Molecular Identification and Antifungal Susceptibility of Clinical Isolates of Sporothrix schenckii Complex in Medellin,Colombia

Background

Sporotrichosis is a subcutaneous mycosis that affects humans and other animals. Infection prevails in tropical and subtropical countries. Until a few years ago, it was considered that two varieties of Sporothrix schenckii caused this mycosis, but by applying molecular taxonomic markers, it has been demonstrated that there are several cryptic species within S. schenckii complex which varies in susceptibility, virulence, and geographic distribution.

Objective

This study aimed to identify the clinical isolates of Sporothrix spp. from patients with sporotrichosis in Medellin, Colombia, using two markers and to evaluate the in vitro susceptibility to itraconazole.

Methods

Thirty-four clinical isolates of Sporothrix spp. from Colombia, three from Mexico, and one from Guatemala were identified through sequencing of the noncoding region ITS-1?+?5.8SDNAr?+?ITS-2 and of the fragment containing exons 3 and 4 of the β-tubulin gene. Clinical isolate sequences were compared with GenBank reference sequences using the BLASTN tool, and then, phylogenetic analysis was performed. Besides, the in vitro susceptibility to itraconazole was evaluated by determining the minimum inhibitory concentrations according to the CLSI M38-A2 method.

Results

Clinical isolates were identified by morphology as Sporothrix spp. Using the molecular markers, ITS and β-tubulin, isolates were identified as S. schenckii sensu stricto (25) and Sporothrix globosa (13). Susceptibility to itraconazole was variable among clinical isolates.

Conclusion

This is the first scientific publication that identifies species that cause sporotrichosis in Colombia, along with the antifungal susceptibility to itraconazole.

     

Isolation and characterization of <Emphasis Type="Italic">Pseudoalteromonas ruthenica</Emphasis> (SBT033), an EPS-producing biofilm bacterium from the seawater intake point of a tropical power station

Biofilm formation in bacteria is closely linked with production of exopolysaccharides (EPS). This study examined the quantitative variations in EPS production and biofilm-forming ability among bacteria isolated from the seawater intake point of a power station located on the east coast of India. Of the 233 isolates obtained from the intake site, 71 bacterial isolates displayed different colony morphological characteristics. Thirteen isolates that produced wide and thick mucoid colonies were further tested for their ability to attach and form biofilms by microtitre plate assay and confocal microscopy. EPS production among the selected bacterial isolates ranged from 826 to 1838 μg ml⁻¹. Strain SBT033, which produced the maximum amount of EPS also displayed the maximum biofilm-forming ability among the 13 isolates. This strain was selected for further characterization using biochemical and molecular methods. The pale orange-pigmented isolate was a Gram negative, aerobic, short rod-shaped and grew well only in the presence of 2% NaCl. On the basis of phenotypic characteristics the isolate SBT033 is shown to belong to the genus Pseudoalteromonas. Analysis of 16S rRNA of the isolate revealed 99% homology with Pseudoalteromonas ruthenica.     

Characterization of a strong CCA-treated wood degrader,unknown <Emphasis Type="Italic">Crustoderma</Emphasis> species

In this study, basidiomycete isolates that possessed a strong ability to degrade chromated copper arsenate (CCA)-treated wood were characterized. These fungal isolates, which were collected from CCA-treated pine log wastes, showed no recognizable morphological properties on culture media. Nucleotide sequence analysis of the large subunit rDNA of the isolates revealed that they were one species. Based on the high sequence similarity (>95%) and close phylogenetic relationship with several known species of Crustoderma, the fungal isolates characterized in this study were classified as a Crustoderma sp. In a wood degradation test, Crustoderma isolate KUC8611 produced a remarkably higher weight loss in CCA-treated Pinus radiata (68.7%), Pseudotsuga menziesii (39.7%), and Tsuga heterophylla (38.5%) wood than other evaluated basidiomycete species, including Crustoderma flavescens and Crustoderma corneum. In addition, extracellular enzymes for cellulose and protein degradation were detected when the isolates were cultured in chromogenic media, which supports the finding that isolate KUC8611 is a wood degrader. Furthermore, an in vitro test for metal tolerance revealed that isolate KUC8611 showed strong arsenic tolerance, but that it could not tolerate copper. Finally, isolate KUC8611 produced lower amounts of oxalic acid than copper-tolerant fungi such as Fomitopsis palustris and Antrodia vaillantii. To the best of our knowledge, this is the first study to report the degradation of CCA-treated wood by a Crustoderma species.     

Chitinolytic Activity of Cold Tolerant Antagonistic Species of <Emphasis Type="Italic">Streptomyces</Emphasis> Isolated from Glacial Sites of Indian Himalaya

Seventy-eight isolates of actinomycetes were isolated from the soil samples collected from alpine zones of Pindari glacier region in Indian Himalaya. Following a plate based rapid screening using two test fungi, five efficient isolates (nos. HA1, HA2, HA6, HA40, and HA142) were selected for further characterization with special reference to their antagonistic properties. Based on phenotypic and genotypic characters, the isolates were identified up to species level. All the isolates belonged to the genus Streptomyces. The isolate nos. HA1 and HA2 were S. sampsonii and HA6, HA40 and HA142 were S. griseobrunneus, S. aurantiacus, and S. griseoluteus, respectively. The isolates showed strong antifungal properties against phytopathogenic test fungi in plate assays. All the isolates hydrolyzed glycol–chitin as a substrate in denaturing conditions showing variable amount of different isoforms.     

Characterization and comparison of toxin‐producing isolates of Dinophysis acuminata from New England and Canada

Following the identification of the first toxic isolate of Dinophysis acuminata from the northwestern Atlantic, we conducted detailed investigations into the morphology, phylogeny, physiology, and toxigenicity of three isolates from three sites within the northeastern U.S./Canada region: Eel Pond and Martha's Vineyard, Massachusetts, and the Bay of Fundy. Another isolate, collected from the Gulf of Mexico, was grown under the same light, temperature, and prey conditions for comparison. Despite observed phenotypic heterogeneity, morphometrics and molecular evidence classified the three northwestern Atlantic isolates as D. acuminata Claparède & Lachmann, whereas the isolate from the Gulf of Mexico was morphologically identified as D. cf. ovum. Physiological and toxin analyses supported these classifications, with the three northwestern Atlantic isolates being more similar to each other with respect to growth rate, toxin profile, and diarrhetic shellfish poisoning (DSP) toxin content (okadaic acid + dinophysistoxin 1/cell) than they were to the isolate from the Gulf of Mexico, which had toxin profiles similar to those published for D. cf. ovum F. Schütt. The DSP toxin content, 0.01–1.8 pg okadaic acid (OA) + dinophysistoxin (DTX1) per cell, of the three northwestern Atlantic isolates was low relative to other D. acuminata strains from elsewhere in the world, consistent with the relative scarcity of shellfish harvesting closures due to DSP toxins in the northeastern U.S. and Canada. If this pattern is repeated with the analyses of more geographically and temporally dispersed isolates from the region, it would appear that the risk of significant DSP toxin outbreaks in the northwestern Atlantic is low to moderate. Finally, the morphological, physiological, and toxicological variability within D. acuminata may reflect spatial (and/or temporal) population structure, and suggests that sub‐specific resolution may be helpful in characterizing bloom dynamics and predicting toxicity.     

Feather degradation by a new keratinolytic <Emphasis Type="Italic">Streptomyces</Emphasis> sp. MS-2

Seventy different actinomycete isolates were evaluated for their ability to produce keratinase using a keratin-salt agar medium containing ball-milled feather as substrate. A novel feather-degrading isolate obtained from marine sediment produced the highest keratinolytic activity when cultured on broth containing whole feather as a primary source of carbon, nitrogen and energy. Based on phenotypic characterization and analysis of 16S rDNA sequencing the isolate was identified as a Streptomyces sp. MS-2. Maximum keratinase activity (11.2 U/mg protein) was achieved when cells were grown on mineral salt liquid medium containing 1% whole chicken feather adjusted to pH 8 and incubated at 35°C for 72 h at 150 rpm. Reduction of disulphide bridges was also detected, increasing with incubation time. Feather degradation led to an increase in free amino acids such as alanine, leucine, valine and isoleucine. Moreover, methionine and phenylalanine were also produced as microbial metabolites.     

<Emphasis Type="Italic">Ophiostoma tsotsi</Emphasis> sp. nov., A Wound-infesting Fungus of Hardwood Trees in Africa

Polymorphic sequence-characterised marker assays from a recent diversity study on the Ascomycete fungus Ophiostoma quercus reported that some isolates from Africa were genetically distinct from O. quercus. In the present study, these African isolates were compared with authentic O. quercus isolates by evaluating morphological characters, growth in culture, mating compatibility and DNA sequence data. The isolates from Africa were morphologically similar to O. quercus, presenting Pesotum and Sporothrix synanamorphs in culture. Phylogenetic analyses of the ribosomal internal transcribed spacer regions 1 and 2, β-tubulin and translation elongation factor 1-α gene regions confirmed that the African group represents a distinct species within the hardwood lineage of the O. piceae complex, closely related to O. ulmi and O. himal-ulmi. Mating studies between O. quercus and the African isolates showed that isolates mated predominantly with those of their own group, although there were rare cases of fertile crosses between the groups. Isolates residing in the African lineage are described here as a new species, O. tsotsi sp. nov.     

New species in the genus <Emphasis Type="Italic">Francisella</Emphasis> (Gammaproteobacteria; Francisellaceae); <Emphasis Type="Italic">Francisella piscicida</Emphasis> sp. nov. isolated from cod (<Emphasis Type="Italic">Gadus morhua</Emphasis>)

A Francisella strain, GM2212, previously isolated from moribund farmed Atlantic cod (Gadus morhua) in Norway, is closely related to Francisella philomiragia among Francisella spp. according to its complete 16S rDNA, 16S-23S intergenic spacer, 23S rDNA, 23S–5S intergenic spacer, 5S rDNA, FopA, lipoprotein TUL4 (LpnA), malate dehydrogenase and hypothetical lipoprotein (LpnB) sequences. A comparison between GM2212 and the type strain of Francisella philomiragia were performed by DNA–DNA hybridization and fatty acid analysis. The DNA–DNA hybridization showed a 70% similarity. The fatty acid analysis showed only minor differences between the Francisella isolates. Due to the inconclusive result from the DNA–DNA hybridisation, major emphasis concerning the status of this isolate is made on previously published molecular, phenotypic and biochemical characters. All characteristics taken together support the establishment of GM2212 as a novel species, for which the name Francisella piscicida sp. nov. is proposed (=CNCM I-3511^T = DSM 18777^T = LMG registration number not yet available).     

Distribution and identification of red yeasts in deep-sea environments around the northwest Pacific Ocean

We isolated 99 yeast strains, including 40 red yeasts, from benthic animals and sediments collected from the deep-sea floor in various areas in the northwest Pacific Ocean. Comparing the yeast isolates from animals and sediments collected from shallow locations, the proportion of red yeasts differed considerably, comprising 81.5% and 10.6% of the isolates from animals and sediments, respectively. All of the red yeast isolates belonged to the genera Rhodotorula and Sporobolomyces. On the basis of morphological and physiological characteristics, the isolates were identified as R. aurantiaca, R. glutinis, R. minuta and R. mucilaginosa of the genus Rhodotorula, and S. salmonicolor and S. shibatanus of the genus Sporobolomyces. Only R. glutinis and R. mucilaginosa were isolated from sediments. All of the others were isolated from animal sources. Phylogenetic analyses based on internal transcribed spacer (ITS) regions and 5.8S rRNA gene sequences allowed us to establish the precise taxonomic placement of each of the isolates and thereby investigate the intraspecific relationships among the isolates. Twenty-two strains identified as members of R. glutinis, which showed a wide distribution in the deep-sea, and five isolates identified as R. minuta, which were isolated only from benthic animals, showed substantial heterogeneity within the species. The isolates phenotypically identified as Sporobolomyces species and R. mucilaginosa phylogenetically occupied the placements corresponding to these species. Some strains assigned to known species on the basis of phenotypic features should be regarded as new species as suggested by the results of molecular analysis.     

Characterization of 1-aminocyclopropane-1-carboxylate deaminase producing methylobacteria from phyllosphere of rice and their role in ethylene regulation

The presence of 1-aminocyclopropane-1-carboxylate deaminase (ACCD) activity among the phyllosphere methylobacteria of rice was detected and its role in regulating plant ethylene level was assessed. Eighteen methylobacterial isolates from four different cultivars of rice were isolated and screened for ACCD. The 16S rRNA homology of ACCD positive methylobacterial isolate closely related to the species Methylobacterium radiotolerans. The accD gene sequence homology of the isolate was 98% similar to Rhizobium leguminosarum. Foliar spray of ACCD positive methylobacterial isolates enhanced the root and shoot length of rice and tomato seedlings under gnotobiotic condition and lower the ethylene level (60–80%) in the plant species.     

Chemical composition and immunological properties of glycoproteins of Sporothrix species

Glycoproteins of 11Sporothrix species were purified from their respective culture filtrates by use of DEAE-Sephadex A-50 and QAE-Sephadex A-25 column chromatography and investigated for their chemical and immunological properties. On the basis of sugar composition, the glycoproteins of the 11Sporothrix species could be divided into two groups, i.e., rhamnose containing (i.e., Rha⁺), and non rhamnose containing (i.e., Rha^–) groups. The species in the former group wereS. curviconia, S. inflata, S. schenckii andS. schenckii var. luriei, and those in the latter group wereS. cyanescens, S. foliorum, S. fungorum, S. ghanensis, S. imectorum, S. luteoalba andS. ramosissima. The glycoproteins of four of the (Rha⁺) species were relatively similar in elution patterns of DEAE-Sephadex A-50 chromatograms, sugar and amino acid compositions, serological reactivity with rabbit andS. schenckii serum and rabbit antiKlebsiella pneumoniae K47 serum, and cutaneous delayed hypersensitivity. In the case of the (Rha^–) species, the glycoproteins of five species cross-reacted with rabbit antiS. schenckii serum and all, but theS. cyanescens, glycoprotein were reactive to some degree in skin tests in sporotrichotic patients. These results strongly suggest that the chemical and immunological properties of these glycoproteins correspond with the morphological observations amongSporothrix species.     

Multiple antibiotic resistances of <Emphasis Type="Italic">Enterococcus</Emphasis> isolates from raw or sand-filtered sewage

Fifty antibiotic-resistant Enterococcus strains were isolated from raw sewage of a wastewater treatment plant and from the same sewage after trickling through a 25-cm sand column, which retained >99% of the initial population. All 50 Enterococcus isolates were resistant against triple sulfa and trimethoprim/sulfamethoxazole and none were resistant against vancomycin. Most of the isolates from raw sewage were resistant to more antibiotics than the isolates from sand column effluent. One Enterococcus isolate from raw sewage (no. 61) and one Enterococcus isolate from sand column effluent (no. 95) had ten antibiotic resistances each. Isolate no. 95 maintained its resistances in the absence of antibiotics during the whole study. It was compared with isolate no. 70, which was one of the isolates, being resistant only against the two sulfonamides. Phenotypically and biochemically, the two organisms were strains of Enterococcus faecalis. Sequence analysis of partical 16S rDNA allowed alignment of isolate no. 95 as a strain of Enterococcus faecium and of isolate no. 70 as a strain of E. faecalis. E. faecium strain no. 95 carried at least six different plasmids, whereas for E. faecalis strain no. 70, no discrete plasmid band was seen on the gels.     

Aggressiveness and Genetic Diversity of Hemileia vastatrix During an Epidemic in Colombia

Since 2008, Colombia has been experiencing an epidemic of the coffee rust Hemileia vastatrix. The altitude range of the disease has expanded, and nursery and young plants that were usually not attacked by the disease are now significantly affected. To determine whether this new epidemic has been caused by a new pathogenic isolate, the molecular diversity of the pathogen causing the epidemic in different regions of the country was assessed, using AFLP molecular markers on isolates collected from coffee fields prior and after the year 2008. We also evaluated the aggressiveness of isolates collected from diverse coffee‐producing areas and from different coffee genotypes. Isolates collected before and during the present epidemic were quite similar both genetically and with regard to their aggressiveness. Out of a total of 349 fragments amplified from 6 AFLP primer combinations, 48 (13.2%) were polymorphic and only 18 were unique among H. vastatrix isolates representative of pre‐2008 and post‐2008 epidemic populations. We conclude that the epidemic was caused by the excessive rainfall that has occurred in Colombia since 2006 and that extended to 2011 and not by the arrival of a new isolate of the pathogen or a change in virulence of the species present in the country.     

Isolation and characterization of polyphenol oxidase- and peroxidase-producing <Emphasis Type="Italic">Bacillus</Emphasis> strains from fully fermented tea (<Emphasis Type="Italic">Camellia sinensis</Emphasis>)

Sixteen aerobic endospore-forming Bacillus spp. were isolated from fully fermented tea leaf samples from 10 tea factories in Lahijan and Langrod cities (Gillan province, Iran). Bacillus spp. isolates were characterized using phenotypic characteristics, antibiotic susceptibility and cellular fatty acid (CFA) patterns. Based on the data obtained, five isolates of tea Bacillus spp. (TB): TB2, TB4, TB6, TB10 and TB12 belonged to the species B. subtilis. Two isolates, TB1 and TB14 were recognized as B. licheniformis. Two Bacillus spp. isolates, TB9 and TB 16 were identified as B. sphaericus. Two isolates, TB5 and TB13 were shown to be B. pumilus. Two isolates, TB7 and TB15 belonged to B. cereus. Amongst the isolates, Bacillus sp. TB3, Bacillus sp. TB8 and Bacillus sp. TB11 showed different phenotypic traits, distinct antibiotic sensitivity and fatty acid profiles, and they may represent novel species. The isolates showed polyphenol oxidase (tyrosinase) and peroxidase activities. The highest polyphenol oxidase and peroxidase activities were observed for Bacillus sp. TB3 and B. licheniformis TB14, respectively, where values of 5.48 and 3.73 units mL⁻¹ were observed.     

Differentiation of α-hemolytic streptococci by direct mass spectrometric profiling

Modern phenotypic and genetic methods (with the exception of multilocus sequence typing) do not provide a reliable differentiation of α-hemolytic streptococci of the Mitis group. In this study, we obtained MALDI mass spectra for 28 clinical isolates that were initially identified by standard methods as Streptococcus pheumoniae. The isolate distribution that was obtained in the course of clustering correlated well with the data on multilocus typing. These studied isolates were attributed by this typing to two closely related species: S. pneumoniae and S. mitis. A classification model that differentiated α-hemolytic streptococci with 100% sensitivity and 94.6% specificity was developed on the basis of a genetic algorithm. Two peaks that were different for the S. mitis and S. pneumoniae isolates were revealed by the analysis of statistical diagrams of peak areas.     

Phylogenetic analysis of Methanobrevibacter isolated from feces of humans and other animals

Comparative 16S rRNA gene sequence and genomic DNA reassociation analyses were used to assess the phylogenetic relationships of Methanobrevibacter fecal isolates. The 16S rRNA gene sequences of Methanobrevibacter smithii strain PS and the human fecal isolates B181 and ALI were essentially identical, and their genomic DNA reassociated at values greater than 94%. The analysis of 16S rRNA sequences of the horse, pig, cow, rat, and goose fecal isolates confirm that they are members of the genus Methanobrevibacter. They had a high degree of sequence similarity (97–98%) with the 16S rRNA gene of M. smithii, indicating that they share a common line of descent. The 16S rRNA genes of the horse and pig isolates had 99.3% sequence similarity. Sequence analysis of the 16S rRNA gene of the sheep fecal isolate showed that it formed a separate line of descent in the genus Methanobrevibacter. Genomic DNA reassociation studies indicate that the horse, pig, cow, and goose fecal isolates represent at least three new species. The horse and pig isolates were the only animal isolates that had > 70% genomic DNA reassociation and represent strains of a single species. The cow, goose, and sheep isolates had little or no genomic DNA reassociation with M. smithii or with each other. The relationship of the rat isolate to the other animal isolates was not determined. An evaluation of the relationship of 16S rRNA gene sequence similarity and genomic DNA reassociation of Methanobrevibacter and other methanogenic archaea indicated that genomic DNA reassociation studies are necessary to establish that two methanogenic organisms belong to the same species. Received: 17 November 1997 / Accepted: 16 January 1998     

Identification of Fusarium species causing basal rot of onion in East Azarbaijan province,Iran and evaluation of their virulence on onion bulbs and seedlings

One of the economically important diseases of onion is the basal rot caused by various Fusarium species. Identification of the pathogenic species prevalent in a region is indispensable for designing management strategies, especially to develop resistant cultivars. Eighty Fusarium isolates are obtained from red onion bulbs on infected fields of East Azarbaijan province. Inoculating the onion bulbs with 38 selective isolates indicated that 17 isolates were pathogenic on onion. According to the morphological and molecular characteristics, these isolates were identified as F. oxysporum, F. solani, F. proliferatum and F. redolens. This is the first report of F. redolens on onion in Iran. On the other hand, the virulence of each pathogenic isolate was evaluated on onion bulbs and seedlings. F. oxysporum which causes severe rot and damping-off was considered as a highly virulent species in both conditions. While, F. proliferatum was considered as the most destructive on onion bulbs. Rot ability of F. solani was not considerable, and only the 4S isolate caused pre- and post-emergence damping-off more than 50%. Finally, F. redolens with less pathogenicity on onion bulbs was identified as the most virulent isolate on onion seedlings, which was explanatory of its importance on farm.