Internal Transcribed Spacer Types Research Articles

Polymorphisms in the ITS rDNA regions for differentiating strains of the Trichophyton mentagrophytes complex in Sfax‐Tunisia

The Trichophyton mentagrophytes complex is the main cause of superficial mycoses in humans and animals. Molecular research has provided useful insights into the taxonomy of this complex to overcome the challenges with conventional diagnostics. The aim of this study was to identify, type and differentiate anthropophilic and zoophilic species of the T. mentagrophytes complex. Sixty clinical samples identified as T. mentagrophytes by morphological characteristics were isolated using polymerase chain reaction-restriction fragment length polymorphism and sequence analysis of the internal transcribed spacer (ITS) regions. The identification of our strains by conventional methods was confirmed using polymerase chain reaction (PCR) sequencing in 93.34% of the cases. The strains under investigation were recategorised as T. rubrum (Tr2711). In addition, PCR products were independently digested with the restriction endonucleases, MvaI and HinfI, to produce a single dominant profile for T. interdigitale. ITS sequence analysis revealed a polymorphism in the ITS1 and 5.8S regions. Analysis of the consensus sequences distinguished four types of genotypes among our T. interdigitale species. Moreover, ITS type I was the dominant genotype characterising the anthropophilic variant of T. interdigitale. The phylogenetic study showed that only 5% of our strains were zoophilic. PCR sequencing was useful for distinguishing anthropophilic and zoophilic species of T. interdigitale, in which the differentiation is relevant because it helps to prescribe the correct treatment and to identify the surrounding source of infection.

Identification, typing, antifungal resistance profile, and biofilm formation of Candida albicans isolates from Lebanese hospital patients.

As leading opportunistic fungal pathogens identification and subtyping of Candida species are crucial in recognizing outbreaks of infection, recognizing particularly virulent strains, and detecting the emergence of drug resistant strains. In this study our objective was to compare identification of Candida albicans by the hospitals through the use of conventional versus identification based on the ITS (Internal Transcribed Spacer) and to assess biofilm forming capabilities, drug resistance patterns and correlate these with MLST typing. ITS typing revealed a 21.2% hospital misidentification rate. Multidrug resistance to three drugs out of four tested was detected within 25% of the isolates raising concerns about the followed treatment regimens. Drug resistant strains as well as biofilm formers were phylogenetically related, with some isolates with significant biofilm forming capabilities being correlated to those that were multidrug resistant. Such isolates were grouped closely together in a neighbor-joining tree generated by MLST typing indicating phylogenetic relatedness, microevolution, or recurrent infection. In conclusion, this pilot study gives much needed insight concerning C. albicans isolates circulating in Lebanese hospitals and is the first study of its kind correlating biofilm formation, antifungal resistance, and evolutionary relatedness.

Should I stay or should I go: biogeographic and evolutionary history of a polyploid complex (Chrysanthemum indicum complex) in response to Pleistocene climate change in China

Quaternary climatic oscillations greatly influenced the distribution and pattern of biodiversity in the Northern Hemisphere. Here we examine how such oscillations in South East Asia may have affected the demographic and evolutionary history of a polyploid plant complex associated with semi-dry habitats. We analyzed plastid and nuclear ribosomal DNA (rDNA) internal transcribed spacer (ITS) sequence variation within the Chrysanthemum indicum complex (Asteraceae), which comprises diploid and polyploid plants distributed throughout China. In total, 368 individuals from 47 populations across the geographical range of the complex were analyzed. We show that the relatively widespread tetraploid form of C.indicum expanded its range southward in the Pleistocene, possibly during the most recent or previous glacial period when conditions became drier and forests retreated in southern China. In marked contrast, diploid and other polyploid members of the complex failed to expand their ranges at these times or have since undergone range contractions in contrast to tetraploid C.indicum. We conclude that hybridization and gene flow between taxa occurred frequently during the evolutionary history of the complex, causing considerable sharing of chlorotypes and ITS types. Nevertheless, taxa within ploidy levels could be largely distinguished according to chlorotype and/or ITS type.

Genetic structure of Ipomoea imperati (Convolvulaceae) in the Mediterranean region and implications for its conservation

In this paper, we studied the relationships of the only surviving Italian population of Ipomoea imperati (Convolvulaceae), a pantropical sandy coastal species, in Sicily and other populations in the Mediterranean region. Herbarium samples which are representative of extinct populations growing in Campania (Italy) were also investigated together with populations from various Atlantic and Mediterranean localities. Chloroplast DNA microsatellites (cp-SSR) and nuclear ribosomal Internal Transcribed Spacer (ITS) sequences were jointly employed, in order to detect relationships among populations. Our aims were several-fold: (1) to clarify if the species is autochthonous in the Mediterranean basin or a post-Columbian introduction; (2) to investigate phylogeographic patterns in the species and (3) to establish the possible role of dispersal in explaining the patterns observed. Chloroplast microsatellite variation indicates that extinct Italian mainland populations of I. imperati from Campania are not closely related to the extant Sicilian one, as they do not share haplotypes. Chloroplast DNA microsatellite variation is largely between populations, and the within populations component accounts for only approximately 10%. CpDNA data is consistent with a single Mediterranean entry point hypothesis or with the notion that some populations display plesiomorphic variability. ITS data is congruent with the possibility that the presence of I. imperati in the Mediterranean is the result of transatlantic dispersal. The population from Sicily and extinct populations from Campania share an ITS type. A Bayesian analysis employing clock calibration data on an expanded ITS dataset with appropriate outgroups indicates that dates of transoceanic distribution are probably earlier than historical times.

Pneumocystis jiroveciihaplotypes at the internal transcribed spacers of the rRNA operon in French HIV-negative patients with diverse clinical presentations ofPneumocystisinfections

Pneumocystis jirovecii, a transmissible fungus, is the causative agent of pulmonary infections. Its genomic diversity has appeared in reports from around the world but data on P. jirovecii genotypes in France are still limited. This study describes the typing of P. jirovecii isolates from 81 HIV-negative patients monitored at Brest University Hospital, Brittany, France, 40 of whom developed Pneumocystis pneumonia (PcP), and remaining 41 patients were colonized by the fungus. The isolates were assayed at the internal transcribed spacer (ITS)1 and ITS2 under improved amplification conditions to avoid in vitro ITS recombination. P. jirovecii ITS haplotypes were identified in 56/81 patients (31 PcP patients and 25 patients who were colonized) which revealed a high diversity in that 27 different haplotypes were identified. Eg was the most frequent haplotype (31/56, 55.3%), followed by Ec and Ai (5/56, 8.9% each). In contrast, Ne, usually the second most frequent haplotype in Europe and the USA, was observed in only 2/56 patients (3.6%). Mixed infections were detected in 18/56 patients (32.1%; 12 PcP patients and six who were colonized). No significant differences were observed in haplotype diversity, frequency of peculiar haplotypes, and mixed infection occurrence, between the two patient populations. The study, conducted with the largest HIV-negative patient population investigated so far, shows that ITS typing remains an efficient method for characterizing P. jirovecii among human populations, whatever their clinical presentation of Pneumocystis infections.

Genetic characterization of UCS region of Pneumocystis jirovecii and construction of allelic profiles of Indian isolates based on sequence typing at three regions

Pneumocystis jirovecii is an opportunistic pathogen that causes severe pneumonia in immunocompromised patients. To study the genetic diversity of P. jirovecii in India the upstream conserved sequence (UCS) region of Pneumocystis genome was amplified, sequenced and genotyped from a set of respiratory specimens obtained from 50 patients with a positive result for nested mitochondrial large subunit ribosomal RNA (mtLSU rRNA) PCR during the years 2005–2008. Of these 50 cases, 45 showed a positive PCR for UCS region. Variations in the tandem repeats in UCS region were characterized by sequencing all the positive cases. Of the 45 cases, one case showed five repeats, 11 cases showed four repeats, 29 cases showed three repeats and four cases showed two repeats. By running amplified DNA from all these cases on a high-resolution gel, mixed infection was observed in 12 cases (26.7%, 12/45). Forty three of 45 cases included in this study had previously been typed at mtLSU rRNA and internal transcribed spacer (ITS) region by our group. In the present study, the genotypes at those two regions were combined with UCS repeat patterns to construct allelic profiles of 43 cases. A total of 36 allelic profiles were observed in 43 isolates indicating high genetic variability. A statistically significant association was observed between mtLSU rRNA genotype 1, ITS type Ea and UCS repeat pattern 4.

Analysis of 16S–23S rRNA internal transcribed spacer regions in Pasteurellaceae isolated from laboratory rodents

The internal transcribed spacer (ITS) regions of members of Pasteurellaceae isolated from rodents, including the [Pasteurella] pneumotropica biotypes Jawetz and Heyl, [Actinobacillus] muris, “Hemophilus influenzaemurium” and Bisgaard taxon 17 were studied and their feasibility to discriminate these species was analyzed. The reference strains of all species analyzed showed unique species‐specific ITS patterns which were further present in 49 clinical isolates of [P.] pneumotropica biotypes Jawetz and Heyl and [A.] muris allowing their identification by comparison to the reference strains pattern. Sequence analysis of the amplified fragments revealed in all species, with exception of “H. influenzaemurium”, a larger ITSile+ala which contained the genes for tRNAIle(GAU) and tRNAAla(UGC) and a smaller ITSglu with the tRNAGlu(UUC) gene. “H. influenzaemurium” revealed two each of the larger and respectively the smaller ITS fragments. Both the length and the sequence of each ITS type were highly conserved within the [P.] pneumotropica biotypes Jawetz and Heyl and [A.] muris strains tested. On the contrary, ITS sequences revealed significant interspecies variations with identity levels ranging from 61.2 to 89.5% for ITSile+ala and 56.5 to 86.8% for ITSglu. Sequences regions with significant interspecies variation but highly conserved within the species were identified and might be used to design probes for the identification of rodent Pasteurellaceae to the species level.

A Cluster of Pneumocystis Infections Among Renal Transplant Recipients: Molecular Evidence of Colonized Patients as Potential Infectious Sources of Pneumocystis jirovecii

Eighteen renal transplant recipients (RTRs) developed Pneumocystis jirovecii infections at the renal transplantation unit of Brest University Hospital (Brest, Brittany, France) from May 2008 through April 2010, whereas no cases of P. jirovecii infection had been diagnosed in this unit since 2002. This outbreak was investigated by identifying P. jirovecii types and analyzing patient encounters. The identification of P. jirovecii internal transcribed spacer (ITS) types was performed on P. jirovecii isolates from the 18 RTRs (12 patients with Pneumocystis pneumonia [PCP], 6 colonized patients), 22 unlinked control patients (18 patients with PCP, 4 colonized patients), and 69 patients (34 patients with PCP, 35 colonized patients) with contemporaneously diagnosed P. jirovecii infections in the Brest geographic area. A transmission map was drawn up. Its analysis was combined with the results of P. jirovecii typing. P. jirovecii ITS type identification was successful in 14 of 18 RTRs, 15 of 22 control patients, and 48 of the 69 patients. Type Eg was the most frequent type in the 3 patient groups. However, its frequency was significantly higher in the first patient group than in the 2 other groups (P < .05 and P < .01, respectively). Fourteen encounters between RTRs who harbored an identical type were observed. Ten patients were considered as possible index patients, of whom 3 were colonized by the fungus, and 7 presented PCP. The results provide to our knowledge the first data on the role of colonized patients as potential sources of P. jirovecii in a context of nosocomial acquisition of the fungus.

Diversity of 16S-23S rDNA Internal Transcribed Spacer (ITS) Reveals Phylogenetic Relationships in Burkholderia pseudomallei and Its Near-Neighbors

Length polymorphisms within the 16S-23S ribosomal DNA internal transcribed spacer (ITS) have been described as stable genetic markers for studying bacterial phylogenetics. In this study, we used these genetic markers to investigate phylogenetic relationships in Burkholderia pseudomallei and its near-relative species. B. pseudomallei is known as one of the most genetically recombined bacterial species. In silico analysis of multiple B. pseudomallei genomes revealed approximately four homologous rRNA operons and ITS length polymorphisms therein. We characterized ITS distribution using PCR and analyzed via a high-throughput capillary electrophoresis in 1,191 B. pseudomallei strains. Three major ITS types were identified, two of which were commonly found in most B. pseudomallei strains from the endemic areas, whereas the third one was significantly correlated with worldwide sporadic strains. Interestingly, mixtures of the two common ITS types were observed within the same strains, and at a greater incidence in Thailand than Australia suggesting that genetic recombination causes the ITS variation within species, with greater recombination frequency in Thailand. In addition, the B. mallei ITS type was common to B. pseudomallei, providing further support that B. mallei is a clone of B. pseudomallei. Other B. pseudomallei near-neighbors possessed unique and monomorphic ITS types. Our data shed light on evolutionary patterns of B. pseudomallei and its near relative species.

Lack of phylogeographic structure in the freshwater cyanobacterium Microcystis aeruginosa suggests global dispersal.

BackgroundFree-living microorganisms have long been assumed to have ubiquitous distributions with little biogeographic signature because they typically exhibit high dispersal potential and large population sizes. However, molecular data provide contrasting results and it is far from clear to what extent dispersal limitation determines geographic structuring of microbial populations. We aimed to determine biogeographical patterns of the bloom-forming freshwater cyanobacterium Microcystis aeruginosa. Being widely distributed on a global scale but patchily on a regional scale, this prokaryote is an ideal model organism to study microbial dispersal and biogeography.Methodology/Principal FindingsThe phylogeography of M. aeruginosa was studied based on a dataset of 311 rDNA internal transcribed spacer (ITS) sequences sampled from six continents. Richness of ITS sequences was high (239 ITS types were detected). Genetic divergence among ITS types averaged 4% (maximum pairwise divergence was 13%). Preliminary analyses revealed nearly completely unresolved phylogenetic relationships and a lack of genetic structure among all sequences due to extensive homoplasy at multiple hypervariable sites. After correcting for this, still no clear phylogeographic structure was detected, and no pattern of isolation by distance was found on a global scale. Concomitantly, genetic differentiation among continents was marginal, whereas variation within continents was high and was mostly shared with all other continents. Similarly, no genetic structure across climate zones was detected.Conclusions/SignificanceThe high overall diversity and wide global distribution of common ITS types in combination with the lack of phylogeographic structure suggest that intercontinental dispersal of M. aeruginosa ITS types is not rare, and that this species might have a truly cosmopolitan distribution.

A limited number of ITS haplotypes defines the diversity of Pneumocystis jirovecii strains in Sweden

The infectious fungus Pneumocystis jirovecii remains an important cause of pneumonia (PCP) in the immunocompromised host. To study the biodiversity of P. jirovecii in Sweden the internal transcribed spacers (ITS) of the rDNA locus were amplified, cloned and sequenced from a set of diagnostic respiratory specimens obtained from 64 patients with P. jirovecii infection during the years 1996–2003. The analysis of 408 cloned sequences from amplified products resulted in the identification of 10 ITS1 and 12 ITS2 established genotypes. Twelve ITS haplotypes (combinations of ITS1 and ITS2) were identified of which nine were found to recur during the time span of the study. Haplotype Eg was the most common, followed by Ne, Bi and Eb. A new ITS2 genotype denoted v was identified in specimens from four patients. There was no association between ITS haplotype and patient age, sex, underlying disease or geographical origin. Shannon and Simpson index analysis revealed no difference in diversity in Sweden compared to other countries studied and no changes in diversity were found during the study period in Sweden. Criteria were defined to enable the discrimination of genuine ITS types and a more accurate assessment of the previously overestimated genetic diversity of P. jirovecii populations. A model depicting the phylogenetic and genealogic relationships in a revised set of global types of this fungus is presented.

Genetic diversity of Microcystis blooms (Cyanobacteria) in recently constructed reservoirs in Tigray (Northern Ethiopia) assessed by rDNA ITS

The cyanobacterium Microcystis is notorious for forming extensive and potentially toxic blooms in nutrient-rich freshwater bodies worldwide. However, little is known about the factors underlying the genetic diversity and structure of natural Microcystis populations, despite the fact that this knowledge is essential to understand the build-up of blooms. Microcystis blooms are common and occur year-round in Africa, but are underinvestigated in this continent. We studied the genetic diversity and structure of Microcystis populations in 30 man-made reservoirs in Tigray (Northern Ethiopia) using Denaturing Gradient Gel Electrophoresis of the 16S–23S rDNA internal transcribed spacer (ITS) region and assessed the importance of local environmental conditions and geographic position of the reservoirs for the observed patterns. The analyses showed that both regional and local Microcystis ITS diversity in these recently constructed reservoirs was relatively low, with several dense blooms containing only a single ITS type. Especially one non-toxic ITS type dominated a considerable fraction of Microcystis blooms, but appeared restricted in its geographic distribution. The relationship between Microcystis ITS population structure and abiotic variables (water clarity, pH) and with zooplankton (Daphnia biomass) indicates a (limited) influence of environmental conditions on Microcystis population structure in the reservoirs of Tigray.

Evolutionary history of an alpine shrub Hippophae tibetana (Elaeagnaceae): allopatric divergence and regional expansion

Increasing evidence suggests that geological or climatic events in the past promoted allopatric speciation of alpine plants in the Qinghai-Tibetan Plateau and adjacent region. However, few studies have been undertaken to examine whether such allopatric divergences also occurred within a morphologically uniform species. In the present study, we report the evolutionary history of an alpine shrub species, Hippophae tibetana, based on examining chloroplast DNA (cpDNA) and nuclear ribosomal internal transcribed spacer (ITS) DNA variations. We sequenced two cpDNA fragments (trnL-F and trnS-G) and the nuclear ITS region in 183 individuals collected from 21 natural populations. Ten chlorotypes and 17 ITS types were identified. Phylogenetic analyses of both chlorotypes and ITS sequence variations suggested two distinct lineages distributed in the eastern and western region, respectively. On the basis of the fast and low plant substitution rates, these two lineages were estimated to have diverged from each other between 1 and 4 million years ago, during the period of the major glaciations and orogenic processes. In addition, ITS has undergone the accelerated evolution in two populations in the southern Himalaya isolated by the high mountains with a surprising accumulation of the private variations. The east-west split was also supported by an analysis of molecular variance, which partitioned around 91% of the total cpDNA variance between these two groups of populations. A single chlorotype was found for most populations in eastern or western region, suggesting a recent postglacial expansion within each region. Star-phylogeny and mismatch analyses of all chlorotypes within the eastern group of populations suggested an earlier regional expansion before the Last Glacial Maximum (LGM). The local fixture of the different chlorotypes in multiple populations suggested more than one refugia remained for eastern or western region. Coalescent tests rejected the hypothesis that all current populations originated from a single refugium during the LGM. Instead, they supported hypothesis that two lineages diverged before the late Pleistocene. These findings, when taken together, suggested that this species had experienced long allopatric divergence and recent regional range expansions in response to orogenic processes and the climate changes. The evolutionary history of this shrub species highlights importance of geographical isolations to the intraspecific divergence of alpine plants occurring in the world's ruff. (c) 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 102, 37-50.

DIVERSITY OF PSEUDO-NITZSCHIA IN THE SOUTHEASTERN BAY OF BISCAY

Ten species of the genus Pseudo-nitzschia were identified in the Nervion River estuary from preserved net tow samples and 31 strains isolated from the estuary. Species identification was performed by means of ultrastructural analysis of valve ornamentation and genetic analysis of selected strains of the P. delicatissima complex. Identified species include: P. australis, P. fraudulenta, P. pungens and P. subpacifica from the P. seriata complex, and P. arenysensis, P. galaxiae, P. multistriata, P. pseudodelicatissima and two uncertain P. pseudodelicatissima-like genotypes from the delicatissima complex. Most clonal strains corresponded to P. pseudodelicatissima-like taxa, P. fraudulenta and P. galaxiae. We were unable to identify the strains of the P. delicatissima-like complex solely on the basis of morphology. A comparison of sequences of the rDNA ribosomal Internal Transcribed Spacers (ITS), including the regions ITS1, 5.8S and ITS2, of four strains of the P. delicatissima complex plus 23 sequences of taxonomically related strains available in GenBank, revealed the presence of P. arenysensis among strains of the P. delicatissima-morphologies and three different ITS types among the three P. pseudodelicatissima-like morphologies analysed. One corresponded to P. pseudodelicatissima sensu stricto (strain Ner-D5) whereas the other two (Ner-D6 and Ner-D8) constituted genetically distinct entities, which appeared in the phylogenetic tree as sister taxa to either P. cuspidata, the former, or to P. calliantha and P. mannii, the latter. The genetic differences among these three strains of P. pseudodelicatissima-like morphologies were corroborated by analysing their ITS2 secondary structure and comparing them with the ITS2 secondary structure of phylogenetically related species from GenBank.

Spatial distribution of lineages of oak powdery mildew fungi in France, using quick molecular detection methods

Keywords: Erysiphe alphitoides / Erysiphe quercicola / cryptic species / Erysiphales / ITS / biological invasion Abstract • Powdery mildew is a major fungal disease of oaks in Europe. Recent studies using internal tran- scribed spacer (ITS) sequences suggested the presence of four different lineages (putative species). The objective of the study was to investigate the spatial distribution of these lineages/species and, in particular, to test the hypothesis of a spatial differentiation, at various scales: regional (France), altitudinal (a Pyrenean transect) and local (within a forest plot). • Detection methods for the four ITS types were developed: (1) single strand conformation poly- morphism analysis (SSCP); (2) PCR amplifications, for which specific primers were designed. SSCP proved to be efficient for the detection of Erysiphe alphitoides and E. quercicola types. In contrast, the rarer ITS types corresponding to E. hypophylla and Phyllactinia guttata (sensu lato) were only detected by specific amplification. • The study confirmed the strong predominance of the ITS sequence associated with E. alphitoides at all spatial scales (with a frequency higher than 80%). Isolates presumably belonging to E. quercicola (i.e. with same ITS type), a recently described species not yet recorded in Europe, were also found in all French regions at a significant frequency (15% at national level). • No pattern of spatial differentiation between the putative species could be demonstrated: E. alphi- toides was often found in association with different ITS types in the same region, the same tree, and even in the same lesion.

Sequence variation of the internal transcribed spacer (ITS) region of ribosomal DNA in Cerastoderma species (Bivalvia: Cardiidae)

The internal transcribed spacer (ITS) region of ribosomal DNA (ITS1, 5.8S rRNA gene and ITS2) constitutes one of the most widely applied molecular markers in phylogenetic studies and species differentiation. Here, the ITS region of the cockles Cerastoderma edule from NW Spain and C. glaucum from the Mediterranean and Baltic Seas was characterized and its variation examined. The length of the ITS was 762–783 bp (ITS1, 226–251 bp; 5.8S rRNA gene, 158–161 bp; ITS2, 305–325 bp) and the GC content ranged between 57% and 60% (ITS1, 52–62%; 5.8S rRNA gene, 56–57%; ITS2, 61–63%). All individuals showed variation among ITS repeats, mostly in the spacers, with nucleotide diversity values of 0.0005–0.07. Among the C. glaucum sequences, two types of ITS (ITSI and ITSII) were distinguished, based on the percentage of differences observed in the spacers, sequence of the 5.8S gene, GC content of the ITS1 and clustering in phylogenetic trees. A PCR assay using specific primers for each ITS type demonstrated that both types coexist in all C. glaucum individuals. While type I seems to contain the bona fide 5.8S rRNA gene, type II could contain a pseudogene. Cerastoderma edule sequences grouped separately on phylogenetic trees, as expected for a differentiated species, but those of C. glaucum from the Mediterranean and Baltic Seas were intermixed in the two clades defining the ITS types, which supports the absence of systematic division previously inferred from other molecular markers.

Cryptic species in the Terfezia boudieri complex

Phylogenetic analyses have corroborated the discovery of three internal transcribed spacer (ITS) Types in Terfezia boudieri isolates in the course of earlier studies and have emphasized the divergence of Type 2 from Types 1 and 3. The application of molecular and physiological tools described below, revealed the existence of cryptic species within T. boudieri. The markers used include sequences taken from the 5' end of the ribosomal large subunit gene, a chitin synthase partial sequence, beta-tubulin partial sequence and amplified fragment length polymorphism (AFLP)-based markers. Following initial sequencing of a single PCR amplified sample for each Type, mass analysis of specimens relied on RFLP differences between the Types. Over 100 fruit bodies, 30 or more specimens for each ITS Type, were tested with each of the markers. The markers analysis divided the isolates into three groups, each correlated to a specific ITS Type. Two of the physiological traits examined: mycelial proliferation and mycorrhiza formation, consistently showed responses paralleling the ITS Types; the data presented suggest that T. boudieri is comprised of three cryptic species.

Analysis of the 16S-23S rRNA gene internal transcribed spacer region in Klebsiella species.

The 16S-23S rRNA gene internal transcribed spacer (ITS) regions of Klebsiella spp., including Klebsiella pneumoniae subsp. pneumoniae, Klebsiella pneumoniae subsp. ozaenae, Klebsiella pneumoniae subsp. rhinoscleromatis, Klebsiella oxytoca, Klebsiella planticola, Klebsiella terrigena, and Klebsiella ornithinolytica, were characterized, and the feasibility of using ITS sequences to discriminate Klebsiella species and subspecies was explored. A total of 336 ITS sequences from 21 representative strains and 11 clinical isolates of Klebsiella were sequenced and analyzed. Three distinct ITS types-ITS(none) (without tRNA genes), ITS(glu) [with a tRNA(Glu (UUC)) gene], and ITS(ile+ala) [with tRNA(Ile (GAU)) and tRNA(Ala (UGC)) genes]-were detected in all species except for K. pneumoniae subsp. rhinoscleromatis, which has only ITS(glu) and ITS(ile+ala). The presence of ITS(none) in Enterobacteriaceae had never been reported before. Both the length and the sequence of each ITS type are highly conserved within the species, with identity levels from 0.961 to 1.000 for ITS(none), from 0.967 to 1.000 for ITS(glu), and from 0.968 to 1.000 for ITS(ile+ala). Interspecies sequence identities range from 0.775 to 0.989 for ITS(none), from 0.798 to 0.997 for ITS(glu), and from 0.712 to 0.985 for ITS(ile+ala). Regions with significant interspecies variations but low intraspecies polymorphisms were identified; these may be targeted in the design of probes for the identification of Klebsiella to the species level. Phylogenetic analysis based on ITS regions reveals the relationships among Klebsiella species similarly to that based on 16S rRNA genes.

Pneumocystis jirovecii multilocus genotyping profiles in patients from Portugal and Spain

Pneumonia caused by the opportunistic organism Pneumocystis jirovecii is a clinically important infection affecting AIDS and other immunocompromised patients. The present study aimed to compare and characterise the frequency pattern of DNA sequences from the P. jirovecii mitochondrial large-subunit rRNA (mtLSU rRNA) gene, the dihydropteroate synthase (DHPS) gene and the internal transcribed spacer (ITS) regions of the nuclear rRNA operon in specimens from Lisbon (Portugal) and Seville (Spain). Total DNA was extracted and used for specific molecular sequence analysis of the three loci. In both populations, mtLSU rRNA gene analysis revealed an overall prevalence of genotype 1. In the Portuguese population, genotype 2 was the second most common, followed by genotype 3. Inversely, in the Spanish population, genotype 3 was the second most common, followed by genotype 2. The DHPS wild-type sequence was the genotype observed most frequently in both populations, and the DHPS genotype frequency pattern was identical to distribution patterns revealed in other European studies. ITS types showed a significant diversity in both populations because of the high sequence variability in these genomic regions. The most prevalent ITS type in the Portuguese population was Eg, followed by Cg. In contrast to other European studies, Bi was the most common ITS type in the Spanish samples, followed by Eg. A statistically significant association between mtLSU rRNA genotype 1 and ITS type Eg was revealed.

An intrafamilial transmission ofArthroderma benhamiaein Canadian porcupines (Erethizon dorsatum) in a Japanese zoo

An intra-familial transmission of Arthroderma benhamiae in Canadian porcupines (Erethizon dorsatum) housed in a Japanese zoo was studied. The family consisted of an adult couple and two offspring (a male and a female). The porcupettes, born in Japan, showed severe hair loss while the parent animals, imported from the USA. (male) and Canada (female), showed mild symptoms or were asymptomatic. Morphologically identical Tricophyton spp. isolates were recovered within seven days from quills of all animals on chloramphenicol-supplemented potato dextrose agar plates incubated at 37 degrees C. Two representative colonies from each animal were identified as Arthroderma benhamiae Americano-European race based on mating type (+) and the internal transcribed spacer (ITS) 1-5.5S-ITS 2 region of the rRNA gene sequences (AB236404-AB236408). The present cases constituted the second isolation of dermatophytes from porcupines. There were two different ITS types, i.e., the predominant one isolated from all animals and a secondary one recovered from only the mother porcupine. The sequences have never been recorded in Japan or in the GenBank database to the best of our knowledge. In addition, they were located at a cluster involving the type strain and mating strains of A. benhamiae Americano-European race and its F1 progeny. In contrast, 28 rodents (eight species) and three insectivora (1 species) exhibited in the petting zoo were negative for any dermatophytes as determined by culture.