Identification and characterization of an Acinetobacter baumannii biofilm-associated protein
Loehfelm, Thomas William
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Acinetobacter baumannii is a Gram-negative bacterium that has emerged recently as an important nosocomial opportunistic pathogen. In 1975, Acinetobacter species accounted for 1.5 and 1.8% of bacterial isolates associated with nosocomial pneumonia and bloodstream infection, respectively, and by 2003, had increased to 6.9 and 2.4% (39). There are an estimated 1.7 million healthcare-associated infections each year in the US, and 67,000 deaths due to hospital-acquired pneumonia and bloodstream infection (61), so Acinetobacter infection is estimated to cause 3200 deaths each year, in addition to significant morbidity costs. With an unusual ability to persist on dry surfaces for months (54, 64) and remarkable intrinsic and acquired resistance to antibiotics (11, 136), Acinetobacter is likely to remain a problem for hospitals and patients worldwide. We have identified a large biofilm-associated protein (Bap) present on the surface of a bloodstream isolate of A. baumannii. We have developed anti-Bap monoclonal antibody (MAb) 6E3, which reacts with 43% of the isolates in our library of clinically-relevant Acinetobacter species, and anti-Bap polyclonal antibody, which reacts with 33% of the isolates. We have analyzed each of the isolates in a semi-quantitative biofilm assay, and demonstrated that for A. baumannii strain 307-0294, Bap is required for wild-type biofilm formation. We have determined that bap transcript abundance is increased during late logarithmic and stationary phases of the bacterial growth curve, likely in response to a diffusible signaling molecule released into the media. Once induced, bap transcript levels are maintained by a cell-density-dependent mechanism, possibly in response to physical cell-cell contact signaling. Consistent with these results, confocal immunofluorescent microscopy of mature biofilms stained with MAb 6E3 demonstrate that Bap is expressed by stationary cells within microcolonies adherent to a glass coverslip, but not by planktonic cells coursing through water channels within and floating above the biofilm structure. Finally, we have identified an outer-membrane protein that co-immunoprecipitates with Bap and is absent from outer-membrane preparations of the Bap-deficient mutant bap1302 ::EZ-Tn5, suggesting that these two proteins interact on the surface of A. baumannii. Acinetobacter colonization of the hospital environment presents a serious threat to vulnerable patients requiring invasive medical devices. Biofilm formation may contribute to the ability of Acinetobacter to persist in the hospital environment, and a thorough understanding of factors that contribute to biofilm formation may facilitate effective disinfection and treatment.