Changes in the proteome of Apis mellifera acutely exposed to sublethal dosage of glyphosate and imidacloprid.

Uncontrolled use of pesticides has caused a dramatic reduction in the number of pollinators, including bees. Studies on the effects of pesticides on bees have reported effects on both metabolic and neurological levels under chronic exposure. In this study, variations in the differential expression of head and thorax-abdomen proteins in Africanized A. mellifera bees treated acutely with sublethal doses of glyphosate and imidacloprid were studied using a proteomic approach. A total of 92 proteins were detected, 49 of which were differentially expressed compared to those in the control group (47 downregulated and 2 upregulated). Protein interaction networks with differential protein expression ratios suggested that acute exposure of A. mellifera to sublethal doses of glyphosate could cause head damage, which is mainly associated with behavior and metabolism. Simultaneously, imidacloprid can cause damage associated with metabolism as well as, neuronal damage, cellular stress, and impairment of the detoxification system. Regarding the thorax-abdomen fractions, glyphosate could lead to cytoskeleton reorganization and a reduction in defense mechanisms, whereas imidacloprid could affect the coordination and impairment of the oxidative stress response.

Metagenomic and genomic analysis of heavy metal-tolerant and -resistant bacteria in resource islands in a semi-arid zone of the Colombian Caribbean.

Bacteria from resource islands can adapt to different extreme conditions in semi-arid regions. We aimed to determine the potential resistance and tolerance to heavy metals from the bacterial community under the canopy of three resource islands in a semi-arid zone of the Colombian Caribbean. Total DNA was extracted from soil and through a metagenomics approach, we identified genes related to heavy metal tolerance and resistance under the influence of drought and humidity conditions, as well as the presence or absence of vegetation. We characterized the genomes of bacterial isolates cultivated in the presence of four heavy metals. The abundances of genes related to heavy metal resistance and tolerance were favored by soil moisture and the presence of vegetation. We observed a high abundance of resistance genes (60.4%) for Cu, Zn, and Ni, while 39.6% represented tolerance. These genes positively correlated with clay and silt content, and negatively correlated with sand content. Resistance and tolerance were associated with detoxification mechanisms involving oxidoreductase enzymes, metalloproteases, and hydrolases, as well as transmembrane proteins involved in metal transport such as efflux pumps and ion transmembrane transporters. The Bacillus velezensis C3-3 and Cytobacillus gottheilii T106 isolates showed resistance to 5 mM of Cd, Co, Mn, and Ni through detoxification genes associated with ABC pumps, metal transport proteins, ion antiporter proteins, and import systems, among others. Overall, these findings highlight the potential of bacteria from resource islands in bioremediation processes of soils contaminated with heavy metals.

Bioinformatic approach for repurposing immunomodulatory drugs for lepromatous leprosy.

Background: The lepromatous leprosy (LL) disease is caused by Mycobacterium leprae and Mycobacterium lepromatosis which is characterized by inadequate response to treatment, a propensity to drug resistance, and patient disability. We aimed to evaluate current immunomodulatory medicines and their target proteins collectively as a drug repurposing strategy to decipher novel uses for LL. Methods: A dataset of human genes associated with LL-immune response was retrieved from public health genomic databases including the Human Genome Epidemiology Navigator and DisGeNET. Retrieved genes were filtered and enriched to set a robust network (≥10, up to 21 edges) and analyzed in the Cytoscape program (v3.9). Drug associations were obtained in the NDEx Integrated Query (v1.3.1) coupled with drug databases such as ChEMBL, BioGRID, and DrugBank. These networks were analyzed in Cytoscape with the CyNDEx-2 plugin and STRING protein network database. Results: Pathways analyses resulted in 100 candidate drugs organized into pharmacological groups with similar targets and filtered on 54 different drugs. Gene-target network analysis showed that the main druggable targets associated with LL were tumoral necrosis factor-alpha, interleukin-1B, and interferon-gamma. Consistently, glucosamine, binimetinib, talmapimod, dilmapimod, andrographolide, and VX-702 might have a possible beneficial effect coupled with LL treatment. Conclusion: Based on our drug repurposing analysis, immunomodulatory drugs might have a promising potential to be explored further as therapeutic options or to alleviate symptoms in LL patients.

Regulations on the Use of Antibiotics in Livestock Production in South America: A Comparative Literature Analysis.

As a global health problem, antimicrobial resistance (AMR) crosses national borders, leading UN (United Nations) multilateral agencies to call for all countries to improve the stewardship of antibiotics in humans and animals. South American countries have changed their regulations regarding antibiotic use in livestock production. This literature review examines how far the five largest meat-producing countries in South America (Argentina, Brazil, Chile, Colombia, and Uruguay) have come in terms of the relevant legislation. Rules on market entry (marketing authorization and official distribution systems) are already set in all countries examined. Four countries do not allow growth promoters based on critically important antibiotics, and countries have also begun to set guidelines and minimum welfare and biosecurity requirements to reduce the therapeutic demand for antibiotics. Nonetheless, there are aspects related to the distribution, use, and disposal of antibiotics that need to be developed further. In conclusion, legislation in South American countries is moving towards the goals set by UN multilateral agencies, but more can be done. Differences between countries' rules and the gold standards set by the World Organization for Animal Health (OIE), World Health Organization (WHO), and Food and Agriculture Organization of the United Nations (FAO) reveal possible adaptations to the countries' realities. Further studies must examine compliance with the legislation already set and investigate other tools that can be used alongside legislation as a driving force to change stakeholder behaviour.

Specific identification of Mycobacterium bovis by Loop-Mediated Isothermal Amplification (LAMP) targeting the Region of Difference 12 (RD12) of the M. tuberculosis complex.

Bovine tuberculosis is a prevalent zoonotic disease that causes high risks for production animals, dairy producers and consumers, together with significant economic losses. Thus, methods for easy, fast and specific detection of Mycobacterium bovis in small and medium-sized livestock under field conditions are very required. In this work, a Loop-Mediated Isothermal Amplification LAMP-PCR targeting the Region of Difference 12 (RD12) of M. bovis genome was designed for the purpose of identification. A set of six primers designed for the isothermal amplification of five different genomic fragments led to the specific identification of M. bovis from other mycobacterial species. A basic colorimetric reaction was clearly observed at first sight under natural light, indicating positive identification of M. bovis in a maximum of 30 min of isothermal amplification at 65 °C. The limit of detection was near 50 fg of M. bovis genomic DNA, corresponding approximately to 10 copies of the genome. •The proposed LAMP-PCR amplification of M. bovis genomic DNA might be performed by untrained laboratory personnel.•Specific identification of M. bovis LAMP is possible in 30 min at 65.. C using a simple water bath.•The basic colorimetric reaction for M. bovis identification could be observed with the naked eye under natural light.

Design of a specific peptide against phenolic glycolipid-1 from Mycobacterium leprae and its implications in leprosy bacilli entry.

BACKGROUND: Mycobacterium leprae, the causative agent of Hansen's disease, causes neural damage through the specific interaction between the external phenolic glycolipid-1 (PGL-1) and laminin subunit alpha-2 (LAMA2) from Schwann cells. OBJECTIVE: To design a LAMA2-based peptide that targets PGL-1 from M. leprae. METHODS: We retrieved the protein sequence of human LAMA2 and designed a specific peptide using the Antimicrobial Peptide Database and physicochemical parameters for antimycobacterial peptide-lipid interactions. We used the AlphaFold2 server to predict its three-dimensional structure, AUTODOCK-VINA for docking, and GROMACS programs for molecular dynamics simulations. FINDINGS: We analysed 52 candidate peptides from LAMA2, and subsequent screening resulted in a single 60-mer peptide. The mapped peptide comprises four ß-sheets and a random coiled region. This peptide exhibits a 45% hydrophobic ratio, in which one-third covers the same surface. Molecular dynamics simulations show that our predicted peptide is stable in aqueous solution and remains stable upon interaction with PGL-1 binding. In addition, we found that PGL-1 has a preference for one of the two faces of the predicted peptide, which could act as the preferential binding site of PGL-1. MAIN CONCLUSIONS: Our LAMA2-based peptide targeting PGL-1 might have the potential to specifically block this key molecule, suggesting that the preferential region of the peptide is involved in the initial contact during the attachment of leprosy bacilli to Schwann cells.

Design of a specific peptide against phenolic glycolipid-1 from Mycobacterium leprae and its implications in leprosy bacilli entry

BACKGROUND Mycobacterium leprae, the causative agent of Hansen's disease, causes neural damage through the specific interaction between the external phenolic glycolipid-1 (PGL-1) and laminin subunit alpha-2 (LAMA2) from Schwann cells. OBJECTIVE To design a LAMA2-based peptide that targets PGL-1 from M. leprae. METHODS We retrieved the protein sequence of human LAMA2 and designed a specific peptide using the Antimicrobial Peptide Database and physicochemical parameters for antimycobacterial peptide-lipid interactions. We used the AlphaFold2 server to predict its three-dimensional structure, AUTODOCK-VINA for docking, and GROMACS programs for molecular dynamics simulations. FINDINGS We analysed 52 candidate peptides from LAMA2, and subsequent screening resulted in a single 60-mer peptide. The mapped peptide comprises four β-sheets and a random coiled region. This peptide exhibits a 45% hydrophobic ratio, in which one-third covers the same surface. Molecular dynamics simulations show that our predicted peptide is stable in aqueous solution and remains stable upon interaction with PGL-1 binding. In addition, we found that PGL-1 has a preference for one of the two faces of the predicted peptide, which could act as the preferential binding site of PGL-1. MAIN CONCLUSIONS Our LAMA2-based peptide targeting PGL-1 might have the potential to specifically block this key molecule, suggesting that the preferential region of the peptide is involved in the initial contact during the attachment of leprosy bacilli to Schwann cells.

Estado de la coinfección tuberculosis/virus de la inmunodeficiencia humana en el municipio de Armenia(Colombia): experiencia de 10 años / Status of tuberculosis/HIV coinfection in the municipality of Armenia (Colombia): Ten years experience

La tuberculosis (TB) representa una de las principales complicaciones clínicas en personas inmunocomprometidas por virus de la inmunodeficiencia humana (VIH). Así, cuando las acciones diagnósticas y terapéuticas no son tempranas, el resultado puede llegar a ser letal. Metodología: Se llevó a cabo un estudio descriptivo y retrospectivo de los casos de coinfección TB/VIH en el período comprendido entre los años 2000 y 2010 en el municipio de Armenia. Se revisaron las fichas de notificación de ambos eventos (Sistema Nacional de Vigilancia en Salud Pública), actas de visitas domiciliarias, tarjetas individuales de tratamiento y certificados de defunción. Resultados: Se incluyeron 113 pacientes con diagnóstico confirmado de coinfección TB/VIH, lo que representa una prevalencia global de 6,8% en 10 años en Armenia. La edad promedio de los pacientes fue 34,3 años (11 a 68 años), y los más comprometidos fueron los del grupo de 14 a 34 años de sexo masculino (91% en formas extrapulmonares y 75% en pulmonares). La TB extrapulmonar o la miliar predominaron con un 50,4% (n = 57), sobre la TB pulmonar. Las formas extrapulmonares más comunes fueron: ganglionar 50% (n = 32), meníngea 19,3% (n = 11), miliar 8,8% (n = 5), pleural 7% (n = 4), peritoneal 7% (n = 4) y vertebral (mal de Pott) 1,8% (n = 1). En 8 casos hubo presentación mixta (pulmonar y extrapulmonar). La letalidad fue del 100% en pacientes infectados por VIH y asociados con cepas resistentes. Conclusiones: Los resultados de este estudio resaltan la importancia de implementar acciones colaborativas entre los programas de TB/VIH para mejorar el diagnóstico y reducir la mortalidad la diseminación de cepas resistentes.

Tuberculosis represents one of the top clinical complications in immunocompromised HIVinfected people. When diagnostic and therapeutic interventions are delayed, outcomes could be fatal due to imminent progress of this deadly combination. Methodology: We carried out a descriptive retrospective study from registered TB/HIV cases from 2000 to 2010 in the municipality of Armenia. Notification forms from both diseases TB and HIV (SIVIGILA), record of home visits, individual treatment cards and death certificates were used as data sources. Results: 113 patients were included with TB/HIV confirmed tests, which represents a global prevalence of 6.8% during 10 years in Armenia. The mean age among patients was 34.3 years (ranging from 11 to 68 years), and the group between 14 and 34 years and male (91% extrapulmonary and 75% pulmonary forms) was the most commonly involved. According to the disease presentation, extrapulmonary TB form (50.4%, n = 57) was more frequent than the pulmonary form. The frequency of extrapulmonary forms was: ganglionar, 50% (n = 32); meningeal, 19.3% (n = 11); miliar, 8.8% (n = 5); pleural, 7% (n = 4); peritoneal, 7% (n = 4), and Pott’s disease, 1.8% (n = 1). In eight cases both forms presented simultaneously (pulmonary and extrapulmonary). Lethality was 100% among VIH positive patients infected with resistant strains. Conclusions: Our results highlight the importance of implementing collaborative actions between TB and HIV programs to improve diagnosis and to reduce mortality and resistant strain dissemination.

Análisis sociodemográfico y espacial de la transmisión de la tuberculosis en la ciudad de Armenia (Colombia) / Sociodemographic and spatial transmission of tuberculosis in the city of Armenia (Colombia)

La tuberculosis es uno de los problemas que demanda atención prioritaria en el municipio de Armenia, capital del departamento del Quindío (Colombia). En este estudio, se encontró que la población afectada, generalmente pertenece al género masculino (razón hombre: mujer 2:1) y en edad económicamente productiva (25-54 años). La mayoría de enfermos son del régimen subsidiado (40%) o no asegurados (26%) y viven en condiciones socioeconómicas críticas. Se encontraron diferencias significativas de carga de enfermedad en las tasas entre comunas: en unas localidades fueron de 10 y en otras de 30 casos por 100.000 habitantes, indicando heterogeneidad de la situación en la ciudad. El análisis geoespacial permite caracterizar y precisar los escenarios donde se deben concentrar las acciones prioritarias del programa de control de la tuberculosis.

Tuberculosis is an important public health problem that demands priority action in the city of Armenia (Colombia). In this study, we found that most cases of TB were males (ratio 2:1) and economically active population (25-54 years). TB patients had subvention health care (40%) or were uncovered by the national health insurance (26%), living in critical socioeconomic condi tions. Significant differences in the TB prevalence rate between town localities were found; in some zones were 10 and 30 cases per 100.000 inhabitants, indicating a heterogeneous situation. Spatial analysis might allow the definition and concentration of early actions in a region for the tuberculosis control.

Características clínicas y sociodemográficas de los casos con tuberculosis resistente en el municipio de Armenia, Quindío (Colombia) / Clinical and socio-demographic characteristics of tuberculosis with antimicrobial resistance in Armenia, Quindio (Colombia)

Objetivo: Analizar los factores sociodemográficos y clínicos de los pacientes con diagnóstico de tuberculosis resistente en el municipio de Armenia durante el período 2006-2009. Métodos: Se realizó un estudio descriptivo y retrospectivo con base en la notificación de pacientes con tuberculosis resistentes durante el período de estudio. Los datos fueron tomados de los registros consignados en las fichas de notificación y las tarjetas individuales de tratamiento categoría IV. Resultados: Entre 2006 y 2009, 678 pacientes ingresaron en el programa de control de la tuberculosis. De ellos, 14 casos (2,0%) fueron resistentes al menos a un medicamento, y entre estos, 7 (50%) fueron multirresistentes. El 21% estuvo asociado a infección por virus de la inmunodeficiencia humana. La tasa de fallecimientos en pacientes con tuberculosis con farmacorresistencia múltiple fue del 71%. Se encontró amplificación de la resistencia en 2 casos, quienes presentaron deterioro en el estado de salud y deceso. Conclusión: La situación en Armenia de resistencia a antituberculosos es de proporción similar a la del resto del país. Se encontraron factores de riesgo conocidos en los casos de resistencia (coinfección con virus de la inmunodeficiencia humana, farmacodependencia, abandono social) en la mayoría de casos, pero también de resistencia primaria y sin factores de riesgo con farmacorresistencia múltiple y una alta mortalidad, lo que llama la atención para mejorar la vigilancia y el control de los casos en tratamiento en la ciudad.

Objective: To analyze the socio-demographic and clinical diagnosis of patients with resistant tuberculosis in the municipality of Armenia in the period 2006-2009. Methods: We performed a retrospective descriptive study based on the notification of patients with resistant tuberculosis during the study period, data were taken from registrations under notification forms and individual treatment cards category IV. Results: 678 patients were admitted to the program of tuberculosis control between 2006 and 2009, 14 cases (2,0%) were resistant to at least one drug and from these cases 7 were multidrug resistant (MDR). In 21% of cases there was co-infection with HIV. The death rate in patients with MDR-TB was 71%. Amplification in resistance was observed in two cases which lead to further deterioration in the health status and death. Conclusion: Armenia has levels of resistance to anti tuberculosis drugs similar to the reported in the rest of Colombia. We found factors already know to be associated with resistance (drug addiction, social abandon, HIV co infection) but there were also cases without these factors and with primary MDR and high mortality, therefore is urgent to improve the public health measures for cases under treatment in this city.

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.

Construcción de una filogenia molecular para las especies de los géneros Klebsiella y Raoultella basada en los genes ARNr 16S y ARN polimerasa subunidad / Construction of a molecular phylogeny for klebsiella and Raoultella SP based on rRNA 165 and RNA polimarase subunit genes

Las bacterias de los géneros Raoultella y Klebsiella son patógenos oportunistas para las cuales no existe un sistema uniforme de clasificación taxonómica internacional. En el presente estudio se propone una filogenia molecular basada en el gen ribosomal 16S (ADNr 16S) y el gen codificante de la subunidad de la ARN polimerasa (rpoB) de los géneros Klebsiella y Raoultella con el fin de establecer relaciones evolutivas entre dichos géneros. Los resultados evidencian una agrupación acorde con la taxonomía y las propiedades bioquímicas características, reportadas en el Genbank. Se estableció una bifurcación en los árboles, lo cual confirma la separación de los géneros Klebsiella y Raoultella. Adicionalmente, se confirmó el carácter polifilético de K. aerogenes por el gen ADNr 16S y la agrupación de R. terrigena y K. oxytoca de acuerdo con el gen rpoB. La comparación entre los árboles obtenidos permitió determinar relaciones evolutivas entre las especies, a partir de los genes evaluados, lo cual refleja cambios aparentes a nivel taxonómico y corrobora la importancia del análisis a nivel de multilocus. Este tipo de estudios permite monitorear la estabilidad de los genotipos microbianos sobre la escala temporal y espacial, mejorar la precisión de las anotaciones taxonómicas (mejor descripción de taxones o subdivisiones genéticas) y evaluar la diversidad genética y adaptabilidad en términos de virulencia o resistencia a drogas.

The bacteria belonging to Raoultella and Klebsiella genera are opportunistic pathogens for which there is no consensus about a unique internationally taxonomic system. In this study, we suggest a molecular phylogeny based on 16S (rDNA 16S) ribosomal gene and the beta subunit RNA polymerase (rpoB) encoding gene of Klebsiella and Raoultella in order to set up evolutionary relationships among these genera. Results showed a cluster with similar biochemical and taxonomy characteristics in agreement to Genbank description, and a tree bifurcation which confirms the Klebsiella and Raoultella genera separation. Moreover, we verified the polyphyletic character of K. aerogenes by rDNA 16S and particularly the clustering for both R. terrigena and K. oxytoca based on rpoB gene. The evolutionary relationships recognition was obtained by comparison among the corresponding trees for both genes unraveling significant changes at taxonomic level and stressing the importance of multilocus analysis approach. These studies are useful for tracking the microbial genotype stability over time and space scale; as well as on improving taxonomic annotations (taxa descriptions and genetic subdivisions) and evaluation of genetic diversity in terms of virulence and drug resistance.

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.