may suggest that P. spinosum has been recently introduced in Oman via a common source.     

Characterization of rabbit Pasteurella multocida isolates by use of whole-cell, outer-membrane, and polymerase chain reaction typing.

PURPOSE: To characterize Pasteurella multocida isolates from laboratory rabbits using serotyping, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins (WCPs) and outer-membrane proteins (OMPs), and polymerase chain reaction (PCR) fingerprinting. METHODS: Fifty isolates were obtained from five sources: ATCC (1), Oklahoma (4), Michigan (9), Minnesota (7), and Texas (29). The PCR fingerprinting was conducted using two minisatellite probes for M13 and a modified M13 core sequence and two microsatellite probes--(GTG)5 and (GACA)4. RESULTS: Forty-five isolates were serogroup A, and five were serogroup D. Ten WCP patterns (W1-W10) with one variation (W1a) and 10 OMP (OM1-OM10) patterns were found. Primers M13 phage, modified M13 phage, (GTG)5, and (GACA)4 generated 7, 9, 5, and 9 fingerprint types, respectively. Combination of WCP, OMP, and PCR fingerprint results yielded 39 groups with a discrimination index of 0.98. The PCR fingerprint results generally indicated clonal association among isolates within geographic locations except for the isolates from Texas, which varied markedly in PCR fingerprint types. CONCLUSION: Single primer PCR fingerprinting provided a simple and rapid means of typing P. multocida isolates from laboratory rabbits. Combinations of conventional and molecular typing enhanced differentiation among P. multocida isolated from rabbits with pasteurellosis.     

<Emphasis Type="Italic">Ochroconis calidifluminalis</Emphasis>, a Sibling of the Neurotropic Pathogen <Emphasis Type="Italic">O. gallopava</Emphasis>, Isolated from Hot Spring

Two strains resembling the neurotropic fungus Ochroconis gallopava were isolated from hot spring river water (IFM 54738 and IFM 54739). The isolates showed optimal growth at 42°C, while the maximum growth temperature was 49°C, thus having temperature relationships similar to those of O. gallopava. Colonies were light olive green, with a color change to dark reddish brown after several passages, which was also observed in O. gallopava. Conidia were indistinguishable from those of O. gallopava. The antifungal susceptibility profile of the isolates was also similar to that of O. gallopava, except for a lower susceptibility to micafungin. The two isolates had 100% homologous rRNA genes including the internal transcribed spacer (ITS) region and the D1/D2 region of the large subunit. The gene fragments, as O. gallopava, could be amplified with species-specific rDNA primers, and loop-mediated isothermal amplification designed for O. gallopava yielded positive results in the two isolates. However, homologies with O. gallopava in ITS and D1/D2 regions were 79.2 and 95.9%, respectively, widely exceeding generally accepted species boundaries. These differences were corroborated in virulence tested in experimental infection. The two isolates did not kill a mouse even until 28 days. However, mortalities of four O. gallopava strains ranged from 40 to 100%. The new isolates mainly affected the kidneys; whereas O. gallopava had a strong preference for the brain. We therefore propose a new species, Ochroconis calidifluminalis, for the two isolates.     

Phenotypic aspects of oral strains of Candida albicans in children with Down's syndrome.

The aim of this article is to characterize the biological aspects of oral strains of C. albicans in children with Down's syndrome. These yeasts were analyzed as to their macromorphological and enzymatic aspects and were tested as to their in vitro susceptibility to antifungal drugs using broth microdilution to determine the minimum inhibitory concentration (MIC). The morphotyping revealed that all oral C. albicans isolates from children with Down's syndrome promoted the formation of fringes regardless of size, while the control group presented smaller fringes. All oral C. albicans strains produced proteinase, but those with phospholipolytic activity showed greater enzyme capacity in the test group. In vitro susceptibility showed that all oral C. albicans isolates were sensitive to the drugs used.     

Cryptococcus neoformans var. grubii - Pathogenicity of environmental isolates correlated to virulence factors, susceptibility to fluconazole and molecular profile

The pathogenicity of Cryptococcus neoformans is heterogeneous and is associated with the expression of virulence factors. This study aimed to correlate the pathogenicity of C. neoformans var. grubii in BALB/c mice with in vitro virulence factors, fluconazole minimal inhibitory concentrations (MICs) and molecular profiles, before and after animal passage. Ten environmental isolates and one ATCC strain of C. neoformans var. grubii mating type α were evaluated. Most isolates (91%) killed 50% or more of the infected animals by day 24 postinfection and were recovered from the lungs and brains of surviving animals on days 7 and 14 postinfection. The burden of yeast in the lungs was more variable than that in the brain. The differences in the expression of virulence factors (growth at 37oC, presence and size of the capsule and production of melanin, urease, proteinase and phospholipase) by most isolates pre and postpassage in animals were not statistically significant. The fluconazole MICs in postpassaged lines differed by a one-dilution from the MIC of the corresponding prepassaged line for six isolates. Using molecular typing [polymerase chain reaction-fingerprinting with (GACA)4 and M13], eight isolates were identified as VNI and three as VNII. We concluded that different isolates with the same molecular and phenotypic profiles, including isolates that are markedly hypervirulent, span a wide range of virulence and there were no changes in virulence factors in the postpassaged lines when compared with the corresponding nonpassaged lines.     

Effects of Antifungal Agents in Sap Activity of <Emphasis Type="Italic">Candida albicans</Emphasis> Isolates

Some antifungal agents have shown to exert effects on expression of virulent factors of Candida as the production of secretory aspartyl proteinase (Sap). In this study, we sought to determine and to compare the influence of fluconazole and voriconazole in proteinase activity of this microorganism. Thirty-one isolates obtained from oral mucosa of human immunodeficiency virus positive (HIV⁺) patients were used in this study. The minimal inhibitory concentrations (MIC) of fluconazole and voriconazole were determined using the broth microdilution method with RPMI 1640 medium and with yeast carbon base–bovine serum albumin (YCB–BSA) medium. The Sap activity following by digestion of BSA as substrate was determined for four Candida albicans strains arbitrarily chosen according to susceptibility (susceptible or resistant) to fluconazole or voriconazole. Besides, the SAP1 to SAP7 genes were screened by PCR for the same isolates that were determined by the Sap activity. In vitro susceptibility testing using the two media presented similar MIC values. Increased Sap activity was observed in resistant isolates on presence of drugs, but the Sap activity by susceptible isolates to azoles showed different behavior on the presence of drug. We detected the presence of SAP1 to SAP7 genes from all susceptible or resistant C. albicans isolates. The present study provides important data about the proteinase activity and the presence of genes of SAP family in fluconazole and voriconazole susceptible or resistant C. albicans isolates.     

Electrophoretic karyotypes of <Emphasis Type="Italic">C. neoformans</Emphasis> serotype A recovered from Thai Patients with AIDS

Thirty-seven clinical isolates of C. neoformans were recovered from AIDS patients and all were serotype A according to standard typing tests. They were further analyzed using RAPD, PCR fingerprinting, and PFGE along with 2 additional reference isolates ATCC 34871 (serotype A) and RV 45981 (serotype D). Using 2 different RAPD primers, all of the clinical isolates and the reference serotype A (ATCC 34871) gave similar RAPD patterns while serotype D (RV 45981) gave distinctive pattern. Corresponding result was also obtained upon PCR by using a primer for microsatellite (GACA)₄. However, using a primer specific to minisatellite M13 + 1, all PCR fingerprinting gave similar gel patterns (M1) for 35/37 of the clinical isolates and the reference serotype A while two clinical isolates generated different patterns called M2 and M3. The reference serotype D gave distinctive pattern called M4. PFGE gave 17 different karyotypes that could be categorized into 4 groups named EKA (1–6), EKB (1–5), EKC (1– 5) and EKD (1). The reference serotype A fell into group EKA as EKA6 while the reference serotype D fell into group EKC as EKC5. Among the clinical isolates, EKA group (20/37 isolates) and type EKA1 (16/20) dominated with only one isolate each for types EKA2 to EKA5. The next most prevalent was group EKB (12/37 isolates) which dominately fell in type EKB1 (8/12) and only one isolate each for types EKB2 to EKB5. Group EKC (4/37 isolates) and group EKD (1/37) had only one isolate for each type (EKC1 to EKC 4 and EKD1). The 2 predominant karyotypes (EKA1, 16/37 and EKB1, 8/37) may represent two originally common clones of C. neoformans expose among the patients. The high discriminatory power of PFGE infers the benefit of subtyping which lead to better understanding on the epidemiology and pathogenic potential of C. neoformans subtypes. Moreover, PCR fingerprinting and RAPD infer the feasibility of detail analysis between serotypes A and D for unencapsulated C. neoformans.     

Molecular genetic identification of Saccharomyces sensu stricto strains from African sorghum beer

Genetic relationships of 24 phenotypically different strains isolated from sorghum beer in West Africa and the type cultures of the Saccharomyces sensu stricto species were investigated by universally primed polymerase chain reaction (PCR) analysis, microsatellite fingerprinting and PCR-restriction fragment length polymorphism (RFLP) of the ribosomal internal transcribed spacers. The results demonstrate that internal transcribed spacer (ITS) PCR-RFLP analysis with the endonucleases HaeIII, HpaII, ScrFI and TaqI is useful for discriminating S. cerevisiae, S. kudriavzevii, S. mikatae from one another and from the S. bayanus/S. pastorianus and S. cariocanus/S. paradoxus pairs. The sorghum beer strains exhibited the same restriction patterns as the type culture of S. cerevisiae CBS 1171. PCR profiles generated with the microsatellite primer (GTG)(5) and the universal primer N21 were almost identical for all isolates and strain CBS 1171. Despite phenotypic peculiarities, the strains involved in sorghum beer production in Ghana and Burkina Faso belong to S. cerevisiae. However, based on sequencing of the rDNA ITS1 region and Southern hybridisation analysis, these strains represent a divergent population of S. cerevisiae.