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.

Acinetobacter baumannii is an emerging bacterial pathogen of considerable health care concern. Nonetheless, relatively little is known about the organism's virulence factors or their regulatory networks. Septicemia and ventilator-associated pneumonia are two of the more severe forms of A. baumannii disease. To identify virulence factors that may contribute to these disease processes, genetically diverse A. baumannii clinical isolates were evaluated for the ability to proliferate in human serum. A transposon mutant library was created in a strain background that propagated well in serum and screened for members with decreased serum growth. The results revealed that disruption of A. baumannii phospholipase D (PLD) caused a reduction in the organism's ability to thrive in serum, a deficiency in epithelial cell invasion, and diminished pathogenesis in a murine model of pneumonia. Collectively, these results suggest that PLD is an A. baumannii virulence factor.

e Multidrug-resistant bacterial pathogens are an increasing threat to public health, and lytic bacteriophages have reemerged as a potential therapeutic option. In this work, we isolated and assembled a five-member cocktail of wild phages against Acinetobacter baumannii and demonstrated therapeutic efficacy in a mouse full-thickness dorsal infected wound model. The cocktail lowers the bioburden in the wound, prevents the spread of infection and necrosis to surrounding tissue, and decreases infection-associated morbidity. Interestingly, this effective cocktail is composed of four phages that do not kill the parent strain of the infection and one phage that simply delays bacterial growth in vitro via a strong but incomplete selection event. The cocktail here appears to function in a combinatorial manner, as one constituent phage targets capsulated A. baumannii bacteria and selects for loss of receptor, shifting the population to an uncapsulated state that is then sensitized to the remaining four phages in the cocktail. Additionally, capsule is a known virulence factor for A. baumannii, and we demonstrated that the emergent uncapsulated bacteria are avirulent in a Galleria mellonella model. These results highlight the importance of anticipating population changes during phage therapy and designing intelligent cocktails to control emergent strains, as well as the benefits of using phages that target virulence factors. Because of the efficacy of this cocktail isolated from a limited environmental pool, we have established a pipeline for developing new phage therapeutics against additional clinically relevant multidrug-resistant pathogens by using environmental phages sourced from around the globe.

bAdenylate kinase (AK) is a ubiquitous intracellular enzyme that is released into the extracellular space upon cell lysis. We have shown that AK release serves as a useful reporter of bactericidal agent activity and can be exploited for antimicrobial screening purposes. The AK assay exhibits improved sensitivity over that of growth-based assays and can detect agents that are active against bacteria in clinically relevant growth states that are difficult to screen using conventional approaches, such as small colony variants (SCV) and bacteria within established biofilms. The usefulness of the AK assay was validated by screening a library of off-patent drugs for agents that exhibit antimicrobial properties toward a variety of bacterial species, including Escherichia coli and all members of the "ESKAPE" pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species). The assay detected antibiotics within the library that were expected to be active against the organism screened. Moreover, 38 drugs with no previously reported antibacterial activity elicited AK release. Four of these were acquired, and all were verified to exhibit antimicrobial activity by standard susceptibility testing. Two of these molecules were further characterized. The antihistamine, terfenadine, was active against S. aureus planktonic, SCV population, and biofilm-associated cells. Tamoxifen, an estrogen receptor antagonist, was active toward E. faecium in vitro and also reduced E. faecium pathogenesis in a Galleria mellonella infection model. Our data demonstrate that the AK assay provides an attractive screening approach for identifying new antimicrobial agents. Further, terfenadine and tamoxifen may represent novel antimicrobial drug development scaffolds.

Acinetobacter baumannii is well adapted to the hospital environment, where infections caused by this organism are associated with significant morbidity and mortality. Genetic determinants of antimicrobial resistance have been described extensively, yet the mechanisms by which A. baumannii regulates antibiotic resistance have not been defined. We sought to identify signals encountered within the hospital setting or human host that alter the resistance phenotype of A. baumannii. In this regard, we have identified NaCl as being an important signal that induces significant tolerance to aminoglycosides, carbapenems, quinolones, and colistin upon the culturing of A. baumannii cells in physiological NaCl concentrations. Proteomic analyses of A. baumannii culture supernatants revealed the release of outer membrane proteins in high NaCl, including two porins (CarO and a 33-to 36-kDa protein) whose loss or inactivation is associated with antibiotic resistance. To determine if NaCl affected expression at the transcriptional level, the transcriptional response to NaCl was determined by microarray analyses. These analyses highlighted 18 genes encoding putative efflux transporters that are significantly upregulated in response to NaCl. Consistent with this, the effect of NaCl on the tolerance to levofloxacin and amikacin was significantly reduced upon the treatment of A. baumannii with an efflux pump inhibitor. The effect of physiological concentrations of NaCl on colistin resistance was conserved in a panel of multidrug-resistant isolates of A. baumannii, underscoring the clinical significance of these observations. Taken together, these data demonstrate that A. baumannii sets in motion a global regulatory cascade in response to physiological NaCl concentrations, resulting in broad-spectrum tolerance to antibiotics.