Design of a molecular method for subspecies specific identification of Klebsiella pneumoniae by using the 16S ribosomal subunit gene.
Artículo de revista
2011-12-19
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. La implementación de esta técnica podría
permitir la diferenciación temprana entre Klebsiella
pneumoniae ozaenae y Klebsiella pneumoniae rhinoscleromatis
que causan dos infecciones tratadas por lo general
de forma empírica y como consecuencia de esto, la terapia
antimicrobiana suele no ser efectiva, en especial en pacientes
crónicos. Se requiere ampliar los estudios con un número
mayor de cepas de referencia y aislados clínicos.
English
- Colombia Médica [855]
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