Detection of Cryptic <Emphasis Type="Italic">Candida</Emphasis> Species Recognized as Human Pathogens Through Molecular Biology Techniques

Purpose of Review

The aim of this review is to evaluate these molecular-based methods able to identify pathogenic cryptic Candida spp. focusing on those that demonstrated to be useful in clinical laboratory settings.

Recent Findings

It is long known that some Candida spp. are genetically heterogeneous. Firstly, individual species were divided into groups based on differences on the sequence of some genes. Later, those groups were designated as cryptic species and defined as phenotypically indistinguishable species that are only identified by their DNA sequences. Many common Candida spp. are now considered complexes formed by several cryptic species. Some of them have been recognized as human pathogens. The identification of these species is problematic but necessary since they have different host range, infection sites, infection severity, and antifungal susceptibility. Several independent DNA markers were proposed as tools for the differentiation of highly related species. We will concentrate on the three species complexes most frequently associated with human infections including Candida albicans, C. glabrata, and C. parapsilosis complexes and a fourth group of less common but multiresistant species including C. haeumulonii complex and C. auris.

Summary

We review the clinically useful molecular tools able to differentiate the cryptic species of C. albicans, C. glabrata, and C. parapsilosis complexes and designated to uncover emerging multiresistant species.

     

Diversity of <Emphasis Type="Italic">Cronobacter</Emphasis> spp. isolates from the vegetables in the middle-east coastline of China

Cronobacter spp. has caused life-threatening neonatal infections mainly resulted from consumption of contaminated powdered infant formula. A total of 102 vegetable samples from retail markets were evaluated for the presence of Cronobacter spp. Thirty-five presumptive Cronobacter isolates were isolated and identified using API 20E and 16S rDNA sequencing analyses. All isolates and type strains were characterized using enterobacterial repetitive intergenic consensus sequence PCR (ERIC–PCR), and genetic profiles of cluster analysis from this molecular typing test clearly showed that there were differences among isolates from different vegetables. A polymerase chain reaction restriction fragment length polymorphism (PCR–RFLP) based on the amplification of the gyrB gene (1258 bp) was developed to differentiate among Cronobacter species. A new PCR–RFLP assay based on the amplification of the gyrB gene using Alu I and Hinf I endonuclease combination is established and it has been confirmed an accurate and rapid subtyping method to differentiate Cronobacter species. Sequence analysis of the gyrB gene was proven to be suitable for the phylogenetic analysis of the Cronobacter strains, which has much better resolution based on SNPs in the identification of Cronobacter species specificity than PCR–RFLP and ERIC–PCR. Our study further confirmed that vegetables are one of the most common habitats or sources of Cronobacter spp. contamination in the middle-east coastline of China.     

Morel Phylogeny and Diagnostics Based on Restriction Fragment Length Polymorphism Analysis of ITS Region of 5.8S Ribosomal RNA Gene

Thirty-six cultures representing eight Morchella and related genera, namely, Morchella esculenta, M. crassipes, M. spongiola, M. vulgaris, M. angusticeps, M. conica, Mitrophora semilibera and Verpa conica were subjected to restriction analysis of ITS1-5.8SITS2 region of rDNA. Six restriction endonuclease enzymes viz TaqI, EcoRl, Mspl, Rsal, Hinfl and BsuRl were used to generate restriction fragments and analysis of phylogenetic relationships among morels. The Amplified Ribosomal DNA Restriction Analysis (ARDRA) not only distinguished yellow morels from black morels but also separated related genera Mitrophora semilibera and Verpa conica from true morels. Simultaneously, each morel species could be separated from each other exhibiting considerable phylogenetic distances. The unique restriction fragment profiles generated by the restriction endonucleases enabled us to identify marker fragments to distinguish each species within and amongst the morel group. Since no intra-specific variation in restriction profiles by the six restriction endonucleases could be visualized among monospores, the technique could be used for rapid identification of wild morel specimens as a cheap alternative to direct sequencing for germplasm cataloguing.     

Toothbrush Contamination by <Emphasis Type="Italic">Candida</Emphasis> spp. and Efficacy of Mouthrinse Spray for Their Disinfection

The aim of the study was to evaluate toothbrush contamination in vivo by Candida spp. and the efficacy of Periogard^® and Neem Sattiva^®, in spray, in the disinfection of these toothbrushes. This study was performed in three phases in which mouthrinses and sterile distilled water (control group) were sprayed six times on toothbrush bristles used by 61 university students. Toothbrushes were then submitted to microbiological processing for the isolation and identification of Candida species. Fifty-nine students completed the three phases of this study, and 22 (37.3%) control group toothbrushes presented growth of Candida species. Periogard^® and Neem Sattiva^® eliminated growth of Candida spp. in 48.1 and 7.4% of toothbrushes, respectively. Contamination by Candida spp. was observed on various toothbrushes of the control group. Periogard^® was more efficacious than Neem Sattiva^® in eliminating growth of Candida spp. on the toothbrush bristles.     

Morphological description of four species belonging to the genus <Emphasis Type="Italic">Nigrobaetis</Emphasis> (Ephemeroptera: Baetidae) from Japan

We associated imagoes and nymphs of four Nigrobaetis species from Japan and described their morphological characters. We found that N. sp. P comprised two Nigrobaetis species in Kanto Plain and in Ishigaki Island, which we described as N. apterus Fujitani n. sp. and N. ishigakiensis Fujitani n. sp., respectively. We also described N. sp. I as N. latus Fujitani n. sp. We recorded N. acinaciger (Kluge), the nymph of which had been confused with N. chocoratus (Gose), from Japan for the first time. We provide identification keys for eight Nigrobaetis species of Japan.     

Usefulness of the Non-conventional <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis> Model to Assess <Emphasis Type="Italic">Candida</Emphasis> Virulence

Invasive candidiasis is caused mainly by Candida albicans, but other Candida species have increasing etiologies. These species show different virulence and susceptibility levels to antifungal drugs. The aims of this study were to evaluate the usefulness of the non-conventional model Caenorhabditis elegans to assess the in vivo virulence of seven different Candida species and to compare the virulence in vivo with the in vitro production of proteinases and phospholipases, hemolytic activity and biofilm development capacity. One culture collection strain of each of seven Candida species (C. albicans, Candida dubliniensis, Candida glabrata, Candida krusei, Candida metapsilosis, Candida orthopsilosis and Candida parapsilosis) was studied. A double mutant C. elegans AU37 strain (glp-4;sek-1) was infected with Candida by ingestion, and the analysis of nematode survival was performed in liquid medium every 24 h until 120 h. Candida establishes a persistent lethal infection in the C. elegans intestinal tract. C. albicans and C. krusei were the most pathogenic species, whereas C. dubliniensis infection showed the lowest mortality. C. albicans was the only species with phospholipase activity, was the greatest producer of aspartyl proteinase and had a higher hemolytic activity. C. albicans and C. krusei caused higher mortality than the rest of the Candida species studied in the C. elegans model of candidiasis.     

New <Emphasis Type="Italic">Penicillium</Emphasis> and <Emphasis Type="Italic">Talaromyces</Emphasis> species from honey,pollen and nests of stingless bees

Penicillium and Talaromyces species have a worldwide distribution and are isolated from various materials and hosts, including insects and their substrates. The aim of this study was to characterize the Penicillium and Talaromyces species obtained during a survey of honey, pollen and the inside of nests of Melipona scutellaris. A total of 100 isolates were obtained during the survey and 82% of those strains belonged to Penicillium and 18% to Talaromyces. Identification of these isolates was performed based on phenotypic characters and β-tubulin and ITS sequencing. Twenty-one species were identified in Penicillium and six in Talaromyces, including seven new species. These new species were studied in detail using a polyphasic approach combining phenotypic, molecular and extrolite data. The four new Penicillium species belong to sections Sclerotiora (Penicillium fernandesiae sp. nov., Penicillium mellis sp. nov., Penicillium meliponae sp. nov.) and Gracilenta (Penicillium apimei sp. nov.) and the three new Talaromyces species to sections Helici (Talaromyces pigmentosus sp. nov.), Talaromyces (Talaromyces mycothecae sp. nov.) and Trachyspermi (Talaromyces brasiliensis sp. nov.). The invalidly described species Penicillium echinulonalgiovense sp. nov. was also isolated during the survey and this species is validated here.     

Detection of pea wilt pathogen <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">pisi</Emphasis> using DNA-based markers

Identification of the fungus Fusarium oxysporum f. sp. pisi (Fop), the causal organism of wilt disease of pea, is a time consuming and arduous task. Diagnosis of Fop by traditional means requires more than 2 months and involves two steps, identification of species using morphological characters and formae specialis ‘pisi’ using pathogenicity assays. The ambiguous morphological differences between F. solani and F. oxysporum further complicate the diagnosis of F. oxysporum. A polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) based method was developed to detect Fop from India. A PCR–RFLP marker, HPACAPS1₃₈₀, generated after restriction of 28S rDNA region with enzyme MvaI, detected accurately the Fop among several other fungi with detection sensitivity of 5 fg of Fop genomic DNA. In a mixture of Fop and pea DNA, the sensitivity was 500 pg of Fop DNA in 50 ng of pea DNA. The assay was further refined to detect the Fop from infected tissues and infested soil. The current assay can detect Fop from culture, plant tissues and soil in a considerably shorter period of time compared to traditional methods.     

How much do we know about hemolytic capability of pathogenic <Emphasis Type="Italic">Candida</Emphasis> species?

Hemolytic factor production by pathogenic Candida species is considered an important attribute in promoting survival within the mammal host through the ability to assimilate iron from the hemoglobin-heme group. Hemolytic capability has been evaluated for Candida species based on hemolysis zones on plate assay, analysis of hemolytic activity in liquid culture medium, and hemolysis from cell-free culture broth. The production of hemolytic factor is variable among Candida species, where C. parapsilosis is the less hemolytic species. In general, no intraspecies differences in beta-hemolytic activities are found among isolates belonging to C. albicans, C. glabrata, C. krusei, C. tropicalis, and C. parapsilosis. The production of hemolytic factor by Candida species is affected by several factors such as glucose supplementation in the culture medium, blood source, presence of erythrocytes and hemoglobin, and presence of electrolytes. On the basis of existing achievements, more researches are still needed in order to extend our knowledge about the biochemical nature of hemolytic molecules produced by distinct Candida species, the mechanism of hemolysis, and the molecular basis of the hemolytic factor expression.     

A re-evaluation of diversity of the Aporocotylidae Odhner, 1912 in <Emphasis Type="Italic">Siganus fuscescens</Emphasis> (Houttuyn) (Perciformes: Siganidae) and associated species

The aporocotylid fauna of the mottled spinefoot, Siganus fuscescens (Houttuyn), from Moreton Bay, Queensland, Australia, was characterised using a combined morphological and molecular approach. Four aporocotylid species were identified, three belonging to the genus Ankistromeces Nolan & Cribb, 2006 and one to Cardicola Short, 1953. Specimens of Cardicola matched an undescribed species from the same host and locality; this species is described as Cardicola mogilae n. sp. Phylogenetic analyses of ITS2 and 28S data showed that C. mogilae n. sp. forms a strongly supported clade with other Cardicola species from siganid fishes. We record Ankistromeces olsoni Nolan & Cribb, 2006 in Moreton Bay for the first time, redescribe A. dunwichensis Nolan & Cribb, 2006 on the basis of new specimens and sequence data and re-report Ankistromeces sp. X from Moreton Bay based on molecular data. We review the status of the ten putative species of aporocotylids reported from siganids. Small variation in ITS2 rDNA sequences, in association with different geographic localities, was previously used to separate Cardicola lafii Nolan & Cribb, 2006 from C. parilus Nolan & Cribb, 2006, C. bartolii Nolan & Cribb, 2006 from C. watsonensis Nolan & Cribb, 2006, C. tantabiddii Nolan & Cribb, 2006 from Cardicola sp. 2, Ankistromeces sp. Y from A. olsoni and Ankistromeces sp. X from Ankistromeces sp. Z. These five combinations are reinterpreted as each representing a single species; Cardicola lafii is recognised as the senior synonym of C. parilus and C. bartolii as the senior synonym of C. watsonensis. This study thus suggests that six, rather than ten, species should be recognised as infecting S. fuscescens. This richness remains greater than is known for any other fish species and siganids are, so far, unique among fishes in harbouring two strongly radiated lineages of aporocotylids.     

<Emphasis Type="Italic">Lepotrema</Emphasis> Ozaki, 1932 (Lepocreadiidae: Digenea) from Indo-Pacific fishes,with the description of eight new species,characterised by morphometric and molecular features

We review species of the genus Lepotrema Ozaki, 1932 from marine fishes in the Indo-West Pacific. Prior to the present study six species were recognised. Here we propose eight new species on the basis of combined morphological and molecular analysis: Lepotrema acanthochromidis n. sp. ex Acanthochromis polyacanthus from the Great Barrier Reef (GBR); Lepotrema hemitaurichthydis n. sp. ex Hemitaurichthys polylepis and H. thompsoni from Palau and French Polynesia; Lepotrema melichthydis n. sp. ex Melichthys vidua from Palau and the GBR; Lepotrema amansis n. sp. ex Amanses scopas from the GBR; Lepotrema cirripectis n. sp. ex Cirripectes filamentosus, C. chelomatus and C. stigmaticus from the GBR; Lepotrema justinei n. sp. ex Sufflamen fraenatum from New Caledonia; Lepotrema moretonense n. sp. ex Prionurus microlepidotus, P. maculatus and Selenotoca multifasciata from Moreton Bay; and Lepotrema amblyglyphidodonis n. sp. ex Amblyglyphidodon curacao and Amphipron akyndynos from the GBR. We also report new host records and provide novel molecular data for two known species: Lepotrema adlardi Bray, Cribb & Barker, 1993 and Lepotrema monile Bray & Cribb, 1998. Two new combinations are formed, Lepotrema cylindricum (Wang, 1989) n. comb. (for Preptetos cylindricus) and Lepotrema navodonis (Shen, 1986) n. comb. (for Lepocreadium navodoni). With the exception of a handful of ambiguous records, the evidence is compelling that the host-specificity of species in this genus is overwhelmingly oioxenous or stenoxenous. This renders the host distribution in three orders and ten families especially difficult to explain as many seemingly suitable hosts are not infected. Multi-loci molecular data (ITS2 rDNA, 28S rDNA and cox1 mtDNA) demonstrate that Lepotrema is a good generic concept, but limited variability in sequence data and differences in phylogenies produced for different gene regions make relationships within the genus difficult to define.     

Recent Taxonomic Developments with <Emphasis Type="Italic">Candida</Emphasis> and Other Opportunistic Yeasts

Increases in susceptible patient populations and advances in identification methods have resulted in the continued recognition of novel yeasts as agents of human infection. Most of these agents are members of the well-recognized genera Candida, Cryptococcus, Trichosporon, and Rhodotorula. Some of these agents are “cryptic species,” members of species complexes, and may not be detectable using classical carbohydrate assimilation-based methods of yeast identification. Such species require DNA- or MALDI-based methods for correct identification, although sporadic isolates may not routinely require delineation to the individual species level. The coming end of the fungal taxonomy rules requiring separate names for sexual and asexual forms of the same fungus will hopefully allow greater clarity, as names for medically important yeast can now be based on the needs of the medical mycology community and the common goal of better communication between laboratory and clinician.     

Comparative Study of the Effects of Fluconazole and Voriconazole on <Emphasis Type="Italic">Candida glabrata</Emphasis>, <Emphasis Type="Italic">Candida parapsilosis</Emphasis> and <Emphasis Type="Italic">Candida rugosa</Emphasis> Biofilms

Infections by non-albicans Candida species are a life-threatening condition, and formation of biofilms can lead to treatment failure in a clinical setting. This study was aimed to demonstrate the in vitro antibiofilm activity of fluconazole (FLU) and voriconazole (VOR) against C. glabrata, C. parapsilosis and C. rugosa with diverse antifungal susceptibilities to FLU and VOR. The antibiofilm activities of FLU and VOR in the form of suspension as well as pre-coatings were assessed by XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] reduction assay. Morphological and intracellular changes exerted by the antifungal drugs on Candida cells were examined by scanning electron microscope (SEM) and transmission electron microscope (TEM). The results of the antibiofilm activities showed that FLU drug suspension was capable of killing C. parapsilosis and C. rugosa at minimum inhibitory concentrations (MICs) of 4× MIC FLU and 256× MIC FLU, respectively. While VOR MICs ranging from 2× to 32× were capable of killing the biofilms of all Candida spp tested. The antibiofilm activities of pre-coated FLU were able to kill the biofilms at ¼× MIC FLU and ½× MIC FLU for C. parapsilosis and C. rugosa strains, respectively. While pre-coated VOR was able to kill the biofilms, all three Candida sp at ½× MIC VOR. SEM and TEM examinations showed that FLU and VOR treatments exerted significant impact on Candida cell with various degrees of morphological changes. In conclusion, a fourfold reduction in MIC₅₀ of FLU and VOR towards ATCC strains of C. glabrata, C. rugosa and C. rugosa clinical strain was observed in this study.     

Development of the St/J and V Genome Specific Molecular Marker Based on 5s rDNA Polymorphism in <Emphasis Type="Italic">Thinopyrum bessarabicum</Emphasis>, <Emphasis Type="Italic">Pseudoroegneria spicata</Emphasis>, and <Emphasis Type="Italic">Dasypyrum villosum</Emphasis>

Nontranscribed spacers (NTS) of 5S rDNA are often polymorphic in closely related species and even in the same genome. The polymorphism of 5S rDNA NTS was shown between genomes St, J, and V of Triticeae species Thinopyrum bessarabicum, Pseudoroegneria spicata, and Dasypyrum villosum, respectively. A molecular genetic marker was designed based on the 5S rDNA NTS polymorphism that allows identification of the St, J, and V genomes. We designed a pair of primers that correspond to the conserved regions of 5S rDNA NTS between the genomes studied. The PCR amplicon length is 158 bp, 171 bp, and 172 bp for V, St, and J genomes, respectively. The fragment of the St genome is characterized by the SmiM I restriction site that enables its differentiation from the J genome fragment that lacks this site. The developed marker showed its efficiency for verification of germplasm accessions and the study of allopolyploids.     

Specific features of ecology of chars of the genus <Emphasis Type="Italic">Salvelinus</Emphasis> (Salmonidae) from the basin of Lake Kronotskoe (Kamchatka) according to parasitological data

A parasitological study was performed of chars of the genus Salvelinus inhabiting Lake Kronotskoe (Kamchatka Peninsula)—S. malma, S. albus, S. schmidtii, and S. kronocius, as well as of juvenile Salvelinus spp. Twenty-three species of parasites, including six species new for the lake, Hennequya zschokkei, Protteocephalus longicollis, Diphyllobothrium dendriticum, Crepidostomum sp., Echinorhynchus salmonis, and Paracanthobdella livanowi, were found. With consideration of published data, in chars of this water body, 28 species of parasites were recorded, including seven species (N. cf. pungitius, B. luciopercae, Crepidostomum sp., Cr. fausti, Cr. cf. cooperi, Eubothrium crassium, and Proteocephalus sp.), whose presence or species identification in the lake ecosystem need confirmation. Two species (N. rutili and Diphyllobothrium sp.) are removed from the list. Parasites common for all species of chars were revealed. They include Myxobolus arcticus, E. salvelini, D. ditretum, Crepidostotum sp., Cr. farionis, Cr. metoecus, Cystidicola farionis, Cucullanus truttae, Philonema oncorhynchi, and Salmincola carpionis. Cluster analysis of the fauna of parasites of different species of chars demonstrated considerable differences in infestation, which indicates differences between them in preference for food items and occupied biotopes and thereby supports the ecological differentiation of chars in the basin of Lake Kronotskoe. S. albus and S. kronocius are most similar in parasitofauna, which is determined by their predation; S. malma as a benthophage is infected by the same species of parasites, but considerably less intensively. Extremely high indices of population numbers of some parasite species are considered as a manifestation of the Krebs cycle in parasites under the conditions of an isolated lake.     

Multigene phylogeny reveals a new species and novel records and hosts in the genus <Emphasis Type="Italic">Ramularia</Emphasis> from Iran

Ramularia is a species-rich genus in the order Capnodiales (Dothideomycetes, Ascomycota) that includes numerous phytopathogenic taxa, several of which are economically important plant pathogens. In this study, six isolates of Ramularia were recovered from leaf spot symptoms on six herbaceous and woody plants from Guilan, East and West Azarbaijan provinces in the north and northwest of Iran. The isolates were studied by a polyphasic approach involving morphological and cultural data, and multi-gene phylogeny (ITS, TEF1-α, ACT, HIS, RPB2 and GAPDH). The isolates were grouped in three species clades of the R. eucalypti species complex. Of these, R. mali is recorded for the first time in Asia and R. glennii represents a new record for the mycobiota of Iran. Ramularia taleshina on Alnus subcordata is described as a new species. Ramularia taleshina is phylogenetically related to R. mali, but they can be differentiated by morphological and cultural characters as well as molecular data. Acalypha australis, Ficus carica and Platanus sp. are reported as new hosts of R. glennii, and Prunus cerasus and Vitis vinifera as new hosts of R. mali.     

Two New Species and New Occurrences of <Emphasis Type="Italic">Syneches</Emphasis> Walker for Brazilian Biome of Caatinga (Diptera: Hybotidae: Hybotinae)

Syneches from Brazilian biome of Caatinga were studied, two new species are described, Syneches atratus sp. nov. and Syneches limeirai sp. nov., and three species, Syneches annulipes Bezzi, 1909, Syneches moraballi Smith, 1963, and Syneches rafaeli Ale-Rocha & Vieira, 2008, are recorded for the biome. An identification key for the species of Syneches from Caatinga biome is provided.