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

Abstract

Acinetobacter baumannii is a successful nosocomial pathogen, causing severe, life-threatening infections in hospitalized patients, including pneumonia and bloodstream infections. The spread of CRAB (carbapenem-resistance Acinetobacter baumannii) strains is a major world-health threat. The successful spread of CRAB is mostly due to it's highly plasticity genome. Although some virulence factors associated with CRAB have been uncovered, many mechanisms contributing to its success are not fully understood. Here we describe strains of CRAB that were isolated from fulminant cases in two hospitals in Israel. These isolates show a rare, hyper mucoid (HM) phenotype and were investigated using phenotypic assays, comparative genomics and in vivo Galleria Mellonella model. The three isolates belonged to the ST3 international clonal type and closely related to each-other as shown by Fourier Transform Infrared Spectroscopy (FTIR) and phylogenetic analyses. These isolates possessed thickened capsules and dense filamentous extracellular polysaccharides (EPS) matrix as shown by transmission electron microscopy (TEM), and overexpress capsule polysaccharide synthesis pathway-related wzc gene. The HM isolates possessed a unique combination of virulence genes involved in iron metabolism, protein secretion, adherence and membrane glycosylation. HM strains were more virulent than control strains in two G. mellonella infection models. In conclusion, our findings demonstrated several virulence factors, all present in three CRAB isolates with rare hyper mucoid phenotypes.

Phenotypic and Genomic Characterization of Nine String-Positive Carbapenem-Resistant Acinetobacter baumannii Isolates from Israel

“…The high plasticity of the CRAB genome could potentially lead to the emergence of novel resistant and virulent isolates, and thus, monitoring new CRAB phenotypes is highly important. CRAB species can form mucoid colonies ( 6 ), and two recent studies linked A. baumannii mucosity and capsule formation to the clinical severity and epidemic potential of the strains ( 6 , 16 ). A. baumannii can undergo a phase variation switch from a matte to an opaque phenotype with different virulence characteristics ( 17 ).…”

“…Isolates were grown overnight at 37°C on TSA blood agar plates. The samples were prepared according to a standard protocol ( 34 ) and as described previously ( 6 ). The samples were fixed for 3 h in Karnovsky fixative (2.5% glutaraldehyde with 2.5% paraformaldehyde in 0.1 M sodium cacodylate buffer [pH 7.4]) and then washed with 0.1 M sodium cacodylate buffer.…”

“…Cultures grown overnight in BHI medium at 37°C with shaking were diluted 1:1,000 in medium (BHI medium). The motility assay was performed on freshly prepared semisolid (0.25% agar) Difco agar (BD, USA) plates using a previously described protocol ( 6 ). The agar was stabbed with approximately 1 μL of a diluted bacterial suspension, followed by a 24-h incubation at 37°C.…”

“…They were stored and used according to the manufacturer’s instructions. The G. mellonella killing assay was previously described by Rakovitsky et al ( 6 ). In brief, bacteria were grown in 5 mL BHI medium overnight at 37°C with shaking, diluted to an OD of 0.1, grown for another 2 to 3 h until they reached logarithmic phase, diluted to 10 5 cells in 20 μL, and injected with a 32-gauge needle (Hamilton, Reno, NV, USA) into larvae (20 larvae per group).…”

“…We recently described mucoid carbapenem-resistant A. baumannii (CRAB) strains with elevated expression levels of the capsular gene wzc , that were associated with fulminant infections in patients and increased virulence in a G. mellonella model ( 6 ). We therefore paid special attention to the mucoid phenotype, and during routine screening of clinical samples, we observed an A. baumannii isolate with a “string test-positive” phenotype.…”