ABSTRACT
Background: Klebsiella pneumoniae is considered an opportunistic pathogen associated with nosocomial infections occurring mainly in pediatric patients, such as premature infants placed in intensive care units. The aim of this study was to characterize K. pneumoniae strains isolated from different clinical sources based on their resistance to antibiotics and the presence of virulence factors associated with their persistence in the hospital environment. Methods: Fifty clinical strains of K. pneumoniae isolated from urine, blood, catheters, and cerebrospinal fluid sources were characterized. Susceptibility testing of antibiotics was performed by the Kirby-Bauer method (Clinical Laboratory Standards Institute, 2010). The ability to form a biofilm was determined by the 96-well microplate method. Capsule and fimbrial structures were visualized by transmission electron microscopy (TEM). Adherence was evaluated on A549 and HT29 cells. Assessment for the presence and expression of the ecpA, fimH, and mrkA genes was performed by PCR and RT-PCR. Results: Clinical strains of K. pneumoniae were isolated from 48% of urine, 24% of blood, 18% of catheters, and 10% of cerebrospinal fluid. Ninety-two percent of the strains showed resistance to cefpodoxime, whereas few strains showed resistance to imipenem and meropenem (4 and 2%, respectively). The extended spectrum-type beta-lactamase (ESBL) phenotype was identified in 97% of the strains positive for resistance to third-generation cephalosporins. In addition, 88% of the strains were multidrug resistant. All strains were able to form biofilms. Capsule and fimbirial structures were visualized by TEM. Based on our adhesion assays, the A549 cell line was more permissive to K. pneumoniae strains than the HT-29 cell line. K. pneumoniae strains amplified and expressed ecpA (100/70%), fimH (98/2%), and mrkA (84/48%) genes, respectively. Conclusion: The K. pneumoniae strains exhibited features that allowed them to survive in the hospital environment (formation of biofilm) and resist antimicrobial therapy (multidrug resistant MDR strains). These strains also possessed a capsule, adhesive properties, and expression of genes encoding colonization factors that favor the selection and persistence of these strains in hospitals.
ABSTRACT
Introducción. A escala mundial, se ha observado la aparición de cepas de Pseudomonas aeruginosa multirresistentes durante las últimas décadas. Este patógeno oportunista produce mecanismos de resistencia a diversos antibióticos. La resistencia a carbapenémicos en cepas de P. aeruginosa se ha asociado con la formación de biopelículas bacterianas, favorecidas por la presencia de exopolisacáridos (EPS) embebidos en una matriz extracelular y por la producción de los pili tipo IV (T4P). El objetivo de este trabajo fue evaluar la formación de biopelículas en cepas clínicas aisladas en el Hospital Infantil de México Federico Gómez de P. aeruginosa resistentes a carbapenémicos, a través de la cuantificación de los expolisacáridos totales-reductores y su asociación con la expresión fenotípica de los T4P. Métodos. Se realizaron ensayos de susceptibilidad antibiótica por el método de Kirby-Bauer en 92 cepas clínicas de P. aeruginosa. Asimismo, se determinó la concentración mínima inhibitoria (MIC) para imipenem (IMP) y meropenem (MEM) por el método de dilución seriada en placas con agar con un replicador de Steers. La producción de metalo-β-lactamasas fue determinada mediante la técnica de difusión en disco y de sinergismo. Las biopelículas fueron realizadas en cepas clínicas de P. aeruginosa resistentes a carbapenémicos, a través de la cuantificación de cristal violeta, azúcares totales (antrona) y azúcares reductores (DNS), además de la expresión fenotípica de los T4P por la actividad de twitching motility . La diversidad genética de las cepas formadoras de biopelículas y productoras de azúcares reductores fue evaluada mediante la técnica de electroforesis de campos pulsados (PFGE). Resultados. El 30.4% (28/92) de las cepas de P. aeruginosa de origen pediátrico fueron recuperadas de la sala de cirugía y el 50% (46/92) de muestras de orina. Los resultados por Kirby-bauer mostraron que más de 50% de la cepas de P. aeruginosa fueron resistentes a 12 diferentes antibióticos. La MIC a los carbapenémicos fue de 64 µg/ml, con 43.1% (25/58) para MEM y 56.8% (33/58) para IMP. Así mismo, la producción de metalo-β-lactamasas fue observada en 43% (25/58) para MEM, 2% (1/58) para IMP y 12% (7/58) para ambas. Los análisis mostraron que 82% (48/58) de las cepas de P. aeruginosa resistentes a carbapenémicos fueron altas formadoras de biopelículas. De éstas, 46.5% (27/58) mostraron concentraciones de EPS totales de 2000 a 6000 µg/ml y 27.5% (16/58) mostraron concentraciones de EPS reductores de 316 a 1108 µg/ml; además, 75% (44/58) de estas cepas mostraron actividad fenotípica de twitching motility . Conclusiones. La detección de azúcares totales, azúcares reductores y el fenómeno de twitching motility son factores que favorecen el desarrollo de las biopelículas en cepas clínicas de P. aeruginosa resistentes a carbapenémicos. Los datos sugieren que estos factores están involucrados en la formación de biopelículas que confieren a la bacteria la capacidad para sobrevivir, persistir y colonizar a su hospedero.
Background. In recent years, the worldwide emergence of multidrug-resistant strains of Pseudomonas aeruginosa has been observed. This opportunistic pathogen produces mechanisms of resistance to several antibiotics. The resistance to carbapenems in P. aeruginosa strains has been associated with bacterial biofilm formation, favored by the presence of exopolysaccharides (EPS) embedded in an extracellular matrix and to the production of type IV pili (T4P). We undertook this study to assess biofilm formation in clinical strains of P. aeruginosa resistant to carbapenems isolated at the Hospital Infantil de Mexico Federico Gomez (HIMFG) through quantification of total-reducing EPS and its association with the phenotypic expression of T4P. Methods. Antibiotic susceptibility tests were performed using the Kirby-Bauer method in 92 clinical isolates of P. aeruginosa ; likewise, the minimum inhibitory concentration (MIC) was determined for imipenem (IMP) and meropenem (MEM) by the serial dilution method in agar plates with a Steers replicator. Production of metallo-β-lactamase (MBL) was determined by the disk diffusion method and synergism. Biofilm formation was performed in clinical isolates of P. aeruginosa resistant to carbapenems through the quantification of crystal violet, total sugar (anthrone), and reducing sugar (DNS), in addition to the phenotypic expression of T4P activity of twitching motility. The genetic diversity of strains forming biofilm and producing reducing sugars was evaluated by pulsed-field gel electrophoresis (PFGE). Results. There were 30.4% (28/92) of P. aeruginosa strains of pediatric origin and 50% (46/92) of urine samples that were recovered from the operating room. The results using the Kirby-Bauer method showed that >50% of P. aeruginosa strains were resistant to 12 different antibiotics. The MIC to carbapenems was 64 µg/ml, with 43.1% (25/58) for MEM and 56.8% (33/58) for IMP. Likewise, MBL production was observed in 43% (25/58) for MEM, 2% (1/58) for IMP, and 12% (7/58) for both. Qualitative and quantitative analysis showed that 82% (48/58) of P. aeruginosa strains resistant to carbapenems were high biofilm formers using the crystal violet method. Of the high biofilm forming strains, 46.5% (27/58) showed concentrations of total EPS between 2000 and 6000 µg/ml and 27.5% (16/58) showed concentrations of reducing EPS between 316 and 1108 µg/ml. In addition, 75% (44/58) of these strains showed phenotypic activity of twitching motility. Conclusions. Detection of total sugars, reducing sugars, and the phenomenon of twitching motility are factors that promote the development of biofilms in clinical strains of P. aeruginosa resistant to MBL producers to carbapenems. Our data suggest that these factors are involved in biofilm formation, which confer bacterium with the ability to survive, persist, and colonize its host.