AB5075, a Highly Virulent Isolate of Acinetobacter baumannii, as a Model Strain for the Evaluation of Pathogenesis and Antimicrobial Treatments.

UNLABELLED: Acinetobacter baumannii is recognized as an emerging bacterial pathogen because of traits such as prolonged survival in a desiccated state, effective nosocomial transmission, and an inherent ability to acquire antibiotic resistance genes. A pressing need in the field of A. baumannii research is a suitable model strain that is representative of current clinical isolates, is highly virulent in established animal models, and can be genetically manipulated. To identify a suitable strain, a genetically diverse set of recent U.S. military clinical isolates was assessed. Pulsed-field gel electrophoresis and multiplex PCR determined the genetic diversity of 33 A. baumannii isolates. Subsequently, five representative isolates were tested in murine pulmonary and Galleria mellonella models of infection. Infections with one strain, AB5075, were considerably more severe in both animal models than those with other isolates, as there was a significant decrease in survival rates. AB5075 also caused osteomyelitis in a rat open fracture model, while another isolate did not. Additionally, a Tn5 transposon library was successfully generated in AB5075, and the insertion of exogenous genes into the AB5075 chromosome via Tn7 was completed, suggesting that this isolate may be genetically amenable for research purposes. Finally, proof-of-concept experiments with the antibiotic rifampin showed that this strain can be used in animal models to assess therapies under numerous parameters, including survival rates and lung bacterial burden. We propose that AB5075 can serve as a model strain for A. baumannii pathogenesis due to its relatively recent isolation, multidrug resistance, reproducible virulence in animal models, and genetic tractability. IMPORTANCE: The incidence of A. baumannii infections has increased over the last decade, and unfortunately, so has antibiotic resistance in this bacterial species. A. baumannii is now responsible for more than 10% of all hospital-acquired infections in the United States and has a >50% mortality rate in patients with sepsis and pneumonia. Most research on the pathogenicity of A. baumannii focused on isolates that are not truly representative of current multidrug-resistant strains isolated from patients. After screening of a panel of isolates in different in vitro and in vivo assays, the strain AB5075 was selected as more suitable for research because of its antibiotic resistance profile and increased virulence in animal models. Moreover, AB5075 is susceptible to tetracycline and hygromycin, which makes it amenable to genetic manipulation. Taken together, these traits make AB5075 a good candidate for use in studying virulence and pathogenicity of this species and testing novel antimicrobials.

Validation of a novel murine wound model of Acinetobacter baumannii infection.

Patients recovering from traumatic injuries or surgery often require weeks to months of hospitalization, increasing the risk for wound and surgical site infections caused by ESKAPE pathogens, which include A. baumannii (the ESKAPE pathogens are Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species). As new therapies are being developed to counter A. baumannii infections, animal models are also needed to evaluate potential treatments. Here, we present an excisional, murine wound model in which a diminutive inoculum of a clinically relevant, multidrug-resistant A. baumannii isolate can proliferate, form biofilms, and be effectively treated with antibiotics. The model requires a temporary, cyclophosphamide-induced neutropenia to establish an infection that can persist. A 6-mm-diameter, full-thickness wound was created in the skin overlying the thoracic spine, and after the wound bed was inoculated, it was covered with a dressing for 7 days. Uninoculated control wounds healed within 13 days, whereas infected, placebo-treated wounds remained unclosed beyond 21 days. Treated and untreated wounds were assessed with multiple quantitative and qualitative techniques that included gross pathology, weight loss and recovery, wound closure, bacterial burden, 16S rRNA community profiling, histopathology, peptide nucleic acid-fluorescence in situ hybridization, and scanning electron microscopy assessment of biofilms. The range of differences that we are able to identify with these measures in antibiotic- versus placebo-treated animals provides a clear window within which novel antimicrobial therapies can be assessed. The model can be used to evaluate antimicrobials for their ability to reduce specific pathogen loads in wounded tissues and clear biofilms. Ultimately, the mouse model approach allows for highly powered studies and serves as an initial multifaceted in vivo assessment prior to testing in larger animals.

Type III secretion systems as targets for novel therapeutics.

Bacterial pathogens use common virulence factors to cause disease. One such virulence factor is the type III secretion system (T3SS), which allows for the direct injection of bacterial proteins, called effector proteins, into the contacting host cell. Pathogens with defective T3SSs are 1000- to 1 million-fold attenuated in animal models of infection. Thus, targeting antivirulence therapies to virulence factors such as the T3SS may provide a novel approach to treating infections. This article provides an overview of approaches used in identifying various classes of T3SS inhibitors, to enable the use of such inhibitors as novel therapeutics.

A cyclic-di-GMP receptor required for bacterial exopolysaccharide production.

Bis-(3',5')-cyclic-dimeric-guanosine monophosphate (c-di-GMP) has been shown to be a global regulatory molecule that modulates the reciprocal responses of bacteria to activate either virulence pathways or biofilm formation. The mechanism of c-di-GMP signal transduction, including recognition of c-di-GMP and subsequent phenotypic regulation, remain largely uncharacterized. The key components of these regulatory pathways are the various adaptor proteins (c-di-GMP receptors). There is compelling evidence suggesting that, in addition to PilZ domains, there are other unidentified c-di-GMP receptors. Here we show that the PelD protein of Pseudomonas aeruginosa is a novel c-di-GMP receptor that mediates c-di-GMP regulation of PEL polysaccharide biosynthesis. Analysis of PelD orthologues identified a number of conserved residues that are required for c-di-GMP binding as well as synthesis of the PEL polysaccharide. Secondary structure similarities of PelD to the inhibitory site of diguanylate cyclase suggest that a common fold can act as a platform to bind c-di-GMP. The combination of a c-di-GMP binding site with a variety of output signalling motifs within one protein domain provides an explanation for the specificity for different cellular responses to this regulatory dinucleotide.