Antifungal susceptibilities of Sporothrix albicans, S. brasiliensis, and S. luriei of the S. schenckii complex identified in Brazil

We studied 40 strains of the species complex formerly classified as the single species Sporothrix schenckii to identify new species within this complex and evaluate their antifungal susceptibility profiles. Based on phenotypic tests (ability to grow at 37°C, colony diameters, and pigmentation of the colonies, as well as assimilation of sucrose and raffinose) and molecular assays (amplification of a fragment of the calmodulin gene), here we report the identification of S. albicans, S. brasiliensis, S. luriei, and S. schenckii; two isolates of these species were detected as itraconazole-resistant strains.     

Microsatellite analysis of three phylogenetic species of Paracoccidioides brasiliensis

Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis, an important human systemic mycosis in Latin America. Recently, the existence of three different phylogenetic species (S1, PS2, and PS3) of P. brasiliensis was demonstrated. Despite being genetically isolated, all three species were capable of inducing disease in both humans and animals, although lower virulence has been found with the PS2 species. The available molecular methods developed to characterize and type strains have not been useful for assigning isolates to the described species, creating the need for molecular markers capable of distinguishing genetically isolated groups. Here, we describe a PCR and sequencing-based microsatellite marker system that is stable, easy to assay, adaptable to large series of isolates, and discriminatory enough to be used as a typing system in identifying the three proposed species of P. brasiliensis. In addition, this system provides an unambiguous tool for strain discrimination between two (S1 and PS2) of the three phylogenetic species.     

Molecular Identification and Phenotypic Characterisation of Sporothrix globosa from Clinical Cases of Eastern Assam,North-east India

Sporotrichosis is known to be endemic in the state of Assam, North-east India, which is situated in the Sub-Himalayan region. This disease is an acute or chronic infection caused by Sporothrix schenckii species complex which currently includes several species of clinical relevance such as Sporothrix brasiliensis, Sporothrix globosa, Sporothrix schenckii sensu stricto, Sporothrix albicans, Sporothrix mexicana, Sporothrix pallida and Sporothrix luriei. S. globosa is the prevalent species in India. Eight culture-positive patients were diagnosed from suspected consecutive cases of two lymphocutaneous and six fixed cutaneous forms over a period of 4 years in a clinical mycology laboratory of a tertiary care centre in Eastern Assam. Phenotypic speciation was inconclusive using the criteria of Marimon et al. because of atypical growth pattern shown by the isolates. Our isolates showed good growth at 37°C ranging from 6 to 27 mm; four of the isolates showed growth of 11–27 mm unlike S. globosa strains reported earlier. Molecular identification was done by sequencing both the internal transcribed spacer (ITS) region and the calmodulin (CAL) protein encoding gene (partial). All the isolates were identified as S. globosa. Molecular confirmation of species using ITS region and CAL protein encoding gene (partial) is necessary for isolates of S. globosa showing atypical biopatterns.     

Serological cross-reactivity between group B Streptococcus and Sporothrix schenckii, Ceratocystis species, and Graphium species.

Serological cross-reactivity of a group B Streptococcus (H36B) with Sporothrix schenckii and 39 different Ceratocystis and Graphium species was investigated by double immunodiffusion. Rabbit anti-H36B serum reacted with antigens from S. schenckii and from 36 of 39 Ceratocystis and Graphium species. It is speculated that low-titer agglutinins to S. schenckii in normal sera are due to antibodies raised against various bacteria which share common antigens with S. schenckii.     

Serological cross-reactivity among Sporothrix schenckii, Ceratocystis, Europhium, and Graphium species.

Ethanol-precipitable culture filtrate antigens of 100 strains of 75 species of the Sporothrix-Ceratocystis-Europhium-Graphium complex and 1 species of Botrytis were examined for neutral sugar components and for serological cross-reactivity with S. schenckii rabbit antiserum and human sporotrichosis sera by capillary precipitin and double immunodiffusion assay. Results revealed that cross-reactive species (60 of 77, ca. 80%) produced exoconidial forms and rhamnose- and mannose-containing polysaccharides and included Ceratocystis, the three known Europhium, and several Graphium-form species. Endoconidial-form Ceratocystis species did not cross-react.     

Molecular and morphological identification of Colletotrichum species of clinical interest

Colletotrichum species have caused human infections in recent years. Because of the difficulties in recognizing them in vitro, we have designed a quick and unambiguous molecular test, based on the amplification of a specific fragment of the internal transcribed spacer 1 region, to distinguish any Colletotrichum isolate from other fungi, including the common pathogenic species. Analysis of the sequences of the ribosomal DNA (rDNA) fragment showed sufficient variability to clearly separate the five species of Colletotrichum that are of clinical interest, i.e., Colletotrichum coccodes, C. crassipes, C. dematium, C. gloeosporioides, and C. graminicola. Sequencing of the D1-D2 region of the large-subunit rDNA gene also supported these results. Additionally, we reviewed the most suitable morphological characteristics for the in vitro identification of these increasingly important opportunistic fungi.     

Pasteurella caballi, a new species from equine clinical specimens.

The name Pasteurella caballi is proposed for a group of organisms represented by 29 strains isolated from respiratory and other infections in horses. P. caballi strains are gram-negative, oxidase-positive, nonmotile, fermentative rods with the key characteristics of the genus Pasteurella. These strains differed from other Pasteurella species in that all were aerogenic and catalase negative, and some strains produced acid from myo-inositol and L-rhamnose. The levels of DNA relatedness of 28 P. caballi strains with labeled DNA from the proposed type strain averaged 91 and 85% (hydroxyapatite method at 55 and 70 degrees C). P. caballi was 13 to 53% related to strains representing 22 other species of the family Pasteurellaceae. The guanine-plus-cytosine content of the DNA of four strains was 41 to 42 mol%. The type strain is 83851 (=ATCC 49197).     

Isolation and characterization of Sporothrix schenckii from clinical and environmental sources associated with the largest U.S. epidemic of sporotrichosis.

The largest recorded epidemic of sporotrichosis in the United States occurred in 1988 and involved a total of 84 cases in 15 states. All cases were associated with Wisconsin-grown sphagnum moss. Twenty-one clinical isolates of Sporothrix schenckii and 69 environmental isolates of Sporothrix spp. from the epidemic were characterized and compared. The environmental isolates were recovered from 102 samples of sphagnum moss and other material by using direct plating techniques. Characteristics examined included macroscopic and microscopic morphology, conversion to a yeast phase, exoantigen reactions, and virulence in mice. On the basis of these studies, eight environmental isolates were identified as S. schenckii, five were identified as Ophiostoma stenoceras, and the remainder were identified as Sporothrix species. The environmental isolates of S. schenckii were recovered from moss samples from one Pennsylvania nursery and from three New York State Soil and Water Conservation districts, but none were recovered from moss directly from the bogs in Wisconsin.     

Relationships among genotypes, virulence and clinical forms of Sporothrix schenckii infection

This study explored the relationships among genotypes, virulence and clinical forms of Sporothrix schenckii. Genomic DNA from isolates of S. schenckii, collected from different clinical forms of sporotrichosis, was amplified by randomly amplified polymorphic DNA (RAPD). Suspensions of different isolates of S. schenckii were inoculated into healthy BALB/c mice to compare their virulence, and the numbers and distribution of spores were determined by histological analysis. RAPD analysis indicated that the isolates from different clinical forms of sporotrichosis belonged to different genotypes. The mice inoculated with isolates from disseminated sporotrichosis showed an earlier onset of illness and more severe lesions than those inoculated with isolates from lymphocutaneous sporotrichosis, which, in turn, showed an earlier onset of illness and more severe lesions than those inoculated with isolates from fixed cutaneous sporotrichosis. Healthy BALB/c mice injected with isolates from disseminated sporotrichosis died within 10 days, whereas isolates from lymphocutaneous sporotrichosis and fixed cutaneous sporotrichosis failed to cause death. Histologically, mice inoculated with isolates from disseminated sporotrichosis had more spores than those inoculated with isolates from lymphocutaneous sporotrichosis and fixed cutaneous sporotrichosis. Thus, different genotypes may be associated closely with the virulence of different clinical forms of S. schenckii infection.     

Delayed hypersensitivity cross-reactions between Sporothrix schenckii and Ceratocystis species in sporotrichotic patients.

Cutaneous delayed hypersensitivity to antigens prepared from Sporothrix schenckii and several Ceratocystis species, including C. stenoceras, C. ulmi, C. ips, and C. minor, was tested in 14 patients with known cutaneous sporotrichosis. Extensive cross-reactions were observed. Nonsporotrichotic people (controls) did not react to these antigens. The correlation coefficient between antigens of S. schenckii and each Ceratocystis species was calculated from the areas of the cutaneous reactions. Among the Ceratocystis species tested, the correlation coefficient between S. schenckii and C. stenoceras was 0.91.     

Aeromonas schubertii, a new mannitol-negative species found in human clinical specimens.

In 1983 the vernacular name Enteric Group 501 was coined for a group of strains that had been referred to our laboratory as "possible Vibrio damsela that does not require NaCl for growth." By DNA-DNA hybridization (hydroxyapatite method, 32P, 60 and 75 degrees C), six strains of Enteric Group 501 were closely related to the labeled strain 2446-81 (70 to 95% at 60 degrees C and 71 to 93% at 75 degrees C; 0 to 1% divergence). Type strains of all Aeromonas species and reference strains of six other Aeromonas DNA hybridization groups were 26 to 42% related (60 degrees C) to strain 2446-81, but type strains of 27 Vibrio and Photobacterium species, including V. damsela, were 0 to 1% (75 degrees C) related. We propose the name Aeromonas schubertii for the highly related group of seven strains formerly known as Enteric Group 501. The type strain is designated as ATCC 43700 (CDC 2446-81). Strains of A. schubertii grew well at 36 degrees C and had positive reactions at this temperature for methyl red, Voges-Proskauer (1% NaCl, Coblentz method), lysine decarboxylase, arginine dihydrolase, motility, lipase, DNase, nitrate reduction to nitrite, oxidase, and growth in nutrient broth with 0 and 1% NaCl. There was no growth in 6% NaCl or on thiosulfate-citrate-bile salts-sucrose agar. The following sugars were fermented: D-glucose, D-galactose, maltose, D-mannose, and trehalose. The following sugars were not fermented: adonitol, L-arabinose, D-arabitol, cellobiose, dulcitol, erythritol, myo-inositol, lactose, D-mannitol, melibiose, alpha-CH3-D-glucoside, raffinose, L-rhamnose, salicin, D-sorbitol, sucrose, and D-xylose. Esculin was not hydrolyzed, and the string test was negative. The mannitol-negative reaction differtiates A. schubertii from other Aeromonas species. The antibiogram of this organism is typical of other Aeromonas strains (resistance to ampicillin and carbenicillin and susceptibility to most other agents). A. schubertii strains have been isolated from abscesses (two strains), wound (one), skin (one), pleural fluid (one), and blood (two). The two blood isolates suggest clinical significance typical of other Aeromonas species , but further information is needed on this group.     

Use of a mouse model to evaluate clinical and environmental isolates of Sporothrix spp. from the largest U.S. epidemic of sporotrichosis.

Five clinical and 69 environmental isolates from the largest U.S. epidemic of sporotrichosis were evaluated in NYLAR male mice following intravenous injection of 5 x 10(6) to 2 x 10(8) conidia per mouse. The clinical isolates and eight environmental isolates produced 100% mortality in groups of three mice each between 12 and 24 days after injection. These virulent isolates grew at 37 degrees C, were dematiaceous by virtue of melanin (melanized) on permissive media (e.g., potato dextrose agar), produced ovoid conidia borne sympodially on lateral conidiophores and pleurogenously about the main hyphal axis, and were identified as Sporothrix schenckii. Two melanized environmental isolates that grew at 35 degrees C but not at 37 degrees C were not virulent and had subtle morphological differences from S. schenckii. The remaining environmental isolates were not melanized, were not virulent, and were not S. schenckii; five were identified as Ophiostoma stenoceras and the remainder were identified as Sporothrix spp. Quantitative organ cultures revealed that clinical isolates grew exponentially in livers and testes, in contrast to an isolate of O. stenoceras that was eliminated from liver, lung, and spleen but that persisted in the testes throughout the 14-day sample period. This model helped to confirm the identification of S. schenckii isolates obtained from the environment.     

Isolation and analysis of a new developmentally regulated gene from amastigotes of Leishmania mexicana mexicana

Leishmania differentiates from the promastigote to the amastigote stage during its digenetic life cycle. Characterization of the developmentally regulated genes during that process would help to elucidate the mechanisms of gene regulation. In this study, specific fragments of mRNAs from the amastigote stage of L. mexicana mexicana were discriminated from those of the promastigote and metacyclic stages by differential display. This technique combined with spliced-leader polymerase chain reaction allowed isolation of the complete gene VG7A5. The sequence of this gene did not align with any published L. mexicana sequence. More than one copy of this gene was identified in the genome by Southern-blot analysis and was transcribed exclusively in the amastigote stage. At 20 bp upstream from the splice AG site it has a trans-splicing polypyrimidine tract. The gene encodes the subcellular localization motifs 5′-GGACT and AAGCT-3′ in the 3′ untranslated region of the mRNA. The open reading frame of the gene VG7A5 predicts a polypeptide of 587 amino acid residues that has a KGRR amidation motif near its carboxyl terminus, suggesting that in the mammalian host this protein may be involved in the process of acute inflammation. Received: 24 June 1999 / Accepted: 10 September 1999     

Escherichia fergusonii and Enterobacter taylorae, two new species of Enterobacteriaceae isolated from clinical specimens.

Escherichia fergusonii (formerly known as Enteric Group 10) and Enterobacter taylorae (formerly known as Enteric Group 19) are proposed as new species in the family Enterobacteriaceae. By DNA hybridization (32P, 60 degrees C, hydroxyapatite), strains of E. fergusonii were 90 to 97% related to the type strain (holotype) ATCC 35469. They were most closely related to Escherichia coli and more distantly related to species in other genera. E. fergusonii strains are positive for indole production, methyl red, lysine decarboxylase, ornithine decarboxylase, and motility. They ferment D-glucose with gas production and also ferment adonitol, L-arabinose, L-rhamnose, maltose, D-xylose, trehalose, cellobiose, and D-arabitol. They are negative for Voges-Proskauer, citrate utilization (17% positive), urea hydrolysis, phenylalanine deamination, arginine dihydrolase, growth in KCN, and fermentation of lactose, sucrose, myo-inositol, D-sorbitol, raffinose, and alpha-methyl-D-glucoside. By DNA hybridization (32P, 60 degrees C, hydroxyapatite), strains of E. taylorae were 84 to 95% related to the type strain (holotype) ATCC 35317. Their nearest relative was E. cloacae, to which they were 61% related. Other named species were more distantly related. Strains of E. taylorae are positive for Voges-Proskauer, citrate utilization, arginine dihydrolase, ornithine decarboxylase, motility, growth in KCN medium, and malonate utilization. They ferment D-glucose with gas production and also ferment D-mannitol, L-arabinose, L-rhamnose, maltose, D-xylose, trehalose, and cellobiose. They are negative for indole production, methyl red, H2S production on triple sugar-iron agar, urea hydrolysis, phenylalanine deamination, lysine decarboxylase, gelatin hydrolysis, and fermentation of adonitol, i-inositol, D-sorbitol, and raffinose. Both new species occur in human clinical specimens. Two strains of E. fergusonii were isolated from blood. Five stains of E. taylorae were isolated from blood, and one was from spinal fluid. These blood and spinal fluid isolates suggest possible clinical significance, but this point requires further study.     

Macrorestriction analysis of clinical and environmental isolates of Sporothrix schenckii

Sporothrix schenckii causes sporotrichosis, a disease that most commonly presents as a subacute or chronic skin infection. An unusually high incidence of clinical cases of sporotrichosis occurred in the southwest of Western Australia over the last 5 years. Anecdotal accounts from patients implicated contact with hay prior to infection. Isolates of S. schenckii from hay and clinical cases were investigated by traditional phenotypic methods and pulsed-field gel electrophoresis (PFGE). The phenotypic evaluation separated S. schenckii from Ophiostoma spp. A DNA macrorestriction method using SfiI and NotI macrorestriction digestion by PFGE was developed to investigate the epidemiological connections. BioNumerics software was used to analyze the results. DNA macrorestriction digestion patterns for the recent Western Australian clinical isolates and four hay isolates were indistinguishable. Eastern state clinical isolates, national Quality Assurance Program isolates, and other environmental isolates gave different macrorestriction patterns. Clinical isolates from the southwest of Western Australia collected in the 1980s and 1990s were also characterized using PFGE. The patterns generated were indistinguishable from those of the recent clinical isolates. PFGE showed that the dominant strain of S. schenckii causing sporotrichosis in Western Australia is present in hay, has caused sporotrichosis for at least 15 years, and is a different strain from the strains found in other parts of Australia.     

Sporotrichosis caused by Sporothrix brasiliensis in Argentina: Case report,molecular identification and in vitro susceptibility pattern to antifungal drugs

Sporotrichosis is considered a neglected disease of humans and animals in many regions of the world and is the most frequent implantation mycosis in Latin America.ObjectivesTo illustrate the zoonotic importance of the disease, describing a case involving a veterinarian and an infant that acquired the disease from a domestic cat and to describe, genotype and characterize these new isolates.MethodsDirect examination of tissue samples from the two patients and feline lesions revealed the presence of Sporothrix yeast-like organisms. Fungal cultures and molecular identification of the strains were performed. Since antifungal susceptibility data of animal-borne isolates are scarce, the in vitro susceptibility testing by a microdilution reference method was determined against azoles, amphotericin B and terbinafine.ResultsFungal culture and sequence analysis of the ITS region of rDNA and calmodulin and β-tubulin genes confirmed the diagnosis and the causative agent as Sporothrix brasiliensis. In all cases, terbinafine was the most active drug, followed by posaconazole, itraconazole and voriconazole; the least active drugs were amphotericine B and fluconazole. Lack of clinical response in the veterinarian and in the infant to itraconazole and potassium iodide, respectively was observed.ConclusionsThis study contributed to the molecular epidemiology of Sporothrix species in Argentina and the characterization of the in vitro susceptibility pattern of S. brasiliensis isolates recovered from a cat and two humans involved in this case of zoonotic sporotrichosis. Bearing in mind the “One Health” concept, the experience described in the present study highlights the need for future strategies for sporotrichosis treatment, control and prevention.     

Taxonomy, biology, and clinical aspects of Fusarium species.

There are several taxonomic systems available for identifying Fusarium species. The philosophy used in each taxonomic system is discussed as well as problems encountered in working with Fusarium species in culture. Fusarium species are toxigenic, and the mycotoxins produced by these organisms are often associated with animal and human diseases. The implications for the association of the carcinogens, fumonisins, produced by Fusarium moniliforme and other Fusarium species with human diseases are discussed. Foreign-body-associated fusarial infection such as keratitis in contact lens wearers, onychomycosis, skin infections, and disseminated multiorgan infections are discussed. Disseminated fusarial hyalohyphomycosis has emerged as a significant, usually fatal infection in the immunocompromised host. Successful outcome is determined by the degree of immunosuppression, the extent of the infection, and the presence of a removable focus such as an indwelling central venous catheter. These infections may be clinically suspected on the basis of a constellation of clinical and laboratory findings, which should lead to prompt therapy, probably with one of the newer antifungal agents. Perhaps the use of such agents or the use of colony-stimulating factors may improve the outcome of this devastating infection. However, until new approaches for treatment develop, effective preventive measures are urgently needed.     

Characterization of three new enterococcal species, Enterococcus sp. nov. CDC PNS-E1, Enterococcus sp. nov. CDC PNS-E2, and Enterococcus sp. nov. CDC PNS-E3, isolated from human clinical specimens

As a reference laboratory, the Streptococcus Laboratory at the Centers for Disease Control and Prevention (CDC) is frequently asked to confirm the identity of unusual or difficult-to-identify catalase-negative, gram-positive cocci. In order to accomplish the precise identification of these microorganisms, we have systematically applied analysis of whole-cell protein profiles (WCPP) and DNA-DNA reassociation experiments, in conjunction with conventional physiological tests. Using this approach, we recently focused on the characterization of three strains resembling the physiological groups I (strain SS-1730), II (strain SS-1729), and IV (strain SS-1728) of enterococcal species. Two strains were isolated from human blood, and one was isolated from human brain tissue. The results of physiological testing were not consistent enough to allow confident inclusion of the strains in any of the known enterococcal species. Resistance to vancomycin was detected in one of the strains (SS-1729). Analysis of WCPP showed unique profiles for each strain, which were not similar to the profiles of any previously described Enterococcus species. 16S ribosomal DNA (rDNA) sequencing results revealed three new taxa within the genus ENTEROCOCCUS: The results of DNA-DNA relatedness experiments were consistent with the results of WCPP analysis and 16S rDNA sequencing, since the percentages of homology with all 25 known species of Enterococcus were lower than 70%. Overall, the results indicate that these three strains constitute three new species of Enterococcus identified from human clinical sources, including one that harbors the vanA gene. The isolates were provisionally designated Enterococcus sp. nov. CDC Proposed New Species of Enterococcus 1 (CDC PNS-E1), type strain SS-1728(T) (= ATCC BAA-780(T) = CCUG 47860(T)); Enterococcus sp. nov. CDC PNS-E2, type strain SS-1729(T) (= ATCC BAA-781(T) = CCUG 47861(T)); and Enterococcus sp. nov. CDC PNS-E3, type strain SS-1730(T) (= ATCC BAA-782(T) = CCUG 47862(T)).     

Sporothrix schenckii sensitivity to voriconazole, itraconazole and amphotericin B.

One hundred clinical isolates of Sporothrix schenckii were tested against voriconazole, itraconazole and amphotericin B using a modification of the NCCLS M27-A in vitro yeast susceptibility testing procedure. NCCLS M38-P for moulds was not used because yeast forms may have been present when the test isolates were incubated at 35 +/- 1 degrees C. The minimum inhibitory concentration (MIC) values were: voriconazole 0.5-8 (geometric mean titer 6.50) microg ml(-1) ; itraconazole 0.03-8 (geometric mean titer 1.56) microg ml(-1); and amphotericin B 0.25-2 (geometric mean titer 1.23) microg ml(-1). The minimum fungicidal concentration (MFC) values were: voriconazole 2-8 (geometric mean titer 7.67) microg ml(-1); itraconazole 0.125-8 (geometric mean titer 7.41) microg ml(-1); and amphotericin B 0.125-2 (geometric mean titer 1.53) microg ml(-1). Based upon MIC values, sensitivity to amphotericin B is strain-dependent. S. schenckii is more sensitive to itraconazole than voriconazole based upon a comparison of MIC geometric means, even though the MIC ranges were essentially the same.