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Phenotypic heterogeneity of Granulibacter spp. isolates obtained from different chronic granulomatous disease patients

Jessica Chu, Anthony M. Pettinato, Larissa S. Rogge, João F. Neves, David E.

Greenberg, Steven M. Holland, Adrian Zelazny, Kol A. Zarember, and John I. Gallin

Abstract (1986/2200 characters): Granulibacter bethesdensis, a member of the Acetobacteraceae family, is an emerging pathogen in patients with Chronic Granulomatous Disease (CGD), a deficiency in the NADPH oxidase. Seven NIH isolates from different CGD patients have identical 16S RNA gene sequences while two lethal cases from Spain and from Portugal differ (only 99.x% ID). Previous studies demonstrated that isolate NIH1.1 was killed by normal, but not CGD, polymorphonuclear leukocytes (PMN) and monocytes and persisted in CGD monocyte-derived macrophages. To better understand clinical differences between Granulibacter infections, we compared available isolates (7 NIH and 1 Portuguese) in terms of bacterial growth rates, ultrastructure, activation of serum complement and the PMN respiratory burst, and sensitivities to normal PMN and monocytes as well as hydrogen peroxide. Growth of the lethal Portuguese isolate was significantly faster than the NIH isolates, of which NIH5.1 grew the slowest. Capsule thickness and bacterial carbohydrate content varied between isolates with the Portuguese isolate showing the presence of high molecular mass carbohydrates that were less abundant in other isolates. Interestingly, the Portuguese isolate did not bind complement proteins C3 and C9 and failed to stimulate a neutrophil respiratory burst, yet could be killed by normal PMN. NIH4.1 was resistant to normal PMN killing and isolates NIH3.1, NIH4.1 and the Portuguese isolate were resistant to normal monocyte killing. In vitro treatment of isolates with hydrogen peroxide demonstrated different sensitivities that may explain differential resistance to PMN. Full genomic sequences available for NIH isolates 1-4 demonstrate significant genomic diversity despite absolute conservation of 16s sequence and sequencing of the remaining isolates is underway. Whether or not different mobile genetic elements between isolates accounts for aspects of microbial pathogenesis observed here is under further study.