Acinetobacter baumannii is a Gram negative opportunistic pathogen that has demonstrated a significant insurgence in the prevalence of infections over recent decades. With only a limited number of "traditional" virulence factors, the mechanisms underlying the success of this pathogen remain of great interest. Major advances have been made in the tools, reagents, and models to study A. baumannii pathogenesis, and this has resulted in a substantial increase in knowledge. This article provides a comprehensive review of the bacterial virulence factors, the host immune responses, and animal models applicable for the study of this important human pathogen. Collating the most recent evidence characterizing bacterial virulence factors, their cellular targets and genetic regulation, we have encompassed numerous aspects important to the success of this pathogen, including membrane proteins and cell surface adaptations promoting immune evasion, mechanisms for nutrient acquisition and community interactions. The role of innate and adaptive immune responses is reviewed and areas of paucity in our understanding are highlighted. Finally, with the vast expansion of available animal models over recent years, we have evaluated those suitable for use in the study of Acinetobacter disease, discussing their advantages and limitations.

Clinical phage therapy is often delivered alongside antibiotics. However, the phenomenon of phage-antibiotic synergy has been mostly studied in vitro. Here, we assessed the in vivo bactericidal effect of a phage-antibiotic combination on Acinetobacter baumannii AB900 using phage øFG02, which binds to capsular polysaccharides and leads to antimicrobial resensitisation in vitro. We performed a two-stage preclinical study using a murine model of severe A. baumannii AB900 bacteraemia. In the first stage, with an endpoint of 11 h, mice (n = 4 per group) were treated with either PBS, ceftazidime, phage øFG02, or the combination of phage and ceftazidime. The second stage involved only the latter two groups (n = 5 per group), with a prolonged endpoint of 16 h. The primary outcome was the average bacterial burden from four body sites (blood, liver, kidney, and spleen). Bacterial colonies from phage-treated mice were retrieved and screened for phage-resistance. In the first stage, the bacterial burden (CFU/g of tissue) of the combination group (median: 4.55 × 10⁵; interquartile range [IQR]: 2.79 × 10⁵–2.81 × 10⁶) was significantly lower than the PBS (median: 2.42 × 10⁹; IQR: 1.97 × 10⁹–3.48 × 10⁹) and ceftazidime groups (median: 3.86 × 10⁸; IQR: 2.15 × 10⁸–6.35 × 10⁸), but not the phage-only group (median: 1.28 × 10⁷; IQR: 4.71 × 10⁶–7.13 × 10⁷). In the second stage, the combination treatment (median: 1.72 × 10⁶; IQR: 5.11 × 10⁵–4.00 × 10⁶) outperformed the phage-only treatment (median: 7.46 × 10⁷; IQR: 1.43 × 10⁷–1.57 × 10⁸). Phage-resistance emerged in 96% of animals receiving phages, and all the tested isolates (n = 11) had loss-of-function mutations in genes involved in capsule biosynthesis and increased sensitivity to ceftazidime. øFG02 reliably drives the in vivo evolution of A. baumannii AB900 towards a capsule-deficient, phage-resistant phenotype that is resensitised to ceftazidime. This mechanism highlights the clinical potential of using phage therapy to target A. baumannii and restore antibiotic activity. National Health and Medical Research Council (Australia).

Acinetobacter baumannii is one of the most notorious hospital-acquired pathogens, and novel treatment strategies are desperately required. Two-component regulatory systems represent potential therapeutic targets as they mediate microorganism adaptation to changing environments, often control virulence, and are specific to bacteria. Here we describe the first global virulence regulator in A. baumannii. Using transcriptional profiling and functional assays of a deletion mutant in the A. baumannii sensor kinase gene, A1S_0574 (termed as gacS), we show that this sensor kinase regulates key virulence characteristics, including pili synthesis, biofilms, and motility, resulting in virulence attenuation in a mammalian septicemia model. Notably, we also identified that GacS regulates an operon novel to A. baumannii (paa operon), which is responsible for the metabolism of aromatic compounds. Deletion of paaE (A1S_1340) confirmed the role of this operon in A. baumannii virulence. Finally, we identified the cognate response regulator (A1S_0236) for GacS and confirmed their interaction. A1S_0236 was shown to regulate 75% of the GacS transcriptome and the same virulence phenotypes. Overexpression of A1S_0236 restored virulence in the gacS mutant. Our study characterizes a global virulence regulator, which may provide an alternate therapeutic target, in one of the most troublesome hospital-acquired pathogens.

Significance: Acinetobacter baumannii is one of the most significant hospital-acquired bacterial pathogens, able to cause life-threatening infections and develop resistance to all currently available antibiotic agents. Here, we established zebrafish as a model to study real-time interactions between innate immune cells and A. baumannii during infection. We identified a bacterial metabolic pathway that, when inhibited, leads to enhanced immune responses toward the bacteria, improving bacterial clearance and reducing severity of disease. The enhanced immune response was secondary to accumulation of a metabolic by-product, which acted as a direct, bacterial-mediated attractant of neutrophils, the key immune cell important in response to bacterial infections. These results pave the way for novel therapeutic targeting of bacterial metabolism to stimulate immune responses to fight off infection.