Isolation of <Emphasis Type="Italic">Cryptococcus gattii</Emphasis> from a <Emphasis Type="Italic">Castanopsis argyrophylla</Emphasis> tree hollow (Mai-Kaw), Chiang Mai,Thailand

The pathogenic yeast Cryptococcus gattii was isolated from a tree hollow of a Castanopsis argyrophylla King ex Hook.f. (Fagaceae) in Chiang Mai, Thailand. Molecular characterization with amplified fragment length polymorphism analysis and multi-locus sequence typing showed that this isolate belonged to genotype AFLP4/VGI representing C. gattii sensu stricto. Subsequent comparison of the environmental isolate with those from clinical samples from Thailand showed that they grouped closely together in a single cluster.     

Antifungal Activity of Chitosan-Coated Poly(lactic-co-glycolic) Acid Nanoparticles Containing Amphotericin B

Amphotericin B (AmB) is one of the most used drugs for the treatment of systemic fungal infections; however, the treatment causes several toxic manifestations, including nephrotoxicity and hemolytic anemia. Chitosan-coated poly(lactide-co-glycolide) (PLGA) nanoparticles containing AmB were developed with the aim to decrease AmB toxicity and propose the oral route for AmB delivery. In this work, the antifungal efficacy of chitosan-coated PLGA nanoparticles containing AmB was evaluated in 20 strains of fungus isolates from patients with vulvovaginal candidiasis (01 Candida glabrata and 03 Candida albicans), bloodstream infections (04 C. albicans and 01 C. tropicalis) and patients with urinary tract infection (04 Candida albicans, 02 Trichosporon asahii, 01 C. guilhermondii, 03 C. glabrata) and 01 Candida albicans ATCC 90028. Moreover, the cytotoxicity over erythrocytes was evaluated. The single-emulsion solvent evaporation method was suitable for obtaining chitosan-coated PGLA nanoparticles containing AmB. Nanoparticles were spherical in shape, presented mean particle size about 460 nm, positive zeta potential and encapsulation efficiency of 42%. Moreover, nanoparticles prolonged the AmB release. All the strains were susceptible to plain AmB and nanostructured AmB, according to EUCAST breakpoint version 8.1 (resistant > 1 μg/mL), using broth microdilution method. In C. albicans (urine, blood, and vulvovaginal secretion isolates, and 1 ATCC), the MIC value of AmB-loaded nanoparticles varied from 0.25 to 0.5 μg/mL and EUCAST varied from 0.03 to 0.5 μg/mL. In urine and vulvovaginal secretion isolates of C. glabrata, the MIC value of AmB-loaded nanoparticles varied from 0.25 to 0.5 μg/mL and EUCAST varied from 0.03 to 0.015 μg/mL. In urine isolates of C. guilhermondii, the MIC value of AmB-loaded nanoparticles was 0.12 μg/mL and EUCAST was 0.06 μg/mL. In blood isolates of C. tropicalis, the MIC value of AmB-loaded nanoparticles was 0.5 μg/mL and EUCAST was 0.25 μg/mL. Finally, in urine isolates of T asahii, the MIC value of AmB-loaded nanoparticles was 1 μg/mL and EUCAST varied from 0.5 to 1 μg/mL. In the cytotoxicity assay, plain AmB was highly hemolytic (100% in 24 h) while AmB-loaded chitosan/PLGA nanoparticles presented negligible hemolysis.     

Extended spectrum beta-lactamase and metallo beta-lactamase production among <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> isolated from different clinical samples in a tertiary care hospital in Kathmandu,Nepal

Background

Extended spectrum beta-lactamase (ESBL) and metallo beta-lactamase (MBL) production in Klebsiella pneumoniae and Escherichia coli are the commonest modes of drug resistance among these commonly isolated bacteria from clinical specimens. So the main purpose of our study was to determine the burden of ESBL and MBL production in E. coli and K. pneumoniae isolated from clinical samples. Further, the antimicrobial susceptibility patterns of E. coli and K. pneumoniae were also determined.

Methods

A cross-sectional study was conducted at Om Hospital and Research Centre, Kathmandu, Nepal by using the E. coli and K. pneumoniae isolated from different clinical samples (urine, pus, body fluids, sputum, blood) from May 2015 to December 2015. Antimicrobial susceptibility testing was performed by Kirby-Bauer disc diffusion technique. Extended spectrum beta-lactamase production was detected by combined disc method using ceftazidime and ceftazidime/clavulanic acid discs and cefotaxime and cefotaxime/clavulanic acid discs. Similarly, metallo beta-lactamase production was detected by combined disc assay using imipenem and imipenem/ethylenediaminetetracetate discs. Bacteria showing resistance to at least three different classes of antibiotics were considered multidrug resistant (MDR).

Results

Of total 1568 different clinical samples processed, 268 (17.1%) samples were culture positive. Among which, E. coli and K. pneumoniae were isolated from 138 (51.5%) and 39 (14.6%) samples respectively. Of the total isolates 61 (34.5%) were ESBL producers and 7 (4%) isolates were found to be MBL producers. High rates of ESBL production (35.9%) was noted among the clinical isolates from outpatients, however no MBL producing strains were isolated from outpatients. Among 138 E. coli and 39 K. pneumoniae, 73 (52.9%) E. coli and 23 (59%) K. pneumoniae were multidrug resistant. The lowest rates of resistance was seen toward imipenem followed by piperacillin/tazobactam, amikacin and cefoperazone/sulbactam.

Conclusions

High rate of ESBL production was found in the E. coli and K. pneumoniae isolated from outpatients suggesting the dissemination of ESBL producing isolates in community. This is very serious issue and can’t be neglected. Regular monitoring of rates of ESBL and MBL production along with multidrug resistance among clinical isolates is very necessary.

     

Fungicidal activity of cellobiose lipids from culture broth of yeast <Emphasis Type="Italic">Cryptococcus humicola</Emphasis> and <Emphasis Type="Italic">Pseudozyma fusiformata</Emphasis>

Cellobiose lipids of yeast fungi Cryptococcus huminola and Pseudozyma fusiformata have similar fungicidal activities against different yeast, including pathogenic Cryptococcus and Candida species. Basidiomycetic yeast reveals maximum sensitivity to these preparations; e.g., cells of cryptococcus Filobasidiella neoformans almost completely die after 30-min incubation in a glycolipid solution at a concentration of 0.02 mg/ml. The same effect toward ascomycetous yeast, including pathogenic Candida species, is achieved only at five to eight times higher concentrations of glycolipids. The cellobiose lipid from P. fusiformata, which, unlike glycolipid from Cr. humicola, has hydroxycaproic acid residue as O-subtituent of cellobiose and additional 15-hydroxy group in aglycone, inhibits the growth of the studied mycelial fungi more efficiently than the cellobiose lipid from Cr. humicola.     

Incidence of metallo-beta-lactamase-producing <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> isolates from hospital setting in Pakistan

The aim of this study was monitoring and surveillance in different wards of the PIMS hospital, Islamabad, to understand emerging challenges of antibiotic resistance in particular association with most virulent serotypes of Klebsiella pneumoniae. The study was conducted during March 2015 to September 2015. The study showed that rate of isolation of K. pneumoniae was 37% (103 positives out of a total of 277 clinical samples) and 7.7% (8) were phenotypically and genotypically confirmed to be metallo-β-lactamase resistant (carbapenem resistant) and all of them were multidrug resistant (MDR). These carbapenem-resistant isolates were isolated from blood, endotracheal tubes, and pus. Molecular screening for the presence of integrons indicated that distribution of class I integrons (87.5% of carbapenem-resistant K. pneumoniae isolates) was higher than class II integrons (1.25%) among given isolates. The study indicated that exposure of metallo-beta-lactamase-producing strains through hospitalizations increases the chances of spread of MDR pathogens. There is an urgent need for effective surveillance and monitoring strategies to control the spread of extremely resistant K. pneumoniae implicated in nosocomial infections leading to the increased health burden and enforcement of policy guideline on appropriate antibiotics usage.     

<Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> as a Probiotic Potential from Kouzeh Cheese (Traditional Iranian Cheese) and Its Antimicrobial Activity

The aim of this study is to isolate and identify Lactobacillus plantarum isolates from traditional cheese, Kouzeh, and evaluate their antimicrobial activity against some food pathogens. In total, 56 lactic acid bacteria were isolated by morphological and biochemical methods, 12 of which were identified as Lactobacillus plantarum by biochemical method and 11 were confirmed by molecular method. For analyzing the antimicrobial activity of these isolates properly, diffusion method was performed. The isolates were identified by 318 bp band dedicated for L. plantarum. The isolated L. plantarum represented an inhibitory activity against four of the pathogenic bacteria and showed different inhibition halos against each other. The larger halos were observed against Staphylococcus aureus and Staphylococcus epidermidis (15 ± 0.3 and 14.8 ± 0.7 mm, respectively). The inhibition halo of Escherichia coli was smaller than that of other pathogen and some L. plantarum did not show any inhibitory activity against E. coli, which were resistant to antimicrobial compounds produced by L. plantarum. The isolated L. plantarum isolates with the antimicrobial activity in this study had strong probiotic properties. These results indicated the nutritional value of Kouzeh cheese and usage of the isolated isolates as probiotic strains.     

Nimesulide inhibits pathogenic fungi: PGE2-dependent mechanisms

Certain non-steroidal anti-inflammatory drugs can inhibit fungal growth, fungal prostaglandin E2 production, and enzyme activation. This study aims to investigate the antifungal effect of nimesulide against pathogenic filamentous fungi and yeast. The experiments detailed below were also designed to investigate whether the action is dependent on E2 fungal prostaglandins. Our data showed that nimesulide exhibited potent antifungal activity, mainly against Trichophyton mentagrophytes (ATCC 9533) and Cryptococcus neoformans with MIC values of 2 and 62 μg/mL, respectively. This drug was also able to inhibit the growth of clinic isolates of filamentous fungi, such as Aspergillus fumigatus, and dermatophytes, such as T. rubrum, T. mentagrophytes, Epidermophyton floccosum, Microsporum canis, and M. gypseum, with MIC values ranging from 112 to 770 μg/mL. Our data also showed that the inhibition of fungal growth by nimesulide was mediated by a mechanism dependent on PGE2, which led to the inhibition of essential fungal enzymes. Thus, we concluded that nimesulide exerts a fungicidal effect against pathogenic filamentous fungi and yeast, involving the inhibition of fungal prostaglandins and fungal enzymes important to the fungal growth and colonization.     

Molecular identification of yeast,lactic and acetic acid bacteria species during spoilage of tchapalo,a traditional sorghum beer from Côte d’Ivoire

Yeasts, lactic and acetic acid bacteria are responsible of microbial spoilage of alcoholic beverages. However species involved in deterioration of sorghum beer produced in Côte d’Ivoire has not been investigated. This study was carried out to identify species of yeast, LAB and AAB during spoilage of tchapalo in order to define the best strategy for beer preservative. Thus, a total of 210 yeasts, LAB and AAB were isolated from samples of tchapalo stored at ambient temperature and at 4 °C for 3 days. Based on PCR–RFLP of the ITS region and the sequencing of D1/D2 domain, yeast isolates were assigned to seven species (Saccharomyces cerevisiae, Candida tropicalis, Rhodotorula mucilaginosa, Trichosporon asahii, Kluyveromyces marxianus, Meyerozyma guilliermondii and Trichosporon coremiiforme). During the storage at ambient temperature and at 4 °C, S. cerevisiae was the predominant species (>?76%). Excepted R. mucilaginosa, occurrence of non-Saccharomyces species was sporadic. LAB species detected in fresh samples using molecular methods were Pediococcus acidilactici, Lactobacillus paracasei, Lb. curvatus, Lb. fermentum and Weisssella paramesenteroides. P. acidilactici was the dominant species (47.8%) followed by Lb. paracasei (17.5%). W. paramesenteroides and Lb. fermentum were not detected during the spoilage at ambient temperature while at 4 °C W. paramesenteroides and Lb. paracasei have not been detected. For AAB, the species found were Acetobacter pasteurianus sub paradoxus and Acetobacter cerevisiae. These species were common to all samples during spoilage and A. pasteurianus sub paradoxus was the most frequently detected.     

Diversity of <Emphasis Type="Italic">Colletotrichum</Emphasis> spp. isolated from chili pepper fruit exhibiting symptoms of anthracnose in Thailand

Colletotrichum spp. are causal agents of anthracnose disease in chili fruits and other tropical crops. The disease is increasing in chili fruits in Thailand and significantly reduces fruit quality and fruit production. Forty-eight isolates of Colletotrichum spp. associated with chili anthracnose were collected from different areas of Thailand during 2010–2015. Based on morphological characteristic identification, 10 isolates were shown to belong to the C. gloeosporioides species complex, 24 isolates belong to the C. acutatum species complex and 14 isolates to C. capsici. For molecular identification, two primer sets, ITS1/ITS4 and ACT528/ACT738, were used for amplification of the internal transcribed spacer of rRNA gene (ITS1–5.8S–ITS2) and partial region actin gene (ACT), respectively. The phylogenetic analysis of individual and combined ITS region and actin nucleotide sequences identified the collected isolates into 4 species: C. gloeosporioides, C. siamense, C. acutatum and C. capsici. The pathogenicity test demonstrated that all four species were pathogenic on intact unwounded and healthy fruits. These results indicated that C. capsici, C. acutatum, C. gloeosporioides and C. siamense were the causal agents of chili anthracnose disease.     

Yeasts from peat in a tropical peat swamp forest in Thailand and their ability to produce ethanol,indole-3-acetic acid and extracellular enzymes

This study aimed to isolate and identify yeasts from peat in To Daeng peat swamp forest in southern of Thailand, and to investigate their ability to produce ethanol from glucose and xylose and to produce indole-3-acetic acid (IAA) and extracellular enzymes. A total of 65 yeast strains were obtained from 15 peat samples using an enrichment technique, and 61 strains were identified to be five species belonging to the phylum Ascomycota, namely Cyberlindnera subsufficiens, Debaryomyces fabryi, Meyerozyma guilliermondii, Saturnispora diversa and Schwanniomyces polymorphus var. africanus, and five species of the phylum Basidiomycota, namely Cryptococcus taiwanensis pro tem, Cutaneotrichosporon mucoides, Papiliotrema flavescens, Papiliotrema laurentii and Rhodotorula mucilaginosa. Four strains were unidentified and require further analysis. They differed from the type strain of P. flavescens by two nucleotide substitutions in the D1/D2 region of the LSU rRNA gene and nine nucleotide substitutions in the ITS region. R. mucilaginosa was the most prevalent yeast species, followed by S. polymorphus var. africanus, Cy. subsufficiens and D. fabryi. None of the yeast strains obtained in this study were able to ferment xylose to ethanol, but all ascomycetous yeast strains produced ethanol from glucose in a range of 9.0–58.0 g/L, with Cy. subsufficiens DMKU-YNB42-1 producing the highest ethanol concentration. A total of 62 strains produced IAA in a range of 9.0 to 66.9 mg/L, with the highest IAA produced by R. mucilaginosa DMKU-Y33-A. Investigation of the production of cellulases, xylanase, pectinase, amylase, protease and lipase revealed that all 65 yeast strains produced at least one extracellular enzyme, a lipase.     

Immunohistochemical Cross-Reactivity Between <Emphasis Type="Italic">Paracoccidioides</Emphasis> sp. from Dolphins and <Emphasis Type="Italic">Histoplasma capsulatum</Emphasis>

Paracoccidioidomycosis ceti is a cutaneous disease of cetaceans caused by uncultivated Paracoccidioides brasiliensis or Paracoccidioides spp. Serological cross-reactions between paracoccidioidomycosis ceti and paracoccidioidomycosis, paracoccidioidomycosis and histoplasmosis, and paracoccidioidomycosis and coccidioidomycosis have been reported before. The present study aimed to detect immunohistochemical cross-reaction between antibodies to Paracoccidioides sp. and Histoplasma capsulatum, and vice versa. Thirty murine sera, obtained from experimental infections of 6 isolates of H. capsulatum, were reacted with paraffin-embedded yeast-form cells of Paracoccidioides sp. derived from a case of paracoccidioidomycosis ceti in Japan. The murine sera were also reacted with human isolates of H. capsulatum yeast cells, with P. brasiliensis yeast cells, and with fungal cells of Coccidioides posadasii. Three dolphins’ sera from cases of paracoccidioidomycosis ceti, two human sera from patients with paracoccidioidomycosis, and a serum from a healthy person with a history of coccidioidomycosis were used in order to determine that the tested fungal cells reacted properly. Sera derived from mice infected with an isolate of H. capsulatum reacted positively against yeast cells of Paracoccidioides sp., yeast cells of P. brasiliensis, and fungal cells of C. posadasii, while those derived from other strains were negative. The present study recorded for the first time the cross-reaction between the yeast cells of H. capsulatum and antibodies against Paracoccidioides spp., the yeast cells of Paracoccidioides sp. and antibodies against H. capsulatum, the yeast cells of Paracoccidioides sp. and antibodies against Coccidioides sp., and fungal cells of C. posadasii and antibodies against Paracoccidioides spp.     

The diversity of culturable yeasts in the phylloplane of rice in Thailand

One hundred and fifty-six yeast strains were obtained by the enrichment technique from the phylloplanes of 85 rice leaf samples collected from seven provinces in Thailand. On the basis of the D1/D2 domain of the large subunit rRNA gene sequence analysis, 156 strains were identified as 34 known species in 18 genera consisting of 25 species in 13 genera of the phylum Ascomycota and nine species in five genera of the phylum Basidiomycota. The species in the phylum Ascomycota comprised 24 species in 12 genera of the order Saccharomycetales and one species viz. Yarrowia lipolytica in Saccharomycetales incertae sedis. The 24 species viz. Candida glabrata in the Nakaseomyces clade of Saccharomycetaceae, Candida jaroonii, Candida membranifaciens and Candida terebra in the Yamadazyma clade of Debaryomycetaceae, Candida pseudolambica in the Pichia clade of Pichiaceae, Candida ruelliae in the Metschnikowia clade of Metschnikowiaceae, and three unaffiliated clade Candida species (Candida catenulata, Candida rugosa and Candida tropicalis); Clavispora lusitaniae, Kodamaea ohmeri, Metschnikowia koreensis and Metschnikowia lopburiensis in Metschnikowiaceae; Cyberlindnera fabianii, Cyberlindnera rhodanensis and Wickerhamomyces ciferrii in Wickerhamomycetaceae; Debaryomyces nepalensis, Meyerozyma caribbica, Meyerozyma guilliermondii, Millerozyma koratensis, and Yamadazyma mexicanum in Debaryomycetaceae; Pichia kudriavzevii in Pichiaceae; and Lachancea thermotolerans in Saccharomycetaceae. The species in Basidiomycota viz. Cryptococcus flavescens, Cryptococcus laurentii, Cryptococcus aff. laurentii and Cryptococcus rajasthanensis in the Tremellales lineage, Bulleromyces clade, Tremellales, Tremellomycetes, Agaricomycotina; Pseudozyma antarctica and Pseudozyma aphidis in Ustilaginales, Ustilaginomycetes, Ustilaginomycotina; Rhodotorula taiwanensis and Sporobolomyces blumeae in Sporidiobolales, Microbotryomycetes, Pucciniomycotina; and Trichosporon asahii in Trichosporonales, Tremellomycetes, Agaricomycotina. The most prevalent species was R. taiwanensis with a 23 % frequency of occurrence followed by Candida tropicalis (16 %) and Cryptococcus fabianii (12 %).     

Diversity,molecular phylogeny and fingerprint profiles of airborne <Emphasis Type="Italic">Aspergillus</Emphasis> species using random amplified polymorphic DNA

In the present study, diversity and phylogenetic relationship of Aspergillus species isolated from Tehran air was studied using random amplified polymorphic DNA (RAPD)–polymerase chain reaction (RAPD-PCR). Thirty-eight Aspergillus isolates belonging to 12 species i.e. A. niger (28.94 %, 11 isolates), A. flavus (18.42 %, 7 isolates), A. tubingensis (13.15 %, 5 isolates), A. japonicus (10.52 %, 4 isolates), A. ochraceus (10.52 %, 4 isolates), and 2.63 %, 1 isolate from each A. nidulans, A. amstelodami, A. oryzae, A. terreus, A. versicolor, A. flavipes and A. fumigatus were obtained by settle plate method which they were distributed in 18 out of 22 sampling sites examined. Fungal DNA was extracted from cultured mycelia of all Aspergillus isolates on Sabouraud Dextrose Agar and used for amplification of gene fragments in RAPD-PCR using 11 primers. RAPD-PCR data was analyzed using UPGMA software. Resulting dendrogram of combined selected primers including PM1, OPW-04, OPW-05, P160, P54, P10 and OPA14 indicated the distribution of 12 Aspergillus species in 8 major clusters. The similarity coefficient of all 38 Aspergillus isolates ranged from 0.02 to 0.40 indicating a wide degree of similarities and differences within and between species. Taken together, our results showed that various Aspergillus species including some important human pathogenic ones exist in the outdoor air of Tehran by different extents in distribution and diversity and suggested inter- and intra-species genetic diversity among Aspergillus species by RAPD-PCR as a rapid, sensitive and reproducible method.     

Time-Kill Curves Studies with Amphotericin B Against <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis>/<Emphasis Type="Italic">C</Emphasis>. <Emphasis Type="Italic">gattii</Emphasis> Species Complex Clinical Isolates

Purpose of Review

We reviewed data on amphotericin B (AmB) tolerance among Cryptococcus neoformans/C. gattii species complex clinical isolates and present our results of large recent study on this issue.

Recent Findings

The standard method to detect antifungal susceptibility is based on MIC (minimal inhibitory concentration) determination; however, there is no interpretative clinical breakpoints defined for antifungal agents against Cryptococcus species, and to date, there is no correlation of MIC and clinical response. The time-kill curves (TKC) methodology seems to provide some correlation with outcome and it could identify distinct profiles of AmB-fungicidal activity.

Summary

Our group analyzed 83 human isolates from cryptococcosis cases. The isolates were tested by TKC and showed up 8.3% of tolerance to AmB. Importantly, the AmB-MIC was low for all isolates, including tolerant ones. Our findings are similar to other authors, due the ability of TKC to identify distinct AmB-fungicidal activity and detecting low susceptible isolates.

     

Dispersal of <Emphasis Type="Italic">Fusarium</Emphasis> spp. by rainwater and pathogenicity on four plant species

Research focused on the occurrence of Fusarium spp. in atmospheric dust or rainwater is not common. Preliminary studies with four sampling dates in 2007 revealed that several species of Fusarium may also be conveyed by rainwater. In order to determine the regular presence of Fusarium spp. in rainfall water, samples were systematically collected for a year (from October 2009 to October 2010) in three points on the Mediterranean coast of the province of Granada (Spain) 10-km distance between them. Throughout the year of sampling, a total of 179 rainwater samples were collected during every significant rainfall event. Eight different Fusarium species were isolated from the rainwater samples: F. oxysporum (32 %), F. proliferatum (26 %) and F. equiseti (20 %) coincide with previous studies, while F. dimerum (3 %), F. semitectum (4.7 %), F. solani (8 %), F. avenaceum (0.5 %) and F. chlamydosporum (3.7 %) were isolated for the first time from rainwater. Results were consistent with previous surveys conducted 100 km away from the sampling sites. Inoculation of 39 different isolates from five different Fusarium species showed pathogenicity on plants. Disease severity differed depending on the inoculated plant species, which means that rain water can be an effective vector to transport new pathogens into new cultivated areas. This work reveals some epidemiological aspects of Fusarium genus in natural environments. Some of the isolated Fusarium spp. are potential mycotoxin producers, such as zearalenone, fumonisin, moniliformin or nivalenol.     

Fungi from leaves of lotus (<Emphasis Type="Italic">Nelumbo nucifera</Emphasis>)

In spite of the self-cleaning property of its leaves called the lotus effect, leaves of lotus (Nelumbo nucifera) provide a habitat for an unknown fungal diversity. The aim of this study was to detect and identify fungi from leaves of N. nucifera, including ectophytic, parasitic and endophytic fungi, in Taiwan using different collection strategies, as well as morphological and diverse molecular markers established in the different systematic groups of fungi. Among ectophytic and parasitic fungi, a new species of Dissoconium and of Pseudocercospora are described, respectively. Phyllosticta nelumbonis Sawada is transferred to Diaporthe. Among plant parasitic fungi, Erysiphe takamatsui and Ps. nymphaeacea are recorded in Taiwan for the first time. Euryale is recorded as a new host genus for Ps. nymphaeacea. The basidiomycetous yeast Fereydounia khargensis is recorded for the first time from living plants and in East Asia. Endophytic fungi from lotus were studied for the first time. From 1002 plant segments, 476 endophytic isolates were produced in culture, comprising 33 typical terrestrial species mainly belonging to the genera Colletotrichum (mainly C. siamense), Diaporthe (D. tulliensis and D. ueckerae) and Fusarium (F. solani species 6, hitherto known from clinical samples), as well as to Xylariaceae, but no Ingoldian fungi. Most isolates were from leaf laminas (71%) compared to those from petioles (29%). From this observation, we conclude that the fungi of the aquatic lotus plant appear to have terrestrial origin and, after dispersal by wind and in spite of the lotus effect, may enter the plant from the lamina. Only three species isolated as endophytes were also found as ectophytic or parasitic fungi.     

Identification of yeasts isolated from raffia wine (<Emphasis Type="Italic">Raphia hookeri</Emphasis>) produced in Côte d’Ivoire and genotyping of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strains by PCR inter-delta

Raffia wine is a traditional alcoholic beverage produced in several African countries where it plays a significant role in traditional customs and population diet. Alcoholic fermentation of this beverage is ensured by a complex natural yeast flora which plays a decisive role in the quality of the final product. This present study aims to evaluate the distribution and the diversity of the yeast strains isolated in raffia wine from four sampling areas (Abengourou, Alépé, Grand-Lahou and Adzopé) in Côte d’Ivoire. Based on the D1/D2 domain of the LSU rDNA sequence analysis, nine species belonging to six genera were distinguished. With a percentage of 69.5 % out of 171 yeast isolates, Saccharomyces cerevisiae was the predominant species in the raffia wine, followed by Kodamaea ohmeri (20.4 %). The other species isolated were Candida haemulonii (4.1 %), Candida phangngensis (1.8 %), Pichia kudriavzevii (1.2 %), Hanseniaspora jakobsenii (1.2 %), Candida silvae (0.6 %), Hanseniaspora guilliermondii (0.6 %) and Meyerozyma caribbica (0.6 %). The molecular characterization of S. cerevisiae isolates at the strain level using the PCR-interdelta method revealed the presence of 21 profiles (named I to XXI) within 115 isolates. Only four profiles (I, III, V and XI) were shared by the four areas under study. Phenotypic characterization of K. ohmeri strains showed two subgroups for sugar fermentation and no diversity for the nitrogen compound assimilations and the growth at different temperatures.     

Development of a Multiplex-PCR probe system for the proper identification of <Emphasis Type="Italic">Klebsiella variicola</Emphasis>

Background

Klebsiella variicola was very recently described as a new bacterial species and is very closely related to Klebsiella pneumoniae; in fact, K. variicola isolates were first identified as K. pneumoniae. Therefore, it might be the case that some isolates, which were initially classified as K. pneumoniae, are actually K. variicola. The aim of this study was to devise a multiplex-PCR probe that can differentiate isolates from these sister species.

Result

This work describes the development of a multiplex-PCR method to identify K. variicola. This development was based on sequencing a K. variicola clinical isolate (801) and comparing it to other K. variicola and K. pneumoniae genomes. The phylogenetic analysis showed that K. variicola isolates form a monophyletic group that is well differentiated from K. pneumoniae. Notably, the isolate K. pneumoniae 342 and K. pneumoniae KP5-1 might have been misclassified because in our analysis, both clustered with K. variicola isolates rather than with K. pneumoniae. The multiplex-PCR (M-PCR-1 to 3) probe system could identify K. variicola with high accuracy using the shared unique genes of K. variicola and K. pneumoniae genomes, respectively. M-PCR-1 was used to assay a collection of multidrug-resistant (503) and antimicrobial-sensitive (557) K. pneumoniae clinical isolates. We found K. variicola with a prevalence of 2.1% (23/1,060), of them a 56.5% (13/23) of the isolates were multidrug resistant, and 43.5% (10/23) of the isolates were antimicrobial sensitive. The phylogenetic analysis of rpoB of K. variicola-positive isolates identified by multiplex-PCR support the correct identification and differentiation of K. variicola from K. pneumoniae clinical isolates.

Conclusions

This multiplex-PCR provides the means to reliably identify and genotype K. variicola. This tool could be very helpful for clinical, epidemiological, and population genetics studies of this species. A low but significant prevalence of K. variicola isolates was found, implying that misclassification had occurred previously. We believe that our multiplex-PCR assay could be of paramount importance to understand the population dynamics of K. variicola in both clinical and environmental settings.

     

Yeasts in Antarctic soils

The yeast flora of 126 soil samples from the Ross Dependency of Antarctica was examined and compared with that of eight samples from East Greenland. Fifty-two Antarctic samples contained yeasts, in numbers ranging from 5 to over 100,000/g; species isolated belonged to the generaDebaryomyces, Cryptococcus, Candida, Trichosporon andRhodotorula. Nearly all isolates ofCandida were obligate psychrophils; they belonged toCandida scottii, C. nivalis, C. gelida andC. frigida. Duplicate samples, taken at the same site within a few yards of one another, contained the same size and kind of yeast populations. Although not all samples which contained plants (algae, lichens or mosses) contained yeasts, almost all samples which contained yeasts contained plants. There was no correlation between yeast population and the pH of the sample, nor between yeast populations and the presence of vertebrate animals. The samples from East Greenland, which were from an area sufficiently warm and moist to support growth of higher plants, all contained yeasts, in numbers from 200 to 56,000/g. Species isolated were similar to those from Antarctic material.