Evaluation of different capsule staining methods and development of an improved capsule staining in Klebsiella pneumoniae

Introduction

The capsule serves as a virulence factor in various bacterial species, playing a crucial role in enabling bacteria to evade the host immune system. Although numerous methods for microscopic capsule visualization have been used for more than a century, the optimal method for capsule staining of Klebsiella pneumoniae has not yet been defined.

Goals

To close this gap, we compared two standard methods and a novel modified protocol to assess capsule production in K. pneumoniae by optical microscopy.

Materials & Methods

Fourteen K. pneumoniae isolates with different phenotypes as well as the reference strain K. pneumoniae ATCC 13883 were cultured overnight on Columbia blood agar at 35°C. Each isolate was stained using Anthony's and Maneval's methods as described by Hughes and Smith (1). Additionally, a novel modification of Maneval's method was used that incorporated an incubation in 10% skim milk broth at 35°C under aerobic conditions for 12 to 18 hours. Microscopy was performed using the Axio Imager.Z2m system equipped with the Plan-APOCHROMAT 100x/1.4 oil immersion objective and the Axiocam 305 camera (Zeiss). For comparison, the mucoid phenotype was also determined in a sedimentation assay as previously described (2).

Results

Robust and reproducible staining protocols were established to directly observe the differences in capsule by light microscopy. The most unambiguous and consistent findings were obtained with the modified Maneval's method including an overnight incubation in skim milk broth. This method effectively differentiated capsule size variations between distinct phenotypes (Figure 1). The concordance of these findings with the results of the sedimentation assay identifying mucoid phenotypes supports the validity of this modified capsule staining technique.

Summary

An improved staining method has been proposed, which can be used for the microscopic assessment of capsule as a virulence factor in K. pneumoniae.

References

Hughes RB, Smith AC. Capsule stain protocols. ASM. 2007.Eger E et al. Hypervirulent Klebsiella pneumoniae sequence type 420 with a chromosomally inserted virulence plasmid. Int J Mol Sci. 2021;22.