Deciphering the multifactorial nature of Acinetobacter baumannii pathogenicity

Abstract

Background: Acinetobacter baumannii is an emerging bacterial pathogen that causes a broad array of infections, particularly in hospitalized patients. Many studies have focused on the epidemiology and antibiotic resistance of A. baumannii, but little is currently known with respect to its virulence potential.

Methodology/principal findings: The aim of this work was to analyze a number of virulence-related traits of four A. baumannii strains of different origin and clinical impact for which complete genome sequences were available, in order to tentatively identify novel determinants of A. baumannii pathogenicity. Clinical strains showed comparable virulence in the Galleria mellonella model of infection, irrespective of their status as outbreak or sporadic strains, whereas a non-human isolate was avirulent. A combined approach of genomic and phenotypic analyses led to the identification of several virulence factors, including exoproducts with hemolytic, phospholipase, protease and iron-chelating activities, as well as a number of multifactorial phenotypes, such as biofilm formation, surface motility and stress resistance, which were differentially expressed and could play a role in A. baumannii pathogenicity.

Conclusion/significance: This work provides evidence of the multifactorial nature of A. baumannii virulence. While A. baumannii clinical isolates could represent a selected population of strains adapted to infect the human host, subpopulations of highly genotypically and phenotypically diverse A. baumannii strains may exist outside the hospital environment, whose relevance and distribution deserve further investigation.

Figure 1. A. baumannii growth kinetics.

(A) Growth (OD₆₀₀) of A. baumannii strains at 37°C in TSBD medium. (B) Temperature-dependent growth kinetics, defined as maximum growth rates (µ_max) of A. baumannii strains grown in TSBD medium at the different temperatures. Values represent the mean (± standard deviation, SD) of three independent experiments. Symbols: AYE, squares; ACICU, circles; ATCC 17978, triangles; SDF, diamonds.

Figure 2. Iron-uptake capability.

(A) Growth of A. baumannii strains on 2,2′-dipyridyl gradient (0–500 µM) TSBD agar plates. The ordinate shows the minimal inhibitory concentration of the iron chelator 2,2′-dipyridyl. (B) Iron-chelating activity in culture supernatants of A. baumannii strains grown in CAA supplemented with either 50 µM 2,2′-dipyridyl or 50 µM FeCl₃ (black and white bars, respectively) and TSBD supplemented with 50 µM 2,2′-dipyridyl or 50 µM FeCl₃ (light and dark gray bars, respectively). Values are expressed as percentage of siderophore units (U) normalized to the cell density (OD₆₀₀) of the bacterial culture. Amount of catechol (C) and hydroxamate (D) groups in the supernatants of A. baumannii cultures. Values are expressed as concentration (µM) of hydroxamate or catechol groups normalized to the OD₆₀₀ of the bacterial culture. Growth conditions and symbols correspond to those described in the legend to panel B. Values represent the mean (±SD) of two independent experiments, each including three biological replicates. ** P<0.01 (ANOVA).

Figure 3. Hemolytic activity.

(A) Hemolysis on 5% horse blood Columbia agar plates. A. baumannii strains are, from top left in clockwise order: AYE, ACICU, ATCC 17978 and SDF. P. aeruginosa PAO1 was spotted on the center of the plate as positive control for hemolysis. Hemolysis is evidenced by the transparent halo around the colony. (B) Hemolytic activity on horse and sheep erythrocytes (black and white bars, respectively) of cell-free supernatants from A. baumannii cultures grown in TSBD medium. Hemolytic activity was normalized to the OD₆₀₀ of the bacterial culture. Values represent the mean (± SD) of two independent experiments, each including three biological replicates.

Figure 4. Production of virulence-related exoproteins.

Phospholipase C (A) and total proteolytic (B) activities in filter-sterilized supernatants of the different A. baumannii strains grown for 14 h in TSBD at 37°C. Activities are expressed in U/L of culture supernatants and normalized to the OD₆₀₀ of the bacterial cultures. P. aeruginosa PAO1 was used as positive control for phospholipase C and proteolytic activities. Values represent the mean (± SD) of three independent experiments. ** P<0.01 (ANOVA).

Figure 5. Biofilm formation.

Biofilm formation on 96-well polystyrene microtiter plates by A. baumannii strains grown statically in LB medium for 24 h. Values represent the mean (± SD) of five independent experiments performed in quadruplicate. * P<0.05, *** P<0.001 (ANOVA).

Figure 6. Twitching motility.

A. baumannii twitching motility after 24 h of growth at 37°C in TSBD medium. A. baumannii strains are, from top-left in clockwise order: AYE, ACICU, SDF and ATCC 17978. The plate shown in the figure is representative of three independent experiments giving similar results.

Figure 7. Resistance to human serum and desiccation.

(A) Serum resistance of A. baumannii strains. Resistance was determined as the percentage of survival in 40% non-heated serum relative to survival in heated serum. Values represent the mean (± SD) of five independent experiments. (B) Resistance to desiccation of A. baumannii strains: AYE (squares), ACICU (circles), ATCC 17978 (triangles) and SDF (diamonds). Strains were inoculated onto 13-mm diameter rounded glass coverslips and incubated at 22°C and 31% relative humidity. A starting bacterial inoculum of 2×10⁷ CFU per coverslip was used. Values represent the mean (± SD) of three independent experiments. The lower detection limit of the assay is 10 CFU per coverslip. ** P<0.01 (ANOVA).