Antibiotic resistant infections lead to 700,000 deaths per year worldwide1. The roles of phenotypically diverse subpopulations of clonal bacteria in the progression of diseases are unclear. We found that the increasingly pathogenic and antibiotic resistant pathogen, Acinetobacter baumannii, harbors a highly virulent subpopulation of cells responsible for disease. This virulent subpopulation possesses a thicker capsule and is resistant to host antimicrobials, which drive its enrichment during infection. Importantly, bacteria harvested from the bloodstream of human patients belong exclusively to this virulent subpopulation. Furthermore, the virulent form exhibits increased resistance to hospital disinfectants and desiccation, indicating a role in environmental persistence and the epidemic spread of disease. We identified a transcriptional “master regulator” of the switch between avirulent and virulent cells, and whose overexpression abrogated virulence. Further, the overexpression strain vaccinated mice against lethal challenge. This work highlights a phenotypic subpopulation of bacteria that drastically alters the outcome of infection, and illustrates how knowledge of the regulatory mechanisms controlling such phenotypic switches can be harnessed to attenuate bacteria and develop translational interventions.

Acinetobacter baumannii strain AB5075 produces colonies with two opacity phenotypes, designated opaque and translucent. These phenotypes were unstable and opaque and translucent colony variants were observed to interconvert at high frequency, suggesting that a phase-variable mechanism was responsible. The frequency of phase variation both within colonies and in broth cultures increased in a cell density-dependent manner and was mediated by the accumulation of an extracellular factor. This factor was distinct from the known A. baumannii signaling molecule 3-OH C 12 -homoserine lactone. Opaque and translucent colony variants exhibited a number of phenotypic differences, including cell morphology, surface motility, biofilm formation, antibiotic resistance, and virulence in a Galleria mellonella model. Additional clinical isolates exhibited a similar phase-variable control of colony opacity, suggesting that this may be a common feature of A. baumannii. IMPORTANCEA novel phase-variable mechanism has been identified in Acinetobacter baumannii that results in an interconversion between opaque and translucent colony phenotypes. This phase variation also coordinately regulates motility, cell shape, biofilm formation, antibiotic resistance, and virulence. The frequency of phase variation is increased at high cell density via a diffusible extracellular signal. To our knowledge, this report presents the first example of phase variation in A. baumannii and also the first example of quorum sensing-mediated control of phase variation in a bacterium. The findings are important, as this phase-variable mechanism can be identified only via changes in colony opacity using oblique light; therefore, many researchers studying A. baumannii may unknowingly be working with different colony variants.

Recently, a novel phase-variable colony opacity phenotype was discovered in Acinetobacter baumannii strain AB5075, where colonies interconvert between opaque and translucent variants. Opaque colonies become mottled or sectored after 24 h of growth due to translucent variants arising within the colony. This easily distinguishable opaque-colony phenotype was used to screen for random transposon insertions that increased the frequency of sectoring at a time point when wild-type colonies were uniformly opaque. A colony was identified that contained multiple papillae of translucent variants, and the insertion in this mutant mapped to an ortholog of the two-component system response regulator ompR. Subsequent investigation of in-frame deletions of ompR and the sensor kinase envZ (located adjacent to ompR) showed that the switching frequency from opaque to translucent was increased 401-and 281-fold, respectively. The ompR mutant also exhibited sensitivity to sodium chloride in growth medium, whereas the envZ mutation did not elicit sensitivity to sodium chloride. Mutation of either gene reduced motility in A. baumannii strain AB5075, but a mutation in both ompR and envZ produced a more profound effect. The ompR and envZ genes were cotranscribed but were not subject to autoregulation by OmpR. Both ompR and envZ mutant opaque variants were attenuated in virulence in the Galleria mellonella infection model, whereas mutation of ompR had no effect on the virulence of the translucent variant.IMPORTANCE Acinetobacter baumannii is a well-known antibiotic-resistant pathogen; many clinical isolates can only be treated by a very small number of antibiotics (including colistin), while some exhibit panresistance. The current antimicrobial arsenal is nearing futility in the treatment of Acinetobacter infections, and new avenues of treatment are profoundly needed. Since phase variation controls the transition between opaque (virulent) and translucent (avirulent) states in A. baumannii, this may represent an "Achilles' heel" that can be targeted via the development of small molecules that lock cells in the translucent state and allow the host immune system to clear the infection. A better understanding of how phase variation is regulated may allow for the development of methods to target this process. The ompR-envZ two-component system ortholog negatively regulates phase variation in A. baumannii, and perturbation of this system leads to the attenuation of virulence in an invertebrate infection model. KEYWORDS Acinetobacter, phase variation, osmotic stress T he Gram-negative bacterium Acinetobacter baumannii is well recognized as an opportunistic pathogen, part of a group of nosocomial pathogens (ESKAPE organisms), which merit increased investigation due to their presence in the nosocomial

Acinetobacter baumannii strain AB5075 forms two cell types distinguished by their opaque (VIR-O) or translucent (AV-T) colonies. VIR-O cells possess a thicker capsule and are more resistant to a variety of stressors than AV-T cells. However, the direct role of the capsule in these stressors was unknown. This study demonstrates that the capsule is required for resistance to disinfectants, lysozyme, and desiccation in Acinetobacter baumannii. In addition, the capsule is required for survival in a mouse lung model of infection.

Colony opacity phase variation in Acinetobacter baumannii strain AB5075 is regulated by a reversible high‐frequency switch. Transposon mutagenesis was used to generate mutations that decreased the opaque to translucent switch and a gene encoding a predicted periplasmic membrane fusion component of a resistance–nodulation–cell division (RND)‐type efflux system was isolated. This gene was designated arpA and immediately downstream was a gene designated arpB that encodes a predicted membrane transporter of RND‐type systems. A nonpolar, in‐frame deletion in arpA resulted in a 70‐fold decrease in the opaque to translucent switch. An arpB::Tc mutant exhibited a 769‐fold decrease in the opaque to translucent switch. However, the translucent to opaque switch was largely unchanged in both the arpA and arpB mutants. The arpA and arpB mutants also exhibited increased surface motility in the opaque form and the arpB mutant exhibited increased susceptibility to aminoglycosides. The arpA and arpB mutants were both attenuated in a Galleria mellonella model of virulence. A divergently transcribed TetR‐type regulator ArpR was capable of repressing the arpAB operon when this TetR regulator was overexpressed. The arpR gene was also involved in regulating the opaque to translucent switch as an in‐frame arpR mutation decreased this switch by 1,916‐fold.