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.

f Staphylococcus pseudintermedius is an opportunistic pathogen in dogs. Four housekeeping genes with allelic polymorphisms were identified and used to develop an expanded multilocus sequence typing (MLST) scheme. The new seven-locus technique shows S. pseudintermedius to have greater genetic diversity than previous methods and discriminates more isolates based upon host origin. Staphylococcus pseudintermedius, recently classified as a member of the Staphylococcus intermedius group (SIG), is the most common opportunistic pathogen in dogs. In this host it is frequently associated with pyoderma and other infections, such as those of the urinary tract, wounds, and otitis externa (1-3). It has also been isolated from infections in cats (4, 5), has zoonotic potential (6-8), and is an important nosocomial pathogen causing postsurgical infections in veterinary clinics (9, 10, 33). The incidence of methicillin-resistant S. pseudintermedius (MRSP) has increased significantly in the past few years (1,5,(11)(12)(13)(14)(15)(16)(17)(18)(19). MRSP has emerged as an important problem worldwide because of multidrug resistance and the limited number of drug choices remaining to treat infections caused by this organism. Multilocus sequence typing (MLST) has been used extensively to define the population genetic structure of Staphylococcus aureus and other bacterial species. This information has been used to predict founder strains as well as track the spread of methicillin resistance and identify epidemic clones (2,20). Likewise, the widespread application of this technique to S. pseudintermedius should help to identify methicillin-sensitive S. pseudintermedius (MSSP) progenitors of MRSP clones and provide a mechanism to track their spatial and temporal distribution (21). The identification of successful and/or virulent clonal populations of S. pseudintermedius may facilitate research into characteristics which provide a selective advantage and inform efforts to develop alternative methods of treatment and control, such as vaccines or phage therapy targeting the major clonal populations of S. pseudintermedius associated with disease.MLST is well established as a valuable method for genotyping bacteria based on the sequence variation of housekeeping genes (9,22). It provides accurate, portable data useful for global epidemiology studies and studies of evolution and population genetics (22)(23)(24)(25)(26). MLST techniques applied to diverse species of bacteria generally use at least seven loci (22,(24)(25)(26)(27). Sequence typing based on four loci (MLST-4) has provided insight into the overall genetic structure of the SIG (1). The development of an MLST method expanded to seven loci (MLST-7) for S. pseudintermedius was undertaken to increase its discriminating power with the same number of loci used for other species of staphylococci (24,32).DNA extracts from 125 previously characterized isolates of S. pseudintermedius from dogs (114 isolates), cats (5 isolates), and human beings (6 isolates) from diverse geographical regions (N...

e 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-mmdiameter, 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.