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

<i>Acinetobacter</i> strains used in this study.

*<p>Classified by ATCC according to U.S. Public Health Service guidelines.</p><p>NA – Not Available.</p

Toxicity of <i>Acinetobacter</i> towards human colonic epithelial cells.

<p>(A) HT29 cell monolayers were exposed to 10⁶ cfu/100 µL for up to 24 h and analyzed for bioreduction activity as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037024#s2" target="_blank">Materials and Methods</a>. (B) Bioreduction activity of HT29 cells following a 24-h exposure to Ab culture fractions in the presence of gentamicin. Bacterial cultures were centrifuged and separated into pellet (P1) and culture filtrate (CF1). The pellet was resuspended in fresh media, vigorously agitated and recentrifuged to yield a secondary pellet (P2) and filtrate (CF2). (C–F) Confocal micrographs of HT29 treated with PBS alone (C, E) or 10⁶ cfu/100 µL of Ab (D) or Ah (E) for 18 h, and then fixed and permeabilized as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037024#s2" target="_blank">Materials and Methods</a>. Cells in C and D were stained with SYTOX™ green and rhodamine-conjugated phalloidin. Panels E and F were stained with SYTOX™ green and Texas Red-conjugated wheat germ agglutinin. Error bars in A and B represent the mean of three exposures ± standard deviation. Bars in C–F represent 10 µm.</p

Mammalian cell inflammatory cytokine levels during exposure to test bacteria.

<p>HT29 (A) and J774A.1 (B) cells were exposed to 10⁶ cfu/100 µL test bacteria for various time intervals. Exposure supernatants were filtered and used in liquid array bead assays for IL-8 (A) or IL-1β, IL-6 and TNF-α (B). Experiments were reproduced twice using both the multiplex liquid bead assay and ELISA, both yielding results within 15% variability of each other. Data points are means of three exposures ∀ standard deviation.</p

<i>Acinetobacter</i> Growth-related Bioreduction Activity.

*<p>Data represent means ± standard deviation from six replicates.</p>†<p>Upper Detection Limit of plate reader.</p

PCR Primers Used in this Study and Resulting Amplicon Sizes.

<p>PCR Primers Used in this Study and Resulting Amplicon Sizes.</p

Phagocytosis and bactericidal activity of J774A.1 macrophage.

<p>Survival of bacteria was monitored by enumerating cfu's following 4-h incubation to 10³ cfu/100 µL with or without monolayers of J774A.1. Bacterial survival is expressed as the ratio cfu = s recovered from incubations with J774A.1 in mammalian culture medium (DMEM+supplements) and from mammalian culture medium alone. Inset: Phagocytosis of bacteria (Ah shown as example) was monitored by exposing J774A.1 monolayers to 10⁶ cfu/100 µL for 60 min, and then fixing and staining with SYTOX nucleic acid stain. Internalization was visualized using a confocal microscope, and artificial graded colour intensity (SYTOX intensity from blue to red) was used to highlight bacteria (arrows) within J774A.1. Data points represent the means of five separate experiments ∀ standard deviation. Bar represents 10 µm.</p

Alignment of Quinolone Resistance Determining Regions.

<p>ClustalW alignment of coding regions of <i>gyrA</i> (<b>A</b>) and <i>parC</i> (<b>B</b>) genes derived from test <i>Acinetobacter</i> strains compared to <i>Ab</i> AYE strain with known fluoroquinolone resistance mutations. The alignment demonstrates that the test strains lack a Ser-Leu transition necessary for fluoroquinolone resistance.</p

Antibiotic Susceptibility Assays.

<p>Antibiograms generated from growing each <i>Acinetobacter</i> strain in trypticase soy broth in the presence of different concentrations and classes of antibiotics for durations and temperatures indicated in the figure. The minimum inhibitory concentration (MIC) is the lowest concentration that is effective in preventing bacterial MTT bioreducton. Data are means of four experiments with vertical bars indicating standard deviation.</p