Exosomes induce endolysosomal permeabilization as a gateway by which exosomal tau seeds escape into the cytosol.

The microtubule-associated protein tau has a critical role in Alzheimer's disease and other tauopathies. A proposed pathomechanism in the progression of tauopathies is the trans-synaptic spreading of tau seeds, with a role for exosomes which are secretory nanovesicles generated by late endosomes. Our previous work demonstrated that brain-derived exosomes isolated from tau transgenic rTg4510 mice encapsulate tau seeds with the ability to induce tau aggregation in recipient cells. We had also shown that exosomes can hijack the endosomal pathway to spread through interconnected neurons. Here, we reveal how tau seeds contained within internalized exosomes exploit mechanisms of lysosomal degradation to escape the endosome and induce tau aggregation in the cytosol of HEK293T-derived 'tau biosensor cells'. We found that the majority of the exosome-containing endosomes fused with lysosomes to form endolysosomes. Exosomes induced their permeabilization, irrespective of the presence of tau seeds, or whether the exosomal preparations originated from mouse brains or HEK293T cells. We also found that permeabilization is a conserved mechanism, operating in both non-neuronal tau biosensor cells and primary neurons. However, permeabilization of endolysosomes only occurred in a small fraction of cells, which supports the notion that permeabilization occurs by a thresholded mechanism. Interestingly, tau aggregation was only induced in cells that exhibited permeabilization, presenting this as an escape route of exosomal tau seeds into the cytosol. Overexpression of RAB7, which is required for the formation of endolysosomes, strongly increased tau aggregation. Conversely, inhibition of lysosomal function with alkalinizing agents, or by knocking-down RAB7, decreased tau aggregation. Together, we conclude that the enzymatic activities of lysosomes permeabilize exosomal and endosomal membranes, thereby facilitating access of exosomal tau seeds to cytosolic tau to induce its aggregation. Our data underscore the importance of endosomal membrane integrity in mechanisms of cellular invasion by misfolded proteins that are resistant to lysosomal degradation.

Extracellular Vesicles Isolated from the Brains of rTg4510 Mice Seed Tau Protein Aggregation in a Threshold-dependent Manner.

The microtubule-associated protein tau has a critical role in Alzheimer disease and related tauopathies. There is accumulating evidence that tau aggregates spread and replicate in a prion-like manner, with the uptake of pathological tau seeds causing misfolding and aggregation of monomeric tau in recipient cells. Here we focused on small extracellular vesicles enriched for exosomes that were isolated from the brains of tau transgenic rTg4510 and control mice. We found that these extracellular vesicles contained tau, although the levels were significantly higher in transgenic mice that have a pronounced tau pathology. Tau in the vesicles was differentially phosphorylated, although to a lower degree than in the brain cells from which they were derived. Several phospho-epitopes (AT8, AT100, and AT180) thought to be critical for tau pathology were undetected in extracellular vesicles. Despite this, when assayed with FRET tau biosensor cells, extracellular vesicles derived from transgenic mice were capable of seeding tau aggregation in a threshold-dependent manner. We also observed that the dye used to label extracellular vesicle membranes was still present during nucleation and formation of tau inclusions, suggesting either a role for membranes in the seeding or in the process of degradation. Together, we clearly demonstrate that extracellular vesicles can transmit tau pathology. This indicates a role for extracellular vesicles in the transmission and spreading of tau pathology. The characteristics of tau in extracellular vesicles and the seeding threshold we identified may explain why tau pathology develops very slowly in neurodegenerative diseases such as Alzheimer disease.

Tau aggregation and its interplay with amyloid-ß.

Neurofibrillary tangles and amyloid plaques constitute the hallmark brain lesions of Alzheimer's disease (AD) patients. Tangles are composed of fibrillar aggregates of the microtubule-associated protein tau, and plaques comprise fibrillar forms of a proteolytic cleavage product, amyloid-ß (Aß). Although plaques and tangles are the end-stage lesions in AD, small oligomers of Aß and tau are now receiving increased attention as they are shown to correlate best with neurotoxicity. One key question of debate, however, is which of these pathologies appears first and hence is upstream in the pathocascade. Studies suggest that there is an intense crosstalk between the two molecules and, based on work in animal models, there is increasing evidence that Aß, at least in part, exerts its toxicity via tau, with the Src kinase Fyn playing a crucial role in this process. In other experimental paradigms, Aß and tau have been found to exert both separate and synergistic modes of toxicity. The challenge, however, is to integrate these different scenarios into a coherent picture. Furthermore, the ability of therapeutic interventions targeting just one of these molecules, to successfully neutralize the toxicity of the other, needs to be ascertained to improve current therapeutic strategies, such as immunotherapy, for the treatment of AD. Although this article is not intended to provide a comprehensive review of the currently pursued therapeutic strategies, we will discuss what has been achieved by immunotherapy and, in particular, how the inherent limitations of this approach can possibly be overcome by novel strategies that involve single-chain antibodies.

Identification of unsafe human induced pluripotent stem cell lines using a robust surrogate assay for pluripotency.

Human induced pluripotent stem cells (hiPSC) have the potential to generate healthy cells and tissues for the study and medical treatment of a large number of diseases. The utility of putative hiPSC-based therapies is constrained by a lack of robust quality-control assays that address the stability of the cells or their capacity to form teratomas after differentiation. Here we report that virally derived hiPSC, but not human embryonic stem cells (hESC) or hiPSC derived using episomal nonintegrating vectors, exhibit a propensity to revert to a pluripotent phenotype following differentiation. This instability was revealed using our published method to identify pluripotent cells undergoing very early-stage differentiation in standard hESC cultures, by fluorescence activated cell sorting (FACS) based on expression of the cell surface markers TG30 (CD9) and GCTM-2. Differentiated cells cultured post-FACS fractionation from virally derived hiPSC lines reacquired immunoreactivity to TG30 (CD9) and GCTM-2, formed stem cell-like colonies, and re-expressed canonical pluripotency markers. Furthermore, differentiated cells from pluripotency-reverting hiPSC lines generated teratomas in immunocompromised mice, raising concerns about their safety in downstream applications. In contrast, differentiated cell populations from hESC and episomally derived hiPSC did not show any of these abnormalities. Our assays may be used to identify "unsafe" hiPSC cell lines and this information should be considered when selecting hiPSC lines for clinical use and indicate that experiments using these "unsafe" hiPSC lines should be interpreted carefully.

Seasonal Stability of the Circadian Rhythm in Patients with Type I Myocardial Infarction.

Background: A circadian rhythm of myocardial infarction has been described but there is little data on its relation with seasons and months. Methods: From June 2013 to June 2018, we analyzed the alerts for acute ST-segment elevation myocardial infarction (STEMI) in a Spanish region with 6.64 million inhabitants, universal health coverage, and an organized STEMI reperfusion network. We selected those patients which an identifiable culprit plaque. Results: We recruited 6765 cases of STEMI due to type I acute myocardial infarction (type-I AMI), with mean age of 63.2 years (range 17-101, standard deviation [SD] 13.7), 5238 were males (77.4%) and 2801 (41.9%) were 65 years or older. The hourly distribution followed a fixed pattern in all months, with most of the events occurring between 6:00 AM and 4:00 PM, a peak at approximately 01:00 PM and a valley between 10:00 PM and 06:00 AM. No significant difference was found when comparing the mean time to first medical contact between July (the month with more daylight hours) and December (the month with shortest days). No significant differences were found between male and female patients, or between patients aged 65 years or older and younger patients. There was a close correlation between the number of events per month and the number of events occurring during the day (6 AM to 6 PM, r = 0.988, p = 0.001) and during the night (6 PM to 6 AM, r = 0.944, p < 0.001), with different slopes of the regression lines (t-test, p < 0.001), so that the difference between day-night occurrences increased with the total incidence. Conclusions: There is a circadian pattern in the presentation of STEMI that is not influenced by sex and age. The different incidence of STEMI at different times of the year does not affect the circadian pattern in terms of the shape of the curve or the mean time of presentation, although diurnal events increase more than nocturnal events, suggesting that triggers are most likely to act during vulnerable periods as determined by a circadian-based rhythm.

CRISPRi screening reveals regulators of tau pathology shared between exosomal and vesicle-free tau.

The aggregation of the microtubule-associated protein tau is a defining feature of Alzheimer's disease and other tauopathies. Tau pathology is believed to be driven by free tau aggregates and tau carried within exosome-like extracellular vesicles, both of which propagate trans-synaptically and induce tau pathology in recipient neurons by a corrupting process of seeding. Here, we performed a genome-wide CRISPRi screen in tau biosensor cells and identified cellular regulators shared by both mechanisms of tau seeding. We identified ANKLE2, BANF1, NUSAP1, EIF1AD, and VPS18 as the top validated regulators that restrict tau aggregation initiated by both exosomal and vesicle-free tau seeds. None of our validated hits affected the uptake of either form of tau seeds, supporting the notion that they operate through a cell-autonomous mechanism downstream of the seed uptake. Lastly, validation studies with human brain tissue also revealed that several of the identified protein hits are down-regulated in the brains of Alzheimer's patients, suggesting that their decreased activity may be required for the emergence or progression of tau pathology in the human brain.

Sox10 gain-of-function causes XX sex reversal in mice: implications for human 22q-linked disorders of sex development.

Male development in mammals is normally initiated by the Y-linked gene Sry, which activates expression of Sox9, leading to a cascade of gene activity required for testis formation. Although defects in this genetic cascade lead to human disorders of sex development (DSD), only a dozen DSD genes have been identified, and causes of 46,XX DSD (XX maleness) other than SRY translocation are almost completely unknown. Here, we show that transgenic expression of Sox10, a close relative of Sox9, in gonads of XX mice resulted in development of testes and male physiology. The degree of sex reversal correlated with levels of Sox10 expression in different transgenic lines. Sox10 was expressed at low levels in primordial gonads of both sexes during normal mouse development, becoming male-specific during testis differentiation. SOX10 protein was able to activate transcriptional targets of SOX9, explaining at a mechanistic level its ability to direct male development. Because over-expression of SOX10 alone is able to mimic the XX DSD phenotypes associated with duplication of human chromosome 22q13, and given that human SOX10 maps to 22q13.1, our results functionally implicate SOX10 in the etiology of these DSDs.

Exosomal and vesicle-free tau seeds-propagation and convergence in endolysosomal permeabilization.

In Alzheimer's disease (AD), ß-amyloid peptides aggregate to form amyloid plaques, and the microtubule-associated protein tau forms neurofibrillary tangles. However, severity and duration of AD correlate with the stereotypical emergence of tau tangles throughout the brain, suggestive of a gradual region-to-region spreading of pathological tau. The current notion in the field is that misfolded tau seeds propagate transsynaptically and corrupt the proper folding of soluble tau in recipient neurons. This is supported by accumulating evidence showing that in AD, functional connectivity and not proximity predicts the spreading of tau pathology. Tau seeds can be found in two flavors, vesicle-free, that is, naked as in oligomers and fibrils, or encapsulated by membranes of secreted vesicles known as exosomes. Both types of seeds have been shown to propagate between interconnected neurons. Here, we describe potential ways of how their propagation can be controlled in several subcellular compartments by manipulating mechanisms affecting production, neuron-to-neuron transmission, internalization, endosomal escape, and autophagy. We emphasize that although vesicle-free tau seeds and exosomes differ, they share the ability to trigger endolysosomal permeabilization. Such a mechanistic convergence in endolysosomal permeabilization presents itself as a unique opportunity to target both types of tau seeding. We discuss the cellular response to endolysosomal damage that might be key to control permeabilization, and the significant overlap in the seeding mechanism of proteopathic agents other than tau, which suggests that targeting the endolysosomal pathway could pave the way toward developing broad-spectrum treatments for neurodegenerative diseases.

Functional analysis of the SRY-KRAB interaction in mouse sex determination.

BACKGROUND INFORMATION: SRY (sex-determining region Y), the master regulator of male development in mammals, has been studied extensively for more than 17 years, but how the SRY protein triggers the chain of events leading to testis development remains unclear. SRY probably requires a partner protein to elicit its molecular function. KRAB-O, a novel protein containing a KRAB (Krüppel-associated box) domain only, was suggested recently as a candidate SRY partner. In order to investigate the possible role of KRAB-O in sex determination, we studied its expression and conducted functional assays of the SRY-KRAB interaction. RESULTS: More than 100 KRAB genes were found to be expressed in mouse developing gonads, including 19 transcripts encoded by the KRAB-O cluster that were found to be expressed in somatic cells at 11.5 dpc (days post-coitum). Loss-of-function analysis in Sry-expressing cultured cells, using shRNA (small hairpin RNA) constructs directed against KRAB-O and its homologous genes, resulted in a reduced ability to up-regulate Sox9 [SRY-related HMG (high-mobility group)-box 9]; however, KRAB-knockdown mice exhibited normal testis development. CONCLUSIONS: Reduced Sox9 expression in KRAB-knockdown cells supports a role for KRAB-O and perhaps other KRAB genes in mediating SRY function. Overlapping expression and potential redundancy between members of the large KRAB-O gene cluster may mask any loss-of-function in vivo, presenting clear challenges for further functional analysis.

Fyn Kinase Controls Tau Aggregation In Vivo.

Alzheimer's disease (AD) is a proteinopathy exhibiting aggregation of ß-amyloid (Aß) as amyloid plaques and tau as neurofibrillary tangles (NFTs), whereas primary tauopathies display only a tau pathology. Aß toxicity is mediated by Fyn kinase in a tau-dependent process; however, whether Fyn controls tau pathology in diseases that lack Aß pathology remains unexplored. To address this, we generate the Tg/Fyn-/- mouse, which couples mutant tau overexpression with Fyn knockout. Surprisingly, Tg/Fyn-/- mice exhibit a near-complete ablation of NFTs, alongside reduced tau hyperphosphorylation, altered tau solubility, and diminished synaptic tau accumulation. Furthermore, Tg/Fyn-/- brain lysates elicit less tau seeding in tau biosensor cells. Lastly, the fibrillization of tau is boosted by its pseudophosphorylation at the Fyn epitope Y18. Together, this identifies Fyn as a key regulator of tau pathology independently of Aß-induced toxicity and thereby represents a potentially valuable therapeutic target for not only AD but also tauopathies more generally.

Exosomes taken up by neurons hijack the endosomal pathway to spread to interconnected neurons.

In Alzheimer disease and related disorders, the microtubule-associated protein tau aggregates and forms cytoplasmic lesions that impair neuronal physiology at many levels. In addition to affecting the host neuron, tau aggregates also spread to neighboring, recipient cells where the misfolded tau aggregates, in a manner similar to prions, actively corrupt the proper folding of soluble tau, and thereby impair cellular functions. One vehicle for the transmission of tau aggregates are secretory nanovesicles known as exosomes. Here, we established a simple model of a neuronal circuit using a microfluidics culture system in which hippocampal neurons A and B were seeded into chambers 1 and 2, respectively, extending axons via microgrooves in both directions and thereby interconnecting. This system served to establish two models to track exosome spreading. In the first model, we labeled the exosomal membrane by coupling tetraspanin CD9 with either a green or red fluorescent tag. This allowed us to reveal that interconnected neurons exchange exosomes only when their axons extend in close proximity. In the second model, we added exosomes isolated from the brains of tau transgenic rTg4510 mice (i.e. exogenous, neuron A-derived) to neurons in chamber 1 (neuron B) interconnected with neuron C in chamber 2. This allowed us to demonstrate that a substantial fraction of the exogenous exosomes were internalized by neuron B and passed then on to neuron C. This transportation from neuron B to C was achieved by a mechanism that is consistent with the hijacking of secretory endosomes by the exogenous exosomes, as revealed by confocal, super-resolution and electron microscopy. Together, these findings suggest that fusion events involving the endogenous endosomal secretory machinery increase the pathogenic potential and the radius of action of pathogenic cargoes carried by exogenous exosomes.

Amyloid-ß and tau complexity - towards improved biomarkers and targeted therapies.

Most neurodegenerative diseases are proteinopathies, which are characterized by the aggregation of misfolded proteins. Although many proteins have an intrinsic propensity to aggregate, particularly when cellular clearance systems start to fail in the context of ageing, only a few form fibrillar aggregates. In Alzheimer disease, the peptide amyloid-ß (Aß) and the protein tau aggregate to form plaques and tangles, respectively, which comprise the histopathological hallmarks of this disease. This Review discusses the complexity of Aß biogenesis, trafficking, post-translational modifications and aggregation states. Tau and its various isoforms, which are subject to a vast array of post-translational modifications, are also explored. The methodological advances that revealed this complexity are described. Finally, the toxic effects of distinct species of tau and Aß are discussed, as well as the concept of protein 'strains', and how this knowledge can facilitate the development of early disease biomarkers for stratifying patients and validating new therapies. By targeting distinct species of Aß and tau for therapeutic intervention, the way might be paved for personalized medicine and more-targeted treatment strategies.

Extracellular Vesicles Containing P301L Mutant Tau Accelerate Pathological Tau Phosphorylation and Oligomer Formation but Do Not Seed Mature Neurofibrillary Tangles in ALZ17 Mice.

In Alzheimer's disease, the distribution of neurofibrillary tangles, a histological hallmark comprised of phosphorylated forms of the protein tau, follows a distinct pattern through anatomically connected brain regions. The well-documented correlation between the severity of tau pathology and disease progression implies a prion-like seeding and spreading mechanism for tau. Experimentally, this has been addressed in transgenic mice by the injection of protein lysates isolated from brains of transgenic mice or patients with tauopathies, including AD, that were shown to behave like seeds, accelerating tau pathology and tangle formation in predisposed mice. More specifically, in vivo data suggest that brain lysates from mice harboring the P301S mutation of tau can seed protein aggregation when injected into the hippocampi of human wild-type tau transgenic ALZ17 mice. Here, we compared the seeding potential of lysates and extracellular vesicles enriched for exosomes (EVs) from wild-type and human P301L tau transgenic rTg4510 mouse brains. We show that transgenic EVs cause increased tau phosphorylation and soluble oligomer formation in a manner comparable to that of freely available proteins in brain lysates, a prerequisite for the formation of mature protein aggregates.

Enrichment and purging of human embryonic stem cells by detection of cell surface antigens using the monoclonal antibodies TG30 and GCTM-2.

Human embryonic stem cells (hESC) can self-renew indefinitely in vitro, and with the appropriate cues can be induced to differentiate into potentially all somatic cell lineages. Differentiated hESC derivatives can potentially be used in transplantation therapies to treat a variety of cell-degenerative diseases. However, hESC differentiation protocols usually yield a mixture of differentiated target and off-target cell types as well as residual undifferentiated cells. For the translation of differentiated hESC-derivatives from the laboratory to the clinic, it is important to be able to discriminate between undifferentiated (pluripotent) and differentiated cells, and generate methods to separate these populations. Safe application of hESC-derived somatic cell types can only be accomplished with pluripotent stem cell-free populations, as residual hESCs could induce tumors known as teratomas following transplantation. Towards this end, here we describe a methodology to detect pluripotency associated cell surface antigens with the monoclonal antibodies TG30 (CD9) and GCTM-2 via fluorescence activated cell sorting (FACS) for the identification of pluripotent TG30(Hi)-GCTM-2(Hi) hESCs using positive selection. Using negative selection with our TG30/GCTM-2 FACS methodology, we were able to detect and purge undifferentiated hESCs in populations undergoing very early-stage differentiation (TG30(Neg)-GCTM-2(Neg)). In a further study, pluripotent stem cell-free samples of differentiated TG30(Neg)-GCTM-2(Neg) cells selected using our TG30/GCTM-2 FACS protocol did not form teratomas once transplanted into immune-compromised mice, supporting the robustness of our protocol. On the other hand, TG30/GCTM-2 FACS-mediated consecutive passaging of enriched pluripotent TG30(Hi)-GCTM-2(Hi) hESCs did not affect their ability to self-renew in vitro or their intrinsic pluripotency. Therefore, the characteristics of our TG30/GCTM-2 FACS methodology provide a sensitive assay to obtain highly enriched populations of hPSC as inputs for differentiation assays and to rid potentially tumorigenic (or residual) hESC from derivative cell populations.

Sry and the hesitant beginnings of male development.

In mammals, Sry (sex-determining region Y gene) is the master regulator of male sex determination. The discovery of Sry in 1990 was expected to provide the key to unravelling the network of gene regulation underlying testis development. Intriguingly, no target gene of SRY protein has yet been discovered, and the mechanisms by which it mediates its developmental functions are still elusive. What is clear is that instead of the robust gene one might expect as the pillar of male sexual development, Sry function hangs by a thin thread, a situation that has profound biological, medical and evolutionary implications.

Plasmodium vivax: parasitemia determination by real-time quantitative PCR in Aotus monkeys.

Plasmodium vivax and Plasmodium falciparum are the two prevalent human malaria species. A Colombian P. vivax wild strain has been adapted in Aotus nancymaae monkeys for use in further biological and immunological studies. We present data validating a real-time PCR assay quantifying P. vivax parasitemia, using the small subunit ribosomal RNA genes as an amplification target. P. vivax species-specific primers were designed on the 18S ribosomal gene V8 region, for amplifying both asexual and sporozoite ssrRNA genes. The assay detects amplification products bound to fluorescent SYBR-Green I dye using Perkin-Elmer GeneAmp-5700-SDS. Linear range standard curves from 6 DNA concentration logs (+0.99 correlation coefficients) were obtained. Standard curves were constructed using a plasmid containing target gene for real-time PCR amplification. This P. vivax specific assay is very sensitive, having a three parasite detection limit, and is reproducible and accurate. It involves a "closed-tube" PCR, avoids time-consuming post-PCR manipulation, and decreases potential PCR contamination.

Design of a molecular method for subspecies specific identification of Klebsiella pneumoniae by using the 16S ribosomal subunit gene / Diseño de un método molecular para la identificación específica de Klebsiella pneumoniae a nivel de subespecie, usando el gen que codifica para la subunidad ribosomal 16S

Introduction: Rhinoscleroma is caused by Klebsiella pneumoniae rhinoscleromatis and the ozena infections caused by K. pneumoniae ozaenae, both infections affect the upper respiratory tract. In the first clinical phases the symptoms are unspecific, and the disease can be misdiagnosed as a common cold, therefore antimicrobial therapy cannot reach effective results and patients must be following up for several years since the infection became chronic. Objective: To identify Klebsiella subspecies using a specific assay based on amplicons restriction of a gene which encodes 16S subunit ribosomal (rDNA16S). Methodology: Specific restriction patterns were generated; using reported sequences from rDNA16S gene and bioinformatics programs MACAW, PFE, GENEDOC and GENE RUNNER. Amplification and restriction assays were standardized. Results: Predictions in silico allowed to propose an algorithm for Klebsiella species and subspecies identification. Two reference strains were included and two clinical isolates which were biotyped and identified by the proposed method. rDNA16S gene restriction patterns showed differences regarding the initially identified species for conventional methods. Additionally two patterns of bands were observed for K. pneumoniae rhinoscleromatis, indicating the polymorphisms presence in the rDNA16S gene. Conclusions: It was confirmed the difficulty to identify K. pneumoniae subspecies by conventional methods. Implementation of this technique could allow an accurate and rapid differentiation among K. pneumoniae ozaenae and K. pneumoniae rhinoscleromatis aetiological agents of two frequently misdiagnosed infections. Antimicrobial therapy usually could be ineffective, especially in chronic patients. Finally it is considered very important to enlarge the study by using more clinical and reference strains.

Introducción: El rinoescleroma es causado por Klebsiella pneumoniae rhinoscleromatis y la ocena por Klebsiella pneumoniae ozaenae respectivamente. Estas infecciones se presentan sobre todo en el tracto respiratorio superior y tienen una sintomatología inespecífica en sus fases iniciales por lo cual se pueden confundir con el catarro común. Las dificultades de establecer un diagnóstico oportuno tienen repercusiones negativas en la terapia antimicrobiana, porque puede no ser efectiva y hacer que la enfermedad evolucione a una fase crónica cuyo seguimiento puede implicar muchos años. Objetivo: Diseñar un ensayo molecular para la identificación a nivel de subespecie de bacterias del género Klebsiella basado en restricción de amplicones del gen que codifica para la subunidad ribosomal 16S (ADNr 16S).Metodología: Se generaron patrones de restricción específicos, utilizando secuencias informadas del gen ADNr 16S y los programas bioinformáticos MACAW, PFE, GENEDOC y GENE RUNNER. Se estandarizaron las condiciones para la amplificación y restricción para el ensayo experimental.Resultados: Las predicciones in silico permitieron proponer un algoritmo para la identificación a nivel de especie y subespecie de las especies del género Klebsiella. Se incluyeron dos cepas de referencia y dos aislados clínicos, que se biotipificaron e identificaron por el método propuesto; los patrones de restricción obtenidos del gen ADNr 16S evidenciaron diferencias con respecto a la especie inicialmente identificada por métodos convencionales. Además se encontraron dos patrones de bandas en Klebsiella pneumoniae rhinoscleromatis, indicando la presencia de polimorfismos en el gen ADNr 16S para esta subespecie. Conclusiones: Se confirmó la dificultad para identificar Klebsiella pneumoniae a nivel de subespecie por métodos convencionales.

Design of a molecular method for subspecies specific identification of Klebsiella pneumoniae by using the 16S ribosomal subunit gene / Diseño de un método molecular para la identificación específica de Klebsiella pneumoniae a nivel de subespecie, usando el gen que codifica para la subunidad ribosomal 16S

Introduction: Rhinoscleroma is caused by Klebsiella pneumoniae subsp. rhinoscleromatis and the ozena infections caused by K. pneumoniae subsp. ozaenae, both infections affect the upper respiratory tract. In the first clinical phases the symptoms are unspecific, and the disease can be misdiagnosed as a common cold, therefore antimicrobial therapy cannot reach effective results and patients must be following up for several years since the infection became chronic. Objective: To identify Klebsiella subspecies using a specific assay based on amplicons restriction of a gene which encodes 16S subunit ribosomal (rDNA16S). Methodology: Specific restriction patterns were generated; using reported sequences from rDNA16S gene and bioinformatics programs MACAW, PFE, GENEDOC and GENE RUNNER. Amplification and restriction assays were standardized. Results: Predictions in silico allowed us to propose an algorithm for Klebsiella species and subspecies identification. Two reference strains were included and two clinical isolates which were biotyped and identified by the proposed method. rDNA16S gene restriction patterns showed differences regarding the initially identified species for conventional methods. Additionally two patterns of bands were observed for K. pneumoniae subsp. rhinoscleromatis, indicating the polymorphisms presence in the rDNA16S gene. Conclusions: We confirmed the difficulty to identify K. pneumoniae subspecies by conventional methods. Implementation of this technique could allow accurate and rapid differentiation among K. pneumoniae subsp. ozaenae and K. pneumoniae subsp. rhinoscleromatis the aetiological agents of two frequently misdiagnosed infections. Antimicrobial therapy usually could be ineffective, especially in chronic patients. Finally we consider very important to enlarge the study by using more clinical and reference strains.

Introducción: El rinoescleroma es causado por Klebsiella pneumoniae subsp. rhinoscleromatis y la ocena por K. pneumoniae subsp. ozaenae, respectivamente. Estas infecciones se presentan sobre todo en el tracto respiratorio superior y originan una sintomatología inespecífica en sus fases iniciales por lo cual se pueden confundir con el catarro común. Las dificultades para establecer un diagnóstico oportuno tienen repercusiones negativas en la terapia antimicrobiana, que puede no ser efectiva y hacer que la enfermedad evolucione a una fase crónica cuyo seguimiento en el paciente puede necesitar muchos años. Objetivo: Diseñar un ensayo molecular para la identificación a nivel de subespecie de bacterias del género Klebsiella basado en restricción de amplicones del gen que codifica para la subunidad ribosomal 16S (ADNr 16S). Metodología: Se generaron patrones de restricción específicos, con secuencias informadas del gen ADNr 16S y los programas bioinformáticos MACAW, PFE, GENEDOC y GENE RUNNER. Se estandarizaron las condiciones para la amplificación y restricción del ensayo experimental.Resultados: Las predicciones in silico permitieron proponer un algoritmo para identificar a nivel de especie y subespecie los miembros del género Klebsiella. Se incluyeron dos cepas de referencia y dos aislamientos clínicos, que fueron biotipificados e identificados por el método propuesto; los patrones de restricción obtenidos del gen ADNr 16S evidenciaron diferencias respecto a la especie inicialmente identificada por métodos convencionales. Además, se encontraron dos patrones de bandas en K. pneumoniae. rhinoscleromatis, que indican la presencia de polimorfismos en el gen ADNr 16S para esta subespecie.