Comparison of the Virulence Potential of Acinetobacter Strains from Clinical and Environmental Sources

Several Acinetobacter strains have utility for biotechnology applications, yet some are opportunistic pathogens. We compared strains of seven Acinetobacter species (baumannii, Ab; calcoaceticus, Ac; guillouiae, Ag; haemolyticus, Ah; lwoffii, Al; junii, Aj; and venetianus, Av-RAG-1) for their potential virulence attributes, including proliferation in mammalian cell conditions, haemolytic/cytolytic activity, ability to elicit inflammatory signals, and antibiotic susceptibility. Only Ah grew at 102 and 104 bacteria/well in mammalian cell culture medium at 37°C. However, co-culture with colonic epithelial cells (HT29) improved growth of all bacterial strains, except Av-RAG-1. Cytotoxicity of Ab and Ah toward HT29 was at least double that of other test bacteria. These effects included bacterial adherence, loss of metabolism, substrate detachment, and cytolysis. Only Ab and Ah exhibited resistance to killing by macrophage-like J774A.1 cells. Haemolytic activity of Ah and Av-RAG-1 was strong, but undetectable for other strains. When killed with an antibiotic, Ab, Ah, Aj and Av-RAG-1 induced 3 to 9-fold elevated HT29 interleukin (IL)-8 levels. However, none of the strains altered levels of J774A.1 pro-inflammatory cytokines (IL-1β, IL-6 and tumor necrosis factor-α). Antibiotic susceptibility profiling showed that Ab, Ag and Aj were viable at low concentrations of some antibiotics. All strains were positive for virulence factor genes ompA and epsA, and negative for mutations in gyrA and parC genes that convey fluoroquinolone resistance. The data demonstrate that Av-RAG-1, Ag and Al lack some potentially harmful characteristics compared to other Acinetobacter strains tested, but the biotechnology candidate Av-RAG-1 should be scrutinized further prior to widespread use

Pseudomonas toxin production.

<p>¹ Room Temperature</p><p>² Trypticase Soy Broth</p><p>Pseudomonas toxin production.</p

Pulmonary Cytokine Levels During Pseudomonas Exposure.

<p>Balb/c mice were endotracheally instilled with saline or 10⁶ cfu of each <i>Pseudomonas</i> strain. At various times following exposure, animals were euthanized and lungs were harvested. Following tissue homogenization, cytokine levels were measured using a multiplex bead array system. Data points represent the mean of three or four treated mice. Asterisks indicate statistically different values compared to saline exposures, as determined using ANOVA and Dunnett’s Multiple Comparison Test (<i>p</i> < 0.05). Bacteria with significant differences are indicated with red boxes in the graph legends.</p

SIR Ranking of <i>Pseudomonas</i> strains used in this study.

<p>SIR Ranking of <i>Pseudomonas</i> strains used in this study.</p

Pseudomonas Strain Information Provided by American Type Culture Collection.

<p>Pseudomonas Strain Information Provided by American Type Culture Collection.</p

Serum Amyloid A during Pseudomonas Exposure.

<p>Balb/c mice were endotracheally instilled with saline or 10⁶ cfu of each <i>Pseudomonas</i> strain. At various times following exposure, blood was collected by cardiac puncture. Blood was processed for SAA detection by ELISA. Data is expressed as the fold-change compared to control values ± relative error (n = 3). For Pa31480 treatment at 48 h, the SAA values for each mouse are indicated by T1, T2, and T3. The variation in these values explains the large relative error observed. Asterisks indicate statistically different values compared to saline exposures, as determined using ANOVA and Dunnett’s Multiple Comparison Test (<i>p</i> < 0.05). Bacteria with significant differences are indicated with red boxes in the graph legends.</p

SIR Ranking of Antibiotics used in this study.

<p>¹S/I/R values correspond to antibiotic susceptible, intermediate and resistant breakpoints as provided by EUCAST (<a href="http://www.bsac.org.uk/" target="_blank">www.bsac.org.uk</a>). Single values represent breakpoints for antibiotic resistance. EUCAST data have been produced in part under ECDC service contracts and made available at no cost by EUCAST and can be accessed freely on the EUCAST website <a href="http://www.eucast.org/" target="_blank">www.eucast.org</a>.</p><p>SIR Ranking of Antibiotics used in this study.</p

Summary of Assays for Comparing Potential Virulence Characteristics of <i>Pseudomonas</i>.

<p>Key:—no response, (+) partial response, + positive response</p><p>Summary of Assays for Comparing Potential Virulence Characteristics of <i>Pseudomonas</i>.</p

Optical density of <i>Pseudomonas</i> cultures in various media measured at 24 h.

<p><u>Key</u>: Positive for Growth (OD>0.1); Delayed Growth (>12h); Negligible Growth (OD<0.1)</p><p>Optical density of <i>Pseudomonas</i> cultures in various media measured at 24 h.</p