Rapid identification of capsulated Acinetobacter baumannii using a density-dependent gradient test

Abstract

Background Gram-negative bacterial capsules are associated with production of carbohydrates, frequently resulting in a mucoid phenotype. Infections caused by capsulated or mucoid A. baumannii are associated with increased clinical severity. Therefore, it is clinically and epidemiologically important to identify capsulated A. baumannii. Here, we describe a density-dependent gradient test to distinguish between capsulated and thin/non-capsulated A. baumannii. Results Thirty-one of 57 A. baumannii isolates displayed a mucoid phenotype. The density-dependent gradient test was comprised of two phases, with silica concentrations of 30% (top phase) and 50% (bottom phase). Twenty-three isolates migrated to the bottom phase, indicating thin or non-capsulated strains, and 34 migrated to the top phase, suggesting strains suspected to be capsulated. There was agreement between the mucoid and the non-mucoid phenotypes and the density-dependent gradient test for all but three isolates. Total carbohydrates extracted from strains suspected to be capsulated were significantly higher. Transmission electron microscopy confirmed the presence of a capsule in the six representative strains suspected to be capsulated. Conclusions The density-dependent gradient test can be used to verify capsule presence in mucoid-appearing A. baumannii strains. Identifying capsulated strains can be useful for directing infection control measures to reduce the spread of hypervirulent strains.

Increased Capsule Thickness and Hypermotility Are Traits of Carbapenem-Resistant Acinetobacter baumannii ST3 Strains Causing Fulminant Infection

“…Isolates were categorized by visual inspection as mucoid or nonmucoid [ 18 ]. The phenotype was also evaluated according to several colony morphology parameters, including texture, elevation, margin, size, and shape [ 19 ].…”

“…The presence of capsule was confirmed using a density-dependent gradient test [ 18 ]. Results were read by visual observation: A bacterial band that migrated to the bottom phase of the gradient was classified as a noncapsulated strain, while a bacterial band that concentrated in the top phase was suspected to be a capsulated strain.…”

DksA is a central regulatory switch for stress protection and virulence in Acinetobacter baumannii

“…For qualitative estimation of capsule levels, we used density gradient centrifugation as previously described 101 , which is based on the effect of cell-associated capsule on bacterial density. Briefly, 1 ml of overnight grown cultures were centrifuged, washed with PBS and resuspended in 1 ml PBS.…”

Presentation_1.pptx

“…Previous work revealed that wild-type VIR-O cells exhibit a 2fold increase in capsule compared with the wild-type AV-T (Chin et al, 2018). We utilized a Percoll density gradient, a method that was recently described as a method to separate A. baumannii strains by capsule level (Kon et al, 2020), to compare capsular polysaccharide levels in AV-T D1132 to wild-type VIR-O and AV-T cells. As seen in Figure 3C, the gradient is able to distinguish between capsule levels of the wild-type VIR-O and AV-T variants, with the AV-T cells migrating significantly further than the VIR-O cells.…”

“…Method slightly modified from Kon et al (2020). Cell cultures of VIR-O, AV-T, and AV-T D1132 were grown in 2 mL LB from pure frozen stocks to OD 600 ~0.6 (shaking, 37°C).…”