Identification of a General O-linked Protein Glycosylation System in Acinetobacter baumannii and Its Role in Virulence and Biofilm Formation

Acinetobacter baumannii is an emerging cause of nosocomial infections. The isolation of strains resistant to multiple antibiotics is increasing at alarming rates. Although A. baumannii is considered as one of the more threatening “superbugs” for our healthcare system, little is known about the factors contributing to its pathogenesis. In this work we show that A. baumannii ATCC 17978 possesses an O-glycosylation system responsible for the glycosylation of multiple proteins. 2D-DIGE and mass spectrometry methods identified seven A. baumannii glycoproteins, of yet unknown function. The glycan structure was determined using a combination of MS and NMR techniques and consists of a branched pentasaccharide containing N-acetylgalactosamine, glucose, galactose, N-acetylglucosamine, and a derivative of glucuronic acid. A glycosylation deficient strain was generated by homologous recombination. This strain did not show any growth defects, but exhibited a severely diminished capacity to generate biofilms. Disruption of the glycosylation machinery also resulted in reduced virulence in two infection models, the amoebae Dictyostelium discoideum and the larvae of the insect Galleria mellonella, and reduced in vivo fitness in a mouse model of peritoneal sepsis. Despite A. baumannii genome plasticity, the O-glycosylation machinery appears to be present in all clinical isolates tested as well as in all of the genomes sequenced. This suggests the existence of a strong evolutionary pressure to retain this system. These results together indicate that O-glycosylation in A. baumannii is required for full virulence and therefore represents a novel target for the development of new antibiotics

Evaluation of liposomal ciprofloxacin formulations in a murine model of anthrax.

The in vivo efficacy of liposomal encapsulated ciprofloxacin in two formulations, lipoquin and apulmiq, were evaluated against the causative agent of anthrax, Bacillus anthracis. Liposomal encapsulated ciprofloxacin is attractive as a therapy since it allows for once daily dosing and achieves higher concentrations of the antibiotic at the site of initial mucosal entry but lower systemic drug concentrations. The in vivo efficacy of lipoquin and apulmiq delivered by intranasal instillation was studied at different doses and schedules in both a post exposure prophylaxis (PEP) therapy model and in a delayed treatment model of murine inhalational anthrax. In the mouse model of infection, the survival curves for all treatment cohorts differed significantly from the vehicle control. Ciprofloxacin, lipoquin and apulmiq provided a high level of protection (87-90%) after 7 days of therapy when administered within 24 hours of exposure. Reducing therapy to only three days still provided protection of 60-87%, if therapy was provided within 24 hours of exposure. If treatment was initiated 48 hours after exposure the survival rate was reduced to 46-65%. These studies suggest that lipoquin and apulmiq may be attractive therapies as PEP and as part of a treatment cocktail for B. anthracis

Single-Nucleotide Repeat Analysis for Subtyping Bacillus anthracis Isolates

Single-nucleotide repeats (SNRs) are variable-number tandem repeats that display very high mutation rates. In an outbreak situation, the use of a marker system that exploits regions with very high mutation rates, such as SNRs, allows the differentiation of isolates with extremely low levels of genetic diversity. This report describes the identification and analysis of SNR loci of Bacillus anthracis. SNR loci were selected in silico, and the loci with the highest diversity were used to design and test locus-specific primers against a number of B. anthracis strains with the same multilocus variable-number tandem repeat analysis (MLVA) genotype. SNR markers that allowed strains with the same MLVA genotype to be differentiated from each other were identified. The resulting SNR marker system can be used as a molecular epidemiological tool in a natural outbreak or bioterrorism event, offering the best chance of distinguishing very closely related isolates

Toxicity of sodium hypochlorite and chlorine dioxide decontaminant in human skin keratinocytes cells.

<p>Toxicity of sodium hypochlorite (0.5%) and chlorine dioxide decontaminant in human skin keratinocytes cells. Just-confluent keratinocyte cultures were treated with varying concentrations of sodium hypochlorite (squares) or chlorine dioxide based decontaminant (circles), diluted in culture medium and incubated for 1 hr before being removed and replaced with fresh culture medium containing no decontaminant. Cytotoxicity was determined at 24 h using the alamarBlue^TM cytotoxicity assay. Results represent the mean +/- SEM of four experiments.</p

Recovery of <i>B</i>. <i>anthracis</i> spores after exposure to decontaminants.

<p>Recovery of <i>B</i>. <i>anthracis</i> Sterne spores (Red) or Ames spores (Yellow) after exposure to test decontaminants over specified periods of time. Sterne spores were quenched using DE broth (blue) after exposure to the decontaminants. Untreated controls (solid bars), chlorine dioxide based test decontaminant (checker bars), 0.5% sodium hypochlorite (striped bars). Quench control samples (Green) were DE broths incubated with each decontaminant prior to Sterne spore addition or in DE broth alone (solid bar). Data are presented as means (n = 3) CFU ml^-1 of spores recovered after specified contact times with decontaminants.</p

Decontamination Efficacy and Skin Toxicity of Two Decontaminants against <i>Bacillus anthracis</i>

<div><p>Decontamination of bacterial endospores such as <i>Bacillus anthracis</i> has traditionally required the use of harsh or caustic chemicals. The aim of this study was to evaluate the efficacy of a chlorine dioxide decontaminant in killing <i>Bacillus anthracis</i> spores in solution and on a human skin simulant (porcine cadaver skin), compared to that of commonly used sodium hypochlorite or soapy water decontamination procedures. In addition, the relative toxicities of these decontaminants were compared in human skin keratinocyte primary cultures. The chlorine dioxide decontaminant was similarly effective to sodium hypochlorite in reducing spore numbers of <i>Bacillus anthracis</i> Ames in liquid suspension after a 10 minute exposure. After five minutes, the chlorine dioxide product was significantly more efficacious. Decontamination of isolated swine skin contaminated with <i>Bacillus anthracis</i> Sterne with the chlorine dioxide product resulted in no viable spores sampled. The toxicity of the chlorine dioxide decontaminant was up to two orders of magnitude less than that of sodium hypochlorite in human skin keratinocyte cultures. In summary, the chlorine dioxide based decontaminant efficiently killed <i>Bacillus anthracis</i> spores in liquid suspension, as well as on isolated swine skin, and was less toxic than sodium hypochlorite in cultures of human skin keratinocytes.</p></div

RODAC plate samplings of swine skin contaminated with <i>B</i>. <i>anthracis</i> spores.

<p>Photos of RODAC plate samplings of swine skin contaminated with <i>B</i>. <i>anthracis</i> spores. a) Pre-soapy water exposure. b) Pre-chlorine dioxide decontaminant exposure. c) Post- 30 second scrub and 5 minute soapy water total contact time. d) Post- 30 second scrub and 5 minute total contact time. e) Soapy water scrub brush after scrubbing skin for 30 seconds with soapy water. f) Chlorine dioxide decontaminant scrub brush after scrubbing skin for 30 seconds with chlorine dioxide decontaminant.</p

MS/MS of A1S_3626 and A1S_3744 showing glycosylation in <i>A. baumannii</i> with a pentasaccharide.

<p>Spots excised from the 2D DIGE, digested with trypsin, and analyzed by MALDI-TOF-MS. Peaks not corresponding to peptide fragmentation were analyzed for glycosylation. A) MS/MS of the precursor ion peak at <i>m/z</i> 2895.165 from A1S_3626 revealed the peptide <b>SAGDQAASDIATATDNASAK</b> with a pentasaccharide of HexNAc-Hex-Hex-(HexNAc)-300 attached. B) MS/MS of the precursor ion peak at <i>m/z</i> 3852.76 from A1S_3744 revealed the peptide <b>ETPKEEEQDKVETAVSEPQPQKPAK</b> with the same pentasaccharide attached. C) MALDI-TOF MS of Pronase E digested membrane proteins showed a precursor ion peak of <i>m/z</i> 1358.4 which MS/MS analysis demonstrated to be the previously identified <i>O</i>-glycan (HexNAc-Hex-Hex-(HexNAc)-300 attached to the peptide fragment “ATD”.</p

Identification of seven <i>O-</i>glycosylated proteins with nine unique glycosylation sites by ZIC HILIC enrichment of tryptically digested <i>A. baumannii</i> ATCC 17978 membrane extracts.

<p>Identification of seven <i>O-</i>glycosylated proteins with nine unique glycosylation sites by ZIC HILIC enrichment of tryptically digested <i>A. baumannii</i> ATCC 17978 membrane extracts.</p