Evaluación del efecto de la exposición in vitro a concentraciones crecientes de imipenem en la resistencia a carbapenémicos, expresión relativa de los genes blaOXA-1, blaOXA-10, blaCTX-M-15, cambios en la formación de biopelículas y costo fisiológico asociado en un aislado clínico de Klebsiella pneumoniae no productora de carbapenemasas inicialmente sensible a imipenem
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Fecha
2022
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es
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Universidad Andrés Bello
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Licencia CC
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Resumen
La resistencia a los antimicrobianos es un problema global que va en aumento a pasos agigantados y es
multifactorial. En Chile la situación no es diferente al resto del mundo, y las principales infecciones intrahospitalarias
están asociadas a Klebsiella pneumoniae (Kpn) resistente a los antibióticos carbapenémicos. Basado en un aislado
clínico proveniente de un paciente con bacteriemia, el cual estaba colonizado con una cepa K. pneumoniae no
productora de carbapenemasas sensible a imipenem (IMI) y meropenem (MEM) pero resistente a ertapenem (ETP)
fue tratado con imipenem, sin embargo terminó adquiriendo resistencia in vivo a los tres carbapenémicos. El
Laboratorio de Genómica y Resistencia Microbiana (GeRM), realizó una secuenciación del genoma completo (WGS)
a estos aislados, y encontraron que la única diferencia entre el aislado sensible y los resistentes, fue la amplificación
en el genoma de los genes blaOXA-1, blaOXA-10 y blaCTX-M-15. Teniendo estos antecedentes se planteó como estrategia
experimental para esta tesis que, K. pneumoniae inicialmente sensible a IMI expuesta a concentraciones crecientes a
dicho antibiótico estaría induciendo un aumento en la resistencia a antibióticos carbapenémicos y por consecuencia,
un aumento en la expresión relativa de los genes blaOXA-1, blaOXA-10 y blaCTX-M-15. Además se propuso que la ganancia
de resistencia impactaría al fenotipo de K. pneumonia en su capacidad de profileración y formación de biopelícula. El
objetivo general fue evaluar el efecto de IMI in vitro, la expresión relativa de los genes blaOXA-1 y blaOXA-10 y blaCTX-M15, junto con explorar la formación de una biopelícula del aislado clínico. Por su parte, los objetivos específicos fueron;
i) obtener K. pneumoniae resistente a IMI a partir del aislado sensible (ACS-Kpn) para luego ii) analizar mediante
qPCR cambios de la expresión relativa de los genes blaOXA-1, blaOXA-10 y blaCTX-M-15, además de iii) asociar cambios en
la formación de biopelículas entre la cepa sensible y las líneas evolucionadas resistentes a carbapenémicos. Mediante
la metodología de evolución adaptativa de laboratorio (ALE), la cual selecciona aquellos linajes mejores adaptados a
la condición estresor/seleccionador, en este caso IMI, se obtuvieron tres líneas evolutivas de K. pneumoniae resistentes
a IMI, MEM y ETP, dicha resistencia fue conservada hasta por 16 días sin el agente estresor en el medio. Respecto a
los niveles de expresión relativa, mediante RT-qPCR sólo se observó una tendencia a la sobreexpresión de blaOXA-1 en
las cepas evolucionadas que fueron expuestas a 32X CMI con respecto al aislado inicial sensible. El estudio del impacto
en el costo fisiológico de las líneas evolutivas se realizó a través desde una mirada a la proliferación, muerte celular y
formación de biopelícula. Las curvas de proliferación demostraron que los cultivos resistentes (CMI de 4 μg/mL) no
presentan costos fisiológicos a diferencia de los cultivos con mayor resistencia a IMI (CMI 8 μg/mL). Por otra parte,
la curva de muerte reveló que IMI es el carbapenémico con mayor efecto bactericida sobre este aislado. Los ensayos
de cuantificación de biopelícula mostraron que el aislado ancestral en sí ya formaba biopelícula. Finalmente, la
hipótesis propuesta fue aceptada parcialmente, dado que la resistencia adquirida fue mediante el aumento de sólo un
gen propuesto, una β-lactamasas de espectro reducido, blaOXA-1, mientras que no se pudo corroborar una relación entre
un aumento de resistencia con el aumento de costo fisiológico y la formación de biopelículas, debido a que solo se
asoció costo fisiológico a aquellos linajes de mayor CMI, mientras que los niveles basales del aislado ancestral sensible
ya eran altos y fuertes.
Antimicrobial resistance is a global problem that is increasing by leaps and bounds and is multifactorial. In Chile, the situation is no different from the rest of the world, and the main nosocomial infections are associated with Klebsiella pneumoniae (Kpn) resistant to carbapenem antibiotics. Based on a clinical isolate from a patient with bacteraemia, which was colonized with a non-carbapenemase-producing strain K. pneumoniae sensitive to imipenem (IMI) and meropenem (MEM) but resistant to ertapenem (ETP), was treated with imipenem, without However, it ended up acquiring resistance in vivo to all three carbapenems. The Laboratory of Genomics and Microbial Resistance (GeRM) performed a whole genome sequencing (WGS) on these isolates, and they found that the only difference between the sensitive isolate and the resistant ones was the amplification of the blaOXA-1 genes in the genome. , blaOXA-10 y blaCTX-M15. Taking this background, it was proposed as an experimental strategy for this thesis that K. pneumoniae initially sensitive to IMI exposed to increasing concentrations of said antibiotic would be inducing an increase in resistance to carbapenem antibiotics and, consequently, an increase in the relative expression of the blaOXA-1, blaOXA-10 y blaCTX-M-15. In addition, it was proposed that the resistance gain would impact the phenotype of K. pneumonia in its profiling capacity and biofilm formation. The general objective was to evaluate the effect of IMI in vitro, the relative expression of the blaOXA-1, blaOXA-10 y blaCTX-M-15genes, together with exploring the formation of a biofilm of the clinical isolate. For their part, the specific objectives were; i) obtain IMI-resistant K. pneumoniae from the sensitive isolate (ACSKpn) and then ii) analyze changes in the relative expression of the blaOXA-1, blaOXA-10 y blaCTX-M-15 genes by qPCR, in addition to iii) associate changes in the formation of biofilms between the sensitive strain and the evolved lines resistant to carbapenems. Using the adaptive laboratory evolution (ALE) methodology, which selects those lineages best adapted to the stressor/selector condition, in this case IMI, three K. pneumoniae evolutionary lines resistant to IMI, MEM and ETP were obtained, said resistance was conserved. for up to 16 days without the stressing agent in between. Regarding the relative expression levels, by means of RT-qPCR a tendency to overexpression of blaOXA-1 was only observed in the evolved strains that were exposed to 32X CMI with respect to the initial sensitive isolate. The study of the impact on the physiological cost of the evolutionary lines was carried out through a look at proliferation, cell death and biofilm formation. The proliferation curves showed that the resistant cultures (MIC of 4 μg/mL) do not present physiological costs, unlike the cultures with greater resistance to IMI (MIC 8 μg/mL). On the other hand, the death curve revealed that IMI is the carbapenem with the greatest bactericidal effect on this isolate. Biofilm quantification assays showed that the ancestral isolate itself already formed a biofilm. Finally, the proposed hypothesis was partially accepted, since the acquired resistance was through the increase of only one proposed gene, a narrow-spectrum βlactamase, blaOXA-1, while a relationship between an increase in resistance with the increase in physiological cost and the formation of biofilms, because physiological cost was only associated with those lineages with the highest MIC, while the basal levels of the sensitive ancestral isolate were already high and strong.
Antimicrobial resistance is a global problem that is increasing by leaps and bounds and is multifactorial. In Chile, the situation is no different from the rest of the world, and the main nosocomial infections are associated with Klebsiella pneumoniae (Kpn) resistant to carbapenem antibiotics. Based on a clinical isolate from a patient with bacteraemia, which was colonized with a non-carbapenemase-producing strain K. pneumoniae sensitive to imipenem (IMI) and meropenem (MEM) but resistant to ertapenem (ETP), was treated with imipenem, without However, it ended up acquiring resistance in vivo to all three carbapenems. The Laboratory of Genomics and Microbial Resistance (GeRM) performed a whole genome sequencing (WGS) on these isolates, and they found that the only difference between the sensitive isolate and the resistant ones was the amplification of the blaOXA-1 genes in the genome. , blaOXA-10 y blaCTX-M15. Taking this background, it was proposed as an experimental strategy for this thesis that K. pneumoniae initially sensitive to IMI exposed to increasing concentrations of said antibiotic would be inducing an increase in resistance to carbapenem antibiotics and, consequently, an increase in the relative expression of the blaOXA-1, blaOXA-10 y blaCTX-M-15. In addition, it was proposed that the resistance gain would impact the phenotype of K. pneumonia in its profiling capacity and biofilm formation. The general objective was to evaluate the effect of IMI in vitro, the relative expression of the blaOXA-1, blaOXA-10 y blaCTX-M-15genes, together with exploring the formation of a biofilm of the clinical isolate. For their part, the specific objectives were; i) obtain IMI-resistant K. pneumoniae from the sensitive isolate (ACSKpn) and then ii) analyze changes in the relative expression of the blaOXA-1, blaOXA-10 y blaCTX-M-15 genes by qPCR, in addition to iii) associate changes in the formation of biofilms between the sensitive strain and the evolved lines resistant to carbapenems. Using the adaptive laboratory evolution (ALE) methodology, which selects those lineages best adapted to the stressor/selector condition, in this case IMI, three K. pneumoniae evolutionary lines resistant to IMI, MEM and ETP were obtained, said resistance was conserved. for up to 16 days without the stressing agent in between. Regarding the relative expression levels, by means of RT-qPCR a tendency to overexpression of blaOXA-1 was only observed in the evolved strains that were exposed to 32X CMI with respect to the initial sensitive isolate. The study of the impact on the physiological cost of the evolutionary lines was carried out through a look at proliferation, cell death and biofilm formation. The proliferation curves showed that the resistant cultures (MIC of 4 μg/mL) do not present physiological costs, unlike the cultures with greater resistance to IMI (MIC 8 μg/mL). On the other hand, the death curve revealed that IMI is the carbapenem with the greatest bactericidal effect on this isolate. Biofilm quantification assays showed that the ancestral isolate itself already formed a biofilm. Finally, the proposed hypothesis was partially accepted, since the acquired resistance was through the increase of only one proposed gene, a narrow-spectrum βlactamase, blaOXA-1, while a relationship between an increase in resistance with the increase in physiological cost and the formation of biofilms, because physiological cost was only associated with those lineages with the highest MIC, while the basal levels of the sensitive ancestral isolate were already high and strong.
Notas
Tesis (Ingeniería en Biotecnología)
Palabras clave
Resistencia a los Antibióticos, Klebsiella Pneumoniae