Exploring Dangerous Connections between Klebsiella pneumoniae Biofilms and Healthcare-Associated Infections

Abstract

Healthcare-associated infections (HAI) are a huge public health concern, particularly when the etiological agents are multidrug resistant. The ability of bacteria to develop biofilm is a helpful skill, both to persist within hospital units and to increase antibiotic resistance. Although the links between antibiotic resistance, biofilms assembly and HAI are consensual, little is known about biofilms. Here, electron microscopy was adopted as a tool to investigate biofilm structures associated with increased antibiotic resistance. The K. pneumoniae strains investigated are able to assemble biofilms, albeit with different kinetics. The biofilm structure and the relative area fractions of bacteria and extracellular matrix depend on the particular strain, as well as the minimal inhibitory concentration (MIC) for the antibiotics. Increased values were found for bacteria organized in biofilms when compared to the respective planktonic forms, except for isolates Kp45 and Kp2948, the MIC values for which remained unchanged for fosfomycin. Altogether, these results showed that the emergence of antimicrobial resistance among bacteria responsible for HAI is a multifactorial phenomenon dependent on antibiotics and on bacteria/biofilm features.

Figure 1

The kinetics of biofilm assembly by 10 strains of K. pneumoniae was followed over 48 h using a spectrophotometric assay (A); in parallel, scanning electron microscopy (B,C) was used to illustrate biofilm assembly. Representative micrographs of 12 h-old biofilms of kp703 (B) and Kp2948 (C) show the different ability to assemble biofilms exhibited by these strains. The presence of organized bacterial structures is highlighted by arrowheads (B). Scale bar: 10 μm.

Figure 2

The biofilms assembled by three K. pneumoniae strains (Kp45, Kp703 and Kp2948) were characterized using SEM in backscattered electron mode. The existence of extracellular matrix (arrow heads) surrounding bacteria from early stages is highlighted by arrow heads in a 4 h-old Kp703 micrograph (A). The relative areas occupied by bacteria (B) and extracellular matrix (C) during the different phases of biofilm assembly were assessed. The differences were considered significant for p < 0.05 (* p < 0.05; ** p < 0.01). Scale bar: 1 μm.