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

Composition and pathogenic potential of a microbial bioremediation product used for crude oil degradation.

A microbial bioremediation product (MBP) used for large-scale oil degradation was investigated for microbial constituents and possible pathogenicity. Aerobic growth on various media yielded >108 colonies mL-1. Full-length 16S rDNA sequencing and fatty acid profiling from morphologically distinct colonies revealed ≥13 distinct genera. Full-length 16S rDNA library sequencing, by either Sanger or long-read PacBio technology, suggested that up to 21% of the MBP was composed of Arcobacter. Other high abundance microbial constituents (>6%) included the genera Proteus, Enterococcus, Dysgonomonas and several genera in the order Bacteroidales. The MBP was most susceptible to ciprofloxacin, doxycycline, gentamicin, and meropenam. MBP exposure of human HT29 and A549 cells caused significant cytotoxicity, and bacterial growth and adherence. An acellular MBP filtrate was also cytotoxic to HT29, but not A549. Both MBP and filtrate exposures elevated the neutrophil chemoattractant IL-8. In endotracheal murine exposures, bacterial pulmonary clearance was complete after one-week. Elevation of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α, and chemokines KC and MCP-1 occurred between 2h and 48h post-exposure, followed by restoration to baseline levels at 96h. Cytokine/chemokine signalling was accompanied by elevated blood neutrophils and monocytes at 4h and 48h, respectively. Peripheral acute phase response markers were maximal at 24h. All indicators examined returned to baseline values by 168h. In contrast to HT29, but similar to A549 observations, MBP filtrate did not induce significant murine effects with the indicators examined. The results demonstrated the potentially complex nature of MBPs and transient immunological effects during exposure. Products containing microbes should be scrutinized for pathogenic components and subjected to characterisation and quality validation prior to commercial release

Changes in acute phase response proteins following exposure.

<p>Mice were exposed to 1.25x10⁷ cfu in 25 μL of MBP. At various times post-exposure, a subset of animals was euthanized. Blood was collected and levels of fibrinogen (A) and serum amyloid A (B) were measured by ELISA. The designation ‘24F’ in the x-axis represents 24h post MBP filtrate exposure. The box-whisker plots show the following: The horizontal line within each box represents the median from six animal treatments. Lower and upper boundaries of the boxes represent the 25th and 75th percentiles, respectively. The whiskers represent the 10th and 90th percentiles. Asterisks indicate statistically different values compared to saline-treated controls (p<0.05).</p

HT29 cytotoxicity and cytokine response.

<p>HT29 cells were exposed to 10⁶ cfu in 100 μL of MBP or its filtrate (1/20 dilution) either in the absence (A) or presence (B) of the broad-spectrum antibiotic, gentamicin. At various times, HT29 cells were assessed for metabolic activity using the MTT bioreduction assay. Supernatants from these exposures were tested for levels of pro-inflammatory cytokines and chemokines. (C) Photomicrograph demonstrating the adherence of bacteria to HT29 cells and the culture well surface. (D) IL-8 was found to be significantly elevated in response to some exposures in comparison to LPS positive controls isolated from <i>Pseudomonas</i> (LPS-P) and <i>Escherichia</i> (LPS-E). Each data point represents a representative experiment done in duplicate ± standard deviation. Asterisks indicate statistically different values compared to saline-treated controls (p<0.05).</p

Identification of selective agar isolates (10 days at 21°C).

<p>Identification of selective agar isolates (10 days at 21°C).</p

Changes in pulmonary cytokines following exposure.

<p>Mice were exposed to 1.25x10⁷ cfu in 25 μL of MBP. At various times post-exposure, a subset of animals was euthanized. Portions of lungs were excised, homogenized, and levels of IL-1β (A), IL-6 (B), TNF-α (C), KC (D), and MCP-1 (E) were measured. The designation ‘24F’ in the x-axis represents 24h post MBP filtrate exposure. The box-whisker plots show the following: The horizontal line within each box represents the median from six animal treatments. Lower and upper boundaries of the boxes represent the 25th and 75th percentiles, respectively. The whiskers represent the 10th and 90th percentiles. Asterisks indicate statistically different values compared to saline-treated controls (p<0.05).</p

Pulmonary clearance of the MBP from Balb/c mice.

<p>Mice were exposed to 1.25x10⁷ cfu in 25 μL of MBP. At various times post-exposure, a subset of animals was euthanized. Portions of lungs were excised, homogenized, and spread plated onto LB-agar plates. The numbers of resulting microbial colonies after 24h incubation is shown. The box-whisker plots show the following: The horizontal line within each box represents the median from six animal treatments. Lower and upper boundaries of the boxes represent the 25th and 75th percentiles, respectively. The whiskers represent the 10th and 90th percentiles.</p

Infections associated with cultivable microbes identified in MBP.

<p>Infections associated with cultivable microbes identified in MBP.</p

Antibiotic susceptibility.

<p>Two different antimicrobial assay formats were tested. Figure A shows resulting microbial growth on quadrant plates (Remel Synergy Quad^®) containing BHI-agar with no antibiotic, 500 μg mL^-1 gentamicin, 2000 μg/mL streptomycin, and 6 μg mL^-1 vancomycin. Figs B and C demonstrate a colorimetric dilution method using MTT to highlight viable microorganisms. Each data point represents the mean of four separate experiments ± standard deviation.</p