Insights on Klebsiella pneumoniae Biofilms Assembled on Different Surfaces Using Phenotypic and Genotypic Approaches

Abstract

Klebsiella pneumoniae is a prominent etiological agent of healthcare associated infections (HAIs). In this context, multidrug-resistant and biofilm-producing bacteria are of special public health concern due to the difficulties associated with treatment of human infections and eradication from hospital environments. Here, in order to study the impact of medical devices-associated materials on the biofilm dynamics, we performed biofilm phenotypic analyses through a classic and a new scanning electron microscopy (SEM) technique for three multidrug-resistant K. pneumoniae isolates growing on polystyrene and silicone. We also applied whole-genome sequencing (WGS) to search for genetic clues underlying biofilm phenotypic differences. We found major differences in the extracellular polymeric substances (EPS) content among the three strains, which were further corroborated by in-depth EPS composition analysis. WGS analysis revealed a high nucleotide similarity within the core-genome, but relevant differences in the accessory genome that may account for the detected biofilm phenotypic dissimilarities, such as genes already associated with biofilm formation in other pathogenic bacteria (e.g., genes coding haemogglutinins and haemolysins). These data reinforce that the research efforts to defeat bacterial biofilms should take into account that their dynamics may be contingent on the medical devices-associated materials.

Keywords: Klebsiella pneumoniae; biofilms; electron microscopy; extracellular polymeric substances (EPS); healthcare associated infections (HAIs); whole genome sequencing (WGS).

Figure 1

K. pneumoniae biofilms assembled on different surfaces: (A–F) representative micrographs of 12 h and 24 h old biofilms assembled on polystyrene; and (G–L) silicone by Kp45, Kp703 and Kp2948 are shown. Twelve hours old polystyrene biofilms: (A) Kp45; (B) Kp703; and (C) Kp2948; 24 h old polystyrene biofilms: (D) Kp45; (E) Kp703; and (F) Kp2948; 12 h old silicone biofilms: (G) Kp45; (H) Kp703; and (I) Kp2948; and 24 h old silicone biofilms: (J) Kp45; (K) Kp703; and (L) Kp2948. The structures identified as mature biofilms are highlighted with a red asterisk (*). Scale bars = 10 µm.

Figure 2

K. pneumoniae biofilms assembled on silicone. The relative areas occupied within biofilms by its major components (bacteria and extracellular polymeric matrix—EPS) were determined using ImageJ software. (A) The total biofilm area was delimited (blue line) as well as the areas occupied by bacteria (purple) and EPS (orange) as schematically shown. Representative micrographs of 4 h old: (B) Kp703; (C) Kp45; and (D) Kp2948 are shown (scale bars = 1 µm).

Figure 3

Characterization of K. pneumoniae biofilms assembled on silicone. (A) Bacteria; and (B) EPS relative areas existent on 4 h, 12 h and 24 h old biofilms assembled on silicone by the three isolates were determined. Bold bars were used for biofilms assembled on silicone and empty bars for biofilms assembled on polystyrene (results from [9]). (A) A significant statistical difference in bacteria relative area was observed for 12 h old for Kp45 biofilms assembled on polystyrene and silicone. (B) For extracellular matrix relative areas, significant statistical differences were observed for 4 h, 12 h and 24 h old Kp2948 assembled on the two surfaces and also between 4 h old Kp45 biofilms assembled on silicone and the other two isolates (* p < 0.05; ** p < 0.01).

Figure 4

The extracellular polymeric matrix of K. pneumoniae biofilm. (A) The results of enzymatic digestion of Kp biofilms are presented (* p < 0.05). In the insets of figures (B–D) are shown CFU of: (B) Kp703; (C) Kp45; and (D) Kp2948 grown on MH-Congo red after recovery from biofilms assembled on silicone. In the TEM micrographs of: (B) Kp703; (C) Kp45; and (D) Kp2948, it is possible to observe the absence of curli (scale bars = 2 µm).

Figure 4

The extracellular polymeric matrix of K. pneumoniae biofilm. (A) The results of enzymatic digestion of Kp biofilms are presented (* p < 0.05). In the insets of figures (B–D) are shown CFU of: (B) Kp703; (C) Kp45; and (D) Kp2948 grown on MH-Congo red after recovery from biofilms assembled on silicone. In the TEM micrographs of: (B) Kp703; (C) Kp45; and (D) Kp2948, it is possible to observe the absence of curli (scale bars = 2 µm).

Figure 5

Pan-genome overview. The Venn diagram shows the number of shared and unique predicted coding sequences (CDSs) among the three K. pneumoniae isolates (Kp703, Kp45 and Kp2948) deduced from a Basic Local Alignment Search Tool (BLASTP) analysis performed using RAST, where protein sequences with more than 75% of sequence identity were considered as homologues. Circles are not drawn to scale.