Quantification of biofilm produced by clinical, environment and hands' isolates Klebsiella species using colorimetric and classical methods

Abstract

Some species of Klebsiella, such as Klebsiella pneumoniae and Klebsiella oxytoca, are important nosocomial pathogens frequently involved in outbreaks in Neonatal Intensive Care Units (NICU) and have the ability to form a biofilm. This study aims to evaluate the biofilm production of K. pneumoniae and K. oxytoca isolates collected from the hands of health professionals, neonates' blood and the environment of a Brazilian NICU, using three colorimetric methods and a classical method of counting the colony-forming units and compare the analysis among these techniques. The biofilm formation was carried out by the microplate technique, using three colorimetric assays: crystal violet, safranin and 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl) -5 [(phenylamino) arbonyl] - 2H-tetrazolium hydroxide (XTT). Also, colony-forming units were determined. Twenty-eight isolates of K. pneumoniae were collected from the blood, hands and environment and five of K. oxytoca from the hands and environment. All of them were strong biofilm producers, but K. pneumoniae isolates produced more biofilm than K. oxytoca when compared to the American Type Culture Collection (ATCC) strains used as positive controls. The number of viable cells in the biofilm produced by K. pneumoniae isolated from blood was significantly higher than in the control sample. Regarding the three colorimetric tests used in the study, the violet crystal obtained a higher absorbance average. The use of crystal-violet and XTT in the evaluation of biofilm in vitro make possible a complete analysis, since that it can quantify the total biomass (including the extracellular matrix) and evaluate the metabolic activity. In conclusion, this study identified isolates of K. pneumoniae and K. oxytoca that produce biofilms in the NICU and the bloodstream of neonates. This fact deserves attention since these patients are immunocompromised. The best methods will be chosen to answer research questions by always adopting more than one method so that more than one parameter or component of the biofilm is analyzed.

Keywords: Biofilm; Klebsiella oxytoca; Klebsiella pneumoniae; Neonatal units.