M13 PCR Fingerprinting Research Articles

The Predominance of a Specific Genotype of Cryptococcus neoformans var. Grubii in China and Japan.

Background The extensive deployment of molecular genotyping methods is the top reliable keyword to characterize the population genetic structure of C. neoformans in the past decade. However, studies involving genotypic analysis of C. neoformans var. grubii from China and Japan are limited. Objectives We address this challenge to determine the genotype distribution of C. neoformans var. grubii strains from China and Japan. Methods Genotypic analysis of 52 C. neoformans var. grubii isolates was performed using multilocus microsatellite typing (MLMT) based on the sequence analysis of 3 functional genes. In order to place the herein-studied strains into the global picture, 22 strains randomly selected from the 52 strains studied by MLMT were also analyzed by restriction fragment length polymorphism analysis of the orotidine monophosphate pyrophosphorylase gene (URA5-RFLP), M13 PCR-fingerprinting, and multilocus sequence typing (MLST). Results MLMT classified 46 (88.5%) of the 52 strains as genotype MLMT-17. The high prevalence of the MLMT-17 type was observed for environmental and clinical isolates from China and Japan. URA5-RFLP analysis, M13 PCR-fingerprinting, and MLST showed that most of these belong to the VNI/ST5 (M5) genotype. Conclusions Our study suggests the predominance of a specific genotype of C. neoformans var. grubii in China and Japan.

Development of Rhodotorula mucilaginosa strain via random mutagenesis for improved lipid production

Oleaginous yeasts are widely used for the production of biodiesel feedstocks because of their high lipid content. In this study, strain improvement was performed on the oleaginous yeast Rhodotorula mucilaginosa to generate mutants with higher lipid yields via random mutagenesis using ethyl methane sulfonate (EMS). Twenty-two mutant isolates prescreened with cerulenin were produced and further characterized via M13 PCR fingerprinting to determine their polymorphism and genetic distances. Eight strains, namely R. mucilaginosa M1, R. mucilaginosa M2, R. mucilaginosa M3, R. mucilaginosa M4, R. mucilaginosa M7, R. mucilaginosa M10, R. mucilaginosa M11 and R. mucilaginosa M18, were chosen based on their genetic distances from the parental strain for biomass production. Six mutants (M1, M2, M3, M4, M7 and M18) showing the highest dry cell weights were further selected for evaluation of lipid production in a laboratory-scale bioreactor using glucose as a carbon source. Results indicated that parental strain exhibited lipid content of 1.83±0.06 g/L, while strains M1, M2, M3, M7 and M18 generated 2.37±0.06 g/L, 2.27±0.06 g/L, 2.27±0.06 g/L, 3.10±0.00 g/L and 3.83±0.15 g/L of intracellular lipid, respectively. These five mutants were identified to have significant increase in lipid production compared to the parental strain. For FAME analysis, two selected strains M7 and M18 comprised of mainly C16 and C18 methyl esters, which are suitable for biodiesel production. This work demonstrated enhanced lipid production in R. mucilaginosa by random mutagenesis. The newly generated strains had higher lipid productivity compared to the parental strain. The application of these strains in industry may reduce the overall cost production of biodiesel.

Molecular typing of clinical isolates of Cryptococcus neoformans/Cryptococcus gattii species complex from Northeast Mexico.

Cryptococcosis is caused by members of the Cryptococcus neoformans/Cryptococcus gattii species complex. Based on molecular identification, these two species have been further differentiated into molecular types. The aim of this work was to characterize clinical cryptococcal isolates recovered from six hospitals in Northeast Mexico from 1995 to 2011. One hundred and sixty-six isolates, which were characterized by biochemical tests and in vitro susceptibility to amphotericin B, fluconazole, and voriconazole, and M13 PCR fingerprinting, were included in this study. Utilizing phenotypic tests, 153 isolates (92.16 %) were identified as C. neoformans and 13 (7.83 %) as C. gattii. All isolates were susceptible to all antifungals tested. Employing M13 PCR fingerprinting, eight molecular types were detected. VNI was the most common genotype (124 cases; 74.6 %), followed by VNII (15 cases; 9 %), VNIII (8 cases; 4.8 %), VNIV (6 cases; 3.6 %), VGI (6 cases; 3.6 %), VGII (3 cases; 1.8 %), and VGIII and VGIV (2 cases, 1.2 % each). We confirm the presence of C. gattii in clinical isolates in Northeast Mexico, and a high clonal diversity in the studied strains of C. neoformans/C. gattii species complex.

Macrobrachium amazonicum: an alternative for microbiological monitoring of aquatic environments in Brazil

This study aimed to evaluate the role of the Amazon River prawn, Macrobrachium amazonicum, as carrier of Candida spp., by analyzing the correlation between Candida spp. from these prawns and their environment (surface water and sediment), through M13-PCR fingerprinting and RAPD-PCR. For this purpose, 27 strains of Candida spp. were evaluated. These strains were recovered from the gastrointestinal tract of adult M. amazonicum (7/27) from Catú Lake, Ceará State, Brazil and from the aquatic environment (surface water and sediment) of this lake (20/27). Molecular comparison between the strains from prawns and the aquatic environment was conducted by M13-PCR fingerprinting and RAPD-PCR, utilizing the primers M13 and OPQ16, respectively. The molecular analysis revealed similarities between the band patterns of eight Candida isolates with the primer M13 and 11 isolates with the primer OPQ16, indicating that the same strains are present in the digestive tract of M. amazonicum and in the aquatic environment where these prawns inhabit. Therefore, these prawns can be used as sentinels for environmental monitoring through the recovery of Candida spp. from the aquatic environment in their gastrointestinal tract

Multilocus sequence typing (MLST) and M13 PCR fingerprinting revealed heterogeneity amongst Cryptococcus species obtained from Italian veterinary isolates.

Cryptococcosis represents a fungal disease acquired from the environment with animals serving as host sentinels for human exposure. The aim of this study was to investigate the genetic characteristics of Cryptococcus isolates from veterinary sources (cats, dogs and birds) to understand their epidemiology and the genetic variability of the casual isolates. Mating-type PCR in connection with MLST analysis using the ISHAM consensus MLST scheme for the C.neoformans/C.gattii species complex was used to genotype 17 C.neoformans isolates. In the absence of an MLST typing scheme Cryptococcus adeliensis, C.albidus, C.aureus, C. carnescens, C.laurentii, C.magnus and C.uniguttulatus strains were typed using M13 PCR fingerprinting. All C.neoformans isolates were MATα mating type, but hybrids possessed αADa and aADα mating and serotypes. Two C.neoformans molecular types VNI, VNIV and VNIII and VNII/VNIV hybrids were identified. Amongst the 66 non-C.neoformans strains investigated 55 M13 PCR fingerprinting types were identified. The wide variety of MLST types of C.neoformans and the occurrence of αADa and aADα hybrids in our study supports the notion of genetic recombination in the area studied. The heterogeneity of the non-C.neoformans isolates remains open to further investigations and should be taken into consideration when identifying emergent pathogens.

Molecular Epidemiology Reveals Genetic Diversity amongst Isolates of the Cryptococcus neoformans/C. gattii Species Complex in Thailand

To gain a more detailed picture of cryptococcosis in Thailand, a retrospective study of 498 C. neoformans and C. gattii isolates has been conducted. Among these, 386, 83 and 29 strains were from clinical, environmental and veterinary sources, respectively. A total of 485 C. neoformans and 13 C. gattii strains were studied. The majority of the strains (68.9%) were isolated from males (mean age of 37.97 years), 88.5% of C. neoformans and only 37.5% of C. gattii strains were from HIV patients. URA5-RFLP and/or M13 PCR-fingerprinting analysis revealed that the majority of the isolates were C. neoformans molecular type VNI regardless of their sources (94.8%; 94.6% of the clinical, 98.8% of the environmental and 86.2% of the veterinary isolates). In addition, the molecular types VNII (2.4%; 66.7% of the clinical and 33.3% of the veterinary isolates), VNIV (0.2%; 100% environmental isolate), VGI (0.2%; 100% clinical isolate) and VGII (2.4%; 100% clinical isolates) were found less frequently. Multilocus Sequence Type (MLST) analysis using the ISHAM consensus MLST scheme for the C. neoformans/C. gattii species complex identified a total of 20 sequence types (ST) in Thailand combining current and previous data. The Thai isolates are an integrated part of the global cryptococcal population genetic structure, with ST30 for C. gattii and ST82, ST83, ST137, ST141, ST172 and ST173 for C. neoformans being unique to Thailand. Most of the C. gattii isolates were ST7 = VGIIb, which is identical to the less virulent minor Vancouver island outbreak genotype, indicating Thailand as a stepping stone in the global spread of this outbreak strain. The current study revealed a greater genetic diversity and a wider range of major molecular types being present amongst Thai cryptococcal isolates than previously reported.

Genetic diversity ofCoccidioides posadasiifrom Brazil

Studies of the genetic variation within populations of Coccidioides posadasii are scarce, especially for those recovered from South America. Understanding the distribution of genotypes among populations is important for epidemiological surveillance. This study evaluated the genetic diversity of 18 Brazilian strains of C. posadasii through the sequencing of the 18-28S region of nuclear rDNA, as well as through RAPD and M13-PCR fingerprinting techniques. The sequences obtained were compared to Coccidioides spp. previously deposited in GenBank. The MEGA5 program was used to perform phylogenetic analyses. Within the C. posadasii clade, a single cluster was observed, containing seven isolates from Ceará, which presented a single nucleotide polymorphism. These isolates were from the same geographical area. The strains of C. posadasii showed a lower rate of genetic diversity in the ITS1 and ITS2 regions. The results of M13 and RAPD-PCR fingerprinting indicated a similar electrophoretic profile. No differences between clinical and environmental isolates were detected. This was the first study assessing the genetic variability of a larger number of C. posadasii isolates from Brazil.

Identification of Novel Hybrids Between Cryptococcus neoformans var. grubii VNI and Cryptococcus gattii VGII

Cryptococcus neoformans and Cryptococcus gattii are pathogenic yeasts causing meningoencephalitis in immunocompromised and immunocompetent hosts. The fungus is typically haploid, and sexual reproduction occurs normally between individuals with opposite mating types, α and a. C. neoformans var. grubii (serotype A) is comprised of molecular types VNI, VNII, and VNB, and C. neoformans var. neoformans (serotype D) contains the molecular type VNIV. Additionally, diploid or aneuploid AD hybrids (VNIII) have been reported. C. gattii contains the molecular types VGI, VGII, VGIII, and VGIV, which encompass both serotypes B and C. To identify possible hybrid strains, URA5-RFLP analysis was performed on 350 globally obtained clinical, environmental, and veterinary isolates. Four clinical isolates from cerebrospinal fluid showed combination patterns of C. neoformans var. grubii and C. gattii: Brazil (n = 2), Colombia (n = 1), and India (n = 1). These strains were monokaryotic and diploid or aneuploid. M13 PCR fingerprinting showed that they contained fragments of both proposed parental groups. Luminex IGS genotyping identified these isolates as hybrids with two different molecular type combinations: three VNI/VGII and one VNI/VGI. Blue color development on CGB agar was delayed in three isolates and absent in one. C. gattii-specific PCR confirmed the presence of C. gattii in the hybrids. CAP59 allele-specific PCR revealed that all the hybrids contained both serotype A and B alleles. Determination of mating-type allelic patterns by PCR revealed that the isolates were αA aB. This is the first study discovering novel natural hybrids between C. neoformans molecular type VNI and C. gattii molecular type VGII.

Genomic diversity of Oenococcus oeni from different winemaking regions of Portugal.

Oenococcus oeni is an alcohol-tolerant, acidophilic lactic acid bacterium that plays an important role in the elaboration of wine. It is often added as a starter culture to carry out malolactic conversion. Given the economic importance of this reaction, the taxonomic structure of this species has been studied in detail. In the present work, phenotypic and molecular approaches were used to identify 121 lactic acid bacteria strains isolated from the wines of three winemaking regions of Portugal. The strains were differentiated at the genomic level by M13-PCR fingerprinting. Twenty-seven genomic clusters represented by two or more isolates and 21 single-member clusters, based on an 85% similarity level, were recognized by hierarchic numerical analysis. M13-PCR fingerprinting patterns revealed a high level of intraspecific genomic diversity in O. oeni. Moreover, this diversity could be partitioned according to the geographical origin of the isolates. Thus, M13-PCR fingerprint analysis may be an appropriate methodology to study the O. oeni ecology of wine during malolactic fermentation as well as to trace new malolactic starter cultures and evaluate their dominance over the native microbiota.

Comparison of Different DNA-Based Methods for Molecular Typing of Histoplasma capsulatum

Histoplasma capsulatum is very prevalent in the environment and is one of the most common causes of mycoses in humans and diverse animals in Brazil. Multiple typing methods have been developed to study H. capsulatum epidemiology; however, there is limited information concerning comparisons of results obtained with different methods using the same set of isolates. To explore the diversity of H. capsulatum in Brazil and to determine correlations between the results of three different molecular typing techniques, we examined 51 environmental, animal, and human isolates by M13 PCR fingerprinting, PCR-restriction fragment length polymorphism (RFLP) analysis of the internal transcribed region 1 (ITS1)-5.8S-ITS2 region of the rDNA locus, and DNA sequencing and phylogenetic analysis of parts of four protein-encoding genes, the Arf (ADP ribosylation factor), H-anti (H antigen precursor), Ole (delta-9 fatty acid desaturase), and Tub1 (alpha-tubulin) genes. Each method identified three major genetic clusters, and there was a high level of concordance between the results of the typing techniques. The M13 PCR fingerprinting and PCR-RFLP analyses produced very similar results and separated the H. capsulatum isolates included in this study into three major groups. An additional approach used was comparison of our Brazilian ITS1-5.8S-ITS2 sequences with the sequences deposited previously in NCBI data banks. Our analyses suggest that H. capsulatum can be divided into different molecular types that are dispersed around the world. Our results indicate that the three methods used in this study are reliable and reproducible and that they have similar sensitivities. However, M13 PCR fingerprinting has some advantages over the other two methods as it is faster, cheaper, and more user friendly, which especially increases its utility for molecular typing of Histoplasma in situations where laboratory facilities are relatively limited.

Identification of Sinorhizobium (Ensifer) medicae based on a specific genomic sequence unveiled by M13-PCR fingerprinting.

A collection of nodule isolates from Medicago polymorpha obtained from southern and central Portugal was evaluated by M13-PCR fingerprinting and hierarchical cluster analysis. Several genomic clusters were obtained which, by 16S rRNA gene sequencing of selected representatives, were shown to be associated with particular taxonomic groups of rhizobia and other soil bacteria. The method provided a clear separation between rhizobia and co-isolated non-symbiotic soil contaminants. Ten M13-PCR groups were assigned to Sinorhizobium (Ensifer) medicae and included all isolates responsible for the formation of nitrogen-fixing nodules upon re-inoculation of M. polymorpha test-plants. In addition, enterobacterial repetitive intergenic consensus (ERIC)-PCR fingerprinting indicated a high genomic heterogeneity within the major M13- PCR clusters of S. medicae isolates. Based on nucleotide sequence data of an M13-PCR amplicon of ca. 1500 bp, observed only in S. medicae isolates and spanning locus Smed_3707 to Smed_3709 from the pSMED01 plasmid sequence of S. medicae WSM419 genome's sequence, a pair of PCR primers was designed and used for direct PCR amplification of a 1399-bp sequence within this fragment. Additional in silico and in vitro experiments, as well as phylogenetic analysis, confirmed the specificity of this primer combination and therefore the reliability of this approach in the prompt identification of S. medicae isolates and their distinction from other soil bacteria.

Grana Padano cheese whey starters: Microbial composition and strain distribution

The aim of this work was to evaluate the species composition and the genotypic strain heterogeneity of dominant lactic acid bacteria (LAB) isolated from whey starter cultures used to manufacture Grana Padano cheese. Twenty-four Grana Padano cheese whey starters collected from dairies located over a wide geographic production area in the north of Italy were analyzed. Total thermophilic LAB streptococci and lactobacilli were quantified by agar plate counting. Population structure of the dominant and metabolically active LAB species present in the starters was profiled by reverse transcriptase, length heterogeneity-PCR (RT–LH–PCR), a culture-independent technique successfully applied to study whey starter ecosystems. The dominant bacterial species were Lactobacillus helveticus, Lactobacillus delbrueckii subsp. lactis, Streptococcus thermophilus, and Lactobacillus fermentum. Diversity in the species composition allowed the whey cultures to be grouped into four main typologies, the one containing L. helveticus, L. delbrueckii subsp. lactis, and S. thermophilus being the most frequent one (45% of the cultures analyzed), followed by that containing only the two lactobacilli (40%). Only a minor fraction of the cultures contained L. helveticus alone (4%) or all the four LAB species (11%). Five hundred and twelve strains were isolated from the 24 cultures and identified by M13-PCR fingerprinting coupled with 16S rRNA gene sequencing. Most of the strains were L. helveticus (190 strains; 37% of the total), L delbrueckii subsp. lactis (90 strains; 18%) and S. thermophilus (215 strains; 42%). This result was in good agreement with the qualitative whey starter composition observed by RT–LH–PCR. M13-PCR fingerprinting indicated a markedly low infra-species diversity, i.e. the same biotypes were often found in more than one culture. The distribution of the biotypes into the different cultures was mainly dairy plant-specific rather than correlated with the different production areas.

Molecular typing of Australian Scedosporium isolates showing genetic variability and numerous S. aurantiacum.

One hundred clinical isolates from a prospective nationwide study of scedosporiosis in Australia (2003-2005) and 46 additional isolates were genotyped by internal transcribed spacer-restriction fragment length polymorphism (ITS-RFLP) analysis, ITS sequencing, and M13 PCR fingerprinting. ITS-RFLP and PCR fingerprinting identified 3 distinct genetic groups. The first group corresponded to Scedosporium prolificans (n = 83), and the other 2 comprised isolates previously identified as S. apiospermum: one of these corresponded to S. apiospermum (n = 33) and the other to the newly described species S. aurantiacum (n = 30). Intraspecies variation was highest for S. apiospermum (58%), followed by S. prolificans (45%) and S. aurantiacum (28%) as determined by PCR fingerprinting. ITS sequence variation of 2.2% was observed among S. apiospermum isolates. No correlation was found between genotype of strains and their geographic origin, body site from which they were cultured, or colonization versus invasive disease. Twelve S. prolificans isolates from 2 suspected case clusters were examined by amplified fragment length polymorphism analysis. No specific clusters were confirmed.

Molecular characterization of Cylindrospermopsis raciborskii strains isolated from Portuguese freshwaters

Cylindrospermopsis raciborskii is a toxic bloom forming cyanobacteria that is a common component of the phytoplankton assemblage in temperate freshwaters, as well as in temperate climates. This species is of major concern in public health, due to its known ability to produce toxins, including cylindrospermopsin and paralytic shellfish poisoning toxin (PSP). In this study, M13 PCR fingerprinting, ERIC PCR fingerprinting and amplification of the internal transcribed spacer (ITS) region were used to characterize nine cultured strains of C. raciborskii, sourced from several freshwater lakes and rivers in Portugal, and two other Australian. Strains belonging to other taxa including Microcystis aeruginosa, Aphanizomenon spp., Planktothrix agardhii and Oscillatoria neglecta were also analysed to evaluate the taxonomical potential of the fingerprinting methods. Data obtained from genomic fingerprinting were used to perform hierarchical cluster analysis and demonstrated ability to differentiate strains at intra-specific level. However, the high level of variability prevents their use as an identification tool. ITS amplification displayed intra-specific polymorphism both in number and length of the obtained amplicons, but revealed itself as a good method for strain clustering. The unsuccessful amplification of peptide synthetase (PS) and polyketide synthase (PKS) genes pointed to the inability of Portuguese C. raciborskii strains to produce cylindrospermopsin. HPLC analysis further confirmed this lack of toxicity, since negative results were obtained for cylindrospermopsin and PSP toxins.

M13 DNA fingerprinting can be used in studies on phenotypic reversions of forest tree mutants

Solitary revertants which have been observed on single mutant tree individuals have up to now been believed to be grow-through cells belonging to the rootstock on which they are commonly grafted. In this study three different phenotypically visible mutants revealing revertant shoots on the same tree were chosen for genetic analysis. The mutant Quercus robur L. ‘argenteomarginata’ was grafted on a normal rootstock, an individual of Carpinus betulus L. var. quercifolia Desf. as well as an individual of Picea glauca (Moench) Voss. ‘conica’ are supposed to have grown from seeds. By means of a highly specific M13 PCR fingerprinting technique the mutant and revertant tissues were analysed in comparison to different individuals of each of the species. With the grafted mutant, cambium tissue of the rootstock was also investigated. Whereas conspecific individuals could be clearly distinguished from each other, mutant and revertant tissues revealed the same banding patterns for each of the three trees. In case of the grafted mutant, the fingerprint obtained from cambium tissue of the rootstock was clearly different from the pattern of mutant and revertant tissue. Results demonstrate the potential of the tool for genetic differentiation between individuals of three tree species hence in the case of the grafted mutant, the hypothesis that the observed reversion is caused by a grow-through of the rootstock is rejected. Furthermore, identical fingerprints of mutant and revertant tissue support identical genetic background of the tissues excluding the gene(s) responsible of the mutation. Possible causes of mutations and reversions regarding the three mutant trees are discussed.