Biofilm Formation of Clinical Klebsiella pneumoniae Strains Isolated from Tracheostomy Tubes and Their Association with Antimicrobial Resistance, Virulence and Genetic Diversity

Abstract

(1) Background: Due to the commonness of tracheotomy procedures and the wide use of biomaterials in the form of tracheostomy tubes (TTs), the problem of biomaterial-associated infections (BAIs) is growing. Bacterial colonization of TTs results in the development of biofilms on the surface of biomaterials, which may contribute to the development of invasive infections in tracheostomized patients. (2) Methods: Clinical strains of K. pneumoniae, isolated from TTs, were characterized according to their ability to form biofilms, as well as their resistance to antibiotics, whether they harbored ESβL genes, the presence of selected virulence factors and genetic diversity. (3) Results: From 53 patients, K. pneumoniae were detected in 18 of the TTs examined, which constituted 34% of all analyzed biomaterials. Three of the strains (11%) were ESβL producers and all had genes encoding CTX-M-1, SHV and TEM enzymes. 44.4% of isolates were biofilm formers, SEM demonstrating that K. pneumoniae formed differential biofilms on the surface of polyethylene (PE) and polyvinyl chloride (PVC) TTs in vitro. A large range of variation in the share of fimbrial genes was observed. PFGE revealed sixteen genetically distinct profiles. (4) Conclusions: Proven susceptibility of TT biomaterials to colonization by K. pneumoniae means that the attention of research groups should be focused on achieving a better understanding of the bacterial pathogens that form biofilms on the surfaces of TTs. In addition, research efforts should be directed at the development of new biomaterials or the modification of existing materials, in order to prevent bacterial adhesion to their surfaces.

Keywords: Klebsiella pneumoniae; PCR; PFGE; SEM; biofilm; tracheostomy tube.

Figure 1

Quantitative assays of biofilm formation on abiotic surface (polystyrene microtiter plate) of clinical strains of K. pneumoniae isolated from patients with tracheostomies. Absorbance values of 0.254–0.509 of OD_595nm were classified as moderate biofilm producers. Absorbance values of 0.127–0.254 were considered as weak biofilm producers and values <0.127 were classed as non-biofilm producers.

Figure 2

Dendrogram representing the genetic relationship between 18 K. pneumoniae isolates after restriction with XbaI enzyme. ESβL-producing strains were part of three different PFGE profiles. DNA fragments were separated by PFGE using a CHEF DR^®II system. PFGE profiles were compared according to their percentage of similarity, estimated by the Dice coefficient and clustered by UPGMA using GelCompar II v.6.5 software. PFGE settings: optimization, 1%; tolerance, 1%. Images were captured and converted to TIFF for computer analysis.

Figure 3

(a) SEM micrograph of a polyethylene tracheostomy tube (PE) showing K. pneumoniae bacterial biofilm on the outer surface. (b) SEM micrograph of a polyethylene tracheostomy tube (PE) showing K. pneumoniae bacterial biofilm on the inner surface. (c) SEM micrograph of a polyvinyl chloride tracheostomy tube (PVC) showing K. pneumoniae bacterial biofilm on the outer surface. (d) SEM micrograph of a polyvinyl chloride tracheostomy tube (PVC) showing K. pneumoniae bacterial biofilm on the inner surface.