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

Multiplicity in cellulases of Schizophyllum commune - derivation partly from heterogeneity in transcription and glycosylation

Peer reviewed: YesNRC publication: Ye

Bipartite organization of the Bacillus subtilis endo-\u3b2-1,4-glucanase revealed by C-terminal mutations

The C-terminal boundary of primary sequence of the Bacillus subtilis PAP115 endo-\u3b2-1,4-glucanase (EG) required for stable catalytic activity has been mapped by site-directed mutagenesis using Escherichia coli as host. The 52 kDa cel gene product, EG470 and a 33 kDa mutant (EG300), lacking 170 residues through a nonsense mutation at the leucine-330 codon of the gene, exhibited similar patterns of enzymatic activity and pH optima using cellooligopentaose as substrate.CD spectra indicated that the bulk of the \u3b1-helical secondary structure in EG470 was contained within EG300. However, relative to EG470, the specific activity of EG300 was 3- to 4-fold lower with amorphous cellulose as substrate and \u2dc4-to5-fold higher with carboxymethylcellulose (soluble cellulose).These results along with data which show that EG470 binding capacity to mirocrystalline cellulose is \u2dc 11 times more than that of EG300, demonstrate the importance of residues 330\u2013499 for non-catalytic binding of cellulose. A construct of the cel gene carrying a deletion of codons 330\u2013499 and an insertion of a nonsense codon at leucine-330, was further used to make mutants EG296 and EG291 with nonsense codon substitutions at arginine and serine-321, respectively.Western analysis using EG-specific antiserum revealed that relative losses in enzymatic activity of EG296 (50%) and EG291 (95%) could be accounted for by the extent of their proteolysis, signifying a marked destabilization of these enzymes by removal of only a few amino acids.Peer reviewed: YesNRC publication: Ye

<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

<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

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

<p>+ signifies substantial growth or activity.</p><p>(+) signifies low level and/or delayed activity.</p><p>− signifies negligible growth or activity</p>*<p>Note that tests for induction of J774A.1 pro-inflammatory cytokines (IL-1β, Il-6, TNF-α) and presence of known virulence gene segments (<i>OmpA</i>, <i>EpsA</i>) were excluded from the summary table since they failed to discriminate between bacterial strains.</p>**<p>Growth for this purpose is defined as MTT bioreduction in the presence of at least 5 µg/mL antibiotic.</p