Genotyping Klebsiella pneumoniae isolated from hepatic abscesses in three patients from Bogota, Colombia / Genotipificación de Klebsiella pneumoniae aislada de abscesos hepáticos de pacientes de Bogotá, Colombia / Genotipificação de Klebsiella pneumoniae isolada de abscessos hepáticos de pacientes de Bogotá, Colômbia

Pyogenic liver abscess caused by Klebsiella pneumoniae represents an ever increasing entity which has mainly been described as occurring in Asia, even though, on a smaller scale, cases are being more frequently described from the USA and Europe, 13% overall mortality being reached worldwide. Affected patients are severely sick, suffering from fever, sweating, having increased acute phase reactants and risk factors such as Diabetes Mellitus, alcoholism and the inherent characteristics of the bacteria causing the disease. Objective: in this work we used a Multilocus Sequencing Typing (MLST), a nucleotide sequence-based method in order to characterize the genetic relationships among bacterial isolates. Materials and methods: the report is focused on three cases involving patients suffering from pyogenic liver abscess caused by Klebsiella pneumoniae in two hospitals in Bogota, Colombia, where phenotyping and hypermucoviscosity studies were carried out, as well as the genotyping of cultured Klebsiella isolates. Results: it was found that the isolated microorganism in cases I and II corresponded to the same K. pneumoniae strain, having 100% sequence identity for the 5 genes being studied while the strain in Case III was genotypically different. Conclusion: it is important to carry out multidisciplinary studies allowing all pyogenic liver abscess cases reported in Colombia to be complied to ascertain the frequency of microorganisms causing this pathology in our country, as well as a genotyping study of different K. pneumoniae strains to compare them and confirm clonal and pathogenicity relationships through housekeeping gene analysis.

El absceso hepático piógeno causado por Klebsiella pneumoniae representa una entidad descrita con frecuencia en los países asiáticos y, en menor escala, también en Estados Unidos y Europa. En el ámbito mundial su mortalidad alcanza a un 13%. Los pacientes pueden presentar un compromiso severo, el cual cursa con fiebre, sudoración y aumento de reactantes de fase aguda, sobre todo en individuos con factores de riesgo como la diabetes mellitus y el alcoholismo, además de las características inherentes de la bacteria responsable de la infección. Objetivo: a continuación se presentan tres casos de pacientes con absceso hepático piógeno causado por Klebsiella pneumoniae en dos hospitales de Bogotá. Materiales y métodos: se realizó caracterización molecular de los aislados de Klebsiella mediante la técnica de Multilocus Secquence Typing (MLST), análisis fenotípicos y de hipermucoviscosidad. Resultado: se encontró que el microorganismo aislado en los casos I y II corresponde a la misma cepa de K. pneumoniae, que tiene 100% de identidad de secuencia de los 5 genes en estudio mientras que la cepa en Case III fue genotípicamente diferente. Conclusión: es importante llevar a cabo estudios multidisciplinarios que incluyan todos los casos de abscesos hepáticos piógenos reportados en Colombia con el objeto de determinar la frecuencia de los microorganismos causantes de esta patología en nuestro país, así como un estudio de genotipificación de diferentes cepas de K. pneumoniae para confirmar las relaciones clonales y de patogenicidad.

O abscesso hepático piogênico causado por Klebsiella pneumoniae representa uma entidade descrita com frequência nos países asiáticos e, em menor escala, também nos Estados Unidos e na Europa. No âmbito mundial sua mortalidade alcança um 13%. Os pacientes podem apresentar um compromisso severo, o qual cursa com febre, sudoração e aumento de reagentes de fase aguda, especialmente em indivíduos com fatores de risco como a diabetes mellitus e o alcoolismo, além das características inerentes da bactéria responsável da infecção. Objetivo: a continuação apresentamse três casos de pacientes com abscesso hepático piogênico causado por Klebsiella pneumoniae em dois hospitais de Bogotá; Materiais e métodos: realizou-se caracterização molecular dos isolados de Klebsiella mediante a técnica de Multilocus Secquence Typing (MLST), análises fenotípicas e de hipermucoviscosidade. Resultado: encontrou-se que o micro-organismo isolado nos casos I e II corresponde à mesma cepa de K. pneumoniae, que tem 100% de identidade de sequencia dos 5 genes em estudo enquanto a cepa no caso III foi genotipicamente diferente. Conclusão: é importante levar a cabo estudos multidisciplinares que incluam todos os casos de abscessos hepáticos piogênicos reportados na Colômbia com o objeto de determinar a frequência dos micro-organismos causadores desta patologia em nosso país, assim como um estudo de genotipificação de diferentes cepas de K. pneumoniae para confirmar as relações clonais e de patogenicidade.

Bacterial translocation in abdominal trauma and postoperative infections.

BACKGROUND: Bacterial translocation (BT) describes the passage of bacteria residing into the gastrointestinal tract, through the intestinal mucosa barrier to sterile tissues such as mesenteric lymph nodes (MLN) and other organs. This phenomenon has not been thoroughly studied in patients with trauma to date, and an association between BT and postoperative infection has not been well established so far. METHODS: MLNs from 36 patients with abdominal trauma were removed during laparotomy and cultured to detect BT. Postoperative infectious complications in these patients were registered, and both phenotypical and molecular typings (through multilocus sequencing) were carried out for microorganisms isolated from MLN and postoperative infection sites. Associations between clinical variables, BT presence, and postoperative infection development were established. RESULTS: BT was detected in 33% of the patients (n = 12). Postoperative infections were present in 22.2% of the patients (n = 8). A significant statistical difference was found between postoperative infections in patients with BT evidence (41.6%), when compared with patients without BT (12.5%; p = 0.047). Bacteria isolated from infection sites were the same as those cultured in MLN in 40% of the cases (n = 2 of 5), allowing us to establish causality between BT and postoperative infection. CONCLUSIONS: There is higher risk of BT in trauma patients, and it is associated with a significant increase of postoperative infections. An abdominal trauma index ≥10 was found to be associated with the development of BT. This is the first study describing BT among patients with abdominal trauma, where causality is confirmed at molecular level.

Molecular modeling and in silico characterization of Mycobacterium tuberculosis TlyA: possible misannotation of this tubercle bacilli-hemolysin.

BACKGROUND: The TlyA protein has a controversial function as a virulence factor in Mycobacterium tuberculosis (M. tuberculosis). At present, its dual activity as hemolysin and RNA methyltransferase in M. tuberculosis has been indirectly proposed based on in vitro results. There is no evidence however for TlyA relevance in the survival of tubercle bacilli inside host cells or whether both activities are functionally linked. A thorough analysis of structure prediction for this mycobacterial protein in this study shows the need for reevaluating TlyA's function in virulence. RESULTS: Bioinformatics analysis of TlyA identified a ribosomal protein binding domain (S4 domain), located between residues 5 and 68 as well as an FtsJ-like methyltranferase domain encompassing residues 62 and 247, all of which have been previously described in translation machinery-associated proteins. Subcellular localization prediction showed that TlyA lacks a signal peptide and its hydrophobicity profile showed no evidence of transmembrane helices. These findings suggested that it may not be attached to the membrane, which is consistent with a cytoplasmic localization. Three-dimensional modeling of TlyA showed a consensus structure, having a common core formed by a six-stranded ß-sheet between two α-helix layers, which is consistent with an RNA methyltransferase structure. Phylogenetic analyses showed high conservation of the tlyA gene among Mycobacterium species. Additionally, the nucleotide substitution rates suggested purifying selection during tlyA gene evolution and the absence of a common ancestor between TlyA proteins and bacterial pore-forming proteins. CONCLUSION: Altogether, our manual in silico curation suggested that TlyA is involved in ribosomal biogenesis and that there is a functional annotation error regarding this protein family in several microbial and plant genomes, including the M. tuberculosis genome.

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.

[Detecting active tuberculosis in Calarcá-Quindío, Colombia , during 2005]. / Búsqueda Activa de Individuos con Tuberculosis Pulmonar y Extrapulmonar en Calarcá-Quindío, Colombia-2005.

OBJECTIVE: Describing socio-demographic characteristics in a population of 195 respiratory symptomatic and 18 tuberculosis patients from Calarcá-Quindío, Colombia during 2005. METHODS: An active search for individuals having symptomatic tuberculosis was carried out in three urban areas, Calarca's municipal prison and the San Juan de Dios teaching hospital. Prior to entering the study, the patients were required to fill out an application form and sign an informed consent form. Sputum samples were taken from each patient for bacilloscopy and culture in Ogawa-Kudoh medium. Phenotypic and genotypic identification were made when growth was observed by using conventional and molecular methods. RESULTS: 18 (9 ,2 %) out of 195 individuals having respiratory symptoms were diagnosed as having tuberculosis. 78 % were male and 22 % female; the average age was 40 for both genders. 174 sputum, 21 bronchial lavage, one urine, one cervical ganglia and pleural liquid sample were analysed; 77 % proved positive by direct smear test and culture, 16,7 % by culture and only 5,5 % by direct smear test. Bacilloscopy average per patient was 2 ,3 . The presence of H1, T1, H3 and LAM9 genotypes was demonstrated by using the spoligotyping test. CONCLUSIONS: The results confirmed the importance of using both direct smear test and/or culture for improving the early detection of pulmonary tuberculosis, its follow-up and control. The existence of four different Mycobacterium tuberculosis genotypes in Calarcá was established .

Búsqueda activa de individuos con tuberculosis pulmonar y extrapulmonar en Calarcá-Quindío, Colombia-2005 / Detecting active tuberculosis in Calarcá-Quindío, Colombia, during 2005

Objetivo Describir características socio demográficas en una muestra de 195 pacientes sintomáticos respiratorios y 18 pacientes tuberculosos de Calarcá - Quindío, Colombia en el 2005. Metodología Se realizó búsqueda activa de tuberculosos en tres zonas urbanas, la cárcel del municipio de Calarcá y el Hospital Universitario San Juan de Dios. Previo ingreso al estudio los pacientes diligenciaron una encuesta y un consentimiento informado. A cada paciente se le tomaron muestras de esputo para baciloscopia y cultivo en medio de Ogawa-Kudoh. Cuando se observó crecimiento se realizó identificación fenotípica por pruebas bioquímicas y genotipificación por spoligotyping. Resultados Se identificaron 195 pacientes sintomáticos respiratorios y 18 (9,2 por ciento) pacientes tuberculosos, de ellos el 78 por ciento corresponde al género masculino y 22 por ciento al femenino con una edad promedio para ambos sexos de 40 años. Se procesaron un total de 174 muestras de esputo, 21 lavados bronco alveolares, una muestra de orina, una muestra de ganglio cervical y un liquido pleural. El 77 por ciento de las muestras analizadas fueron positivos por examen directo y cultivo, 16,7 por ciento por cultivo y el 5,5 por ciento por baciloscopia únicamente. El promedio de baciloscopias por paciente fue 2,3. La prueba de spoligotyping realizada evidenció la presencia de los genotipos H1, T1, H3 y LAM9. Conclusión Los resultados obtenidos demuestran la importancia de realizar simultáneamente baciloscopia y cultivo para aumentar la detección de pacientes bacilíferos sintomáticos respiratorios, al igual que en el seguimiento y control de estos pacientes. Se demostró la presencia de cuatro genotipos diferentes de Mycobacterium tuberculosis en Calarcá.

Objective Describing socio-demographic characteristics in a population of 195 respiratory symptomatic and 18 tuberculosis patients from Calarcá-Quindío, Colombia during 2005. Methods An active search for individuals having symptomatic tuberculosis was carried out in three urban areas, Calarca's municipal prison and the San Juan de Dios teaching hospital. Prior to entering the study, the patients were required to fill out an application form and sign an informed consent form. Sputum samples were taken from each patient for bacilloscopy and culture in Ogawa-Kudoh medium. Phenotypic and genotypic identification were made when growth was observed by using conventional and molecular methods. Results 18 (9 ,2 percent) out of 195 individuals having respiratory symptoms were diagnosed as having tuberculosis. 78 percent were male and 22 percent female; the average age was 40 for both genders. 174 sputum, 21 bronchial lavage, one urine, one cervical ganglia and pleural liquid sample were analysed; 77 percent proved positive by direct smear test and culture, 16,7 percent by culture and only 5,5 percent by direct smear test. Bacilloscopy average per patient was 2 ,3 . The presence of H1, T1, H3 and LAM9 genotypes was demonstrated by using the spoligotyping test. Conclusions The results confirmed the importance of using both direct smear test and/or culture for improving the early detection of pulmonary tuberculosis, its follow-up and control. The existence of four different Mycobacterium tuberculosis genotypes in Calarcá was established.