Avaliação in vitro da formação de biofilme de klebsiella pneumoniae em cateteres impregnados com substâncias com atividade antimicrobiana

Abstract

Klebsiella pneumoniae is responsible for several infections in impaired and immunosuppressed patients, especially those associated with long-term of hospital stay and the use of invasive medical devices. The major implication of this microorganism in catheters - associated infections involve bacterial’ colonization and biofilm formation. Biofilm is a complex structure that could detach from the abiotic surface and circulate in body fluids with their resistance profile. The use of catheters impregnated with antimicrobial substances could effectively reduce blood stream infections, inhibiting microbial adherence and consequently the biofilm formation. This study aims to evaluate in vitro activity of antimicrobial drugs and the effectivity of the central venous catheter impregnated with antimicrobial and antiseptic regarding the biofilm formation and gene expression involved. Fragments of catheter venous central impregnated with N-acetylcysteine / levofloxacin (NAC / LEV) and Chlorhexidine / silver sulfadiazine (CH/SS) were assayed in triplicate against a standard strain K. pneumoniae ATCC 13883 and a clinical isolate of K. pneumoniae carbapenemase – producer. The activity and durability of antimicrobial substances on catheter’s surface were tested in three, seven, ten, 15 and 30 days. The catheters were separately in tubes containing human serum. After that, they were removed and the sizes of the zones of inhibition evaluated using a modified Kirby-Bauer Disk diffusion test. In vitro biofilm colonization was assessed by quantitative culture and scanning electron microscopy. The knowledge of the activity and durability of antimicrobials in these devices allowed to evaluate their effectiveness against the adhesion of the microorganism to the impregnated abiotic surface. The NAC / LEV impregnated catheter obtained longer durability than the CH/SS catheter, but the second catheter was more effective in inhibiting biofilm but with low durability. The emergence of resistant subpopulations in the NAC / LEV impregnated catheter was demonstrated to the clinical isolated from the third day of segment incubation in the serum. As for the presence of zones of inhibition, the clinical isolated had lower inhibition halo than the reference sample. Scanning electron microscopy confirmed the results of the quantitative culture and showed 8 some features of the K. pneumoniae biofilm such as the exopolysaccharide matrix - EPS, structures that promote the adhesion (curli) to the surface, besides its organization, in both samples. Human serum contributed to the formation of this community. Biofilm formation of carbapenemase-producing K. pneumoniae can occur on catheters impregnated as early as third day, although the biofilm is less thick. The emergence of resistant subpopulations can compromise catheter activity and therapy, becoming a real problem, especially when considering its clinical use in immunosuppressed patients. The present study contributed to demonstrate that impregnated catheters have limited antimicrobial activity over time and may vary according to the microorganism that colonizes the medical device. However, more studies are needed to evaluate the intrinsic features of the structure, the mechanism of adaptability K.pneumoniae KPC+ biofilm to other microorganisms, transfer of resistance genes and gene expression.