Interaction of <i>Klebsiella pneumoniae</i> with tissue macrophages in a mouse infection model and ex-vivo pig organ perfusions: an exploratory investigation.

Abstract

Hypervirulent <i>Klebsiella pneumoniae</i> (hv<i>Kp</i>) strains of capsule type K1 and K2 cause invasive infections associated with hepatic abscesses, which can be difficult to treat and are frequently associated with relapsing infections. Other <i>K pneumoniae</i> strains (non-hv<i>Kp</i>), including lineages that have acquired carbapenem resistance, do not manifest this pathology. In this work we aimed to test the hypothesis that within-macrophage replication is a key mechanism underpinning abscess formation in hv<i>Kp</i> infections. In this exploratory investigation, to study the pathophysiology of abscess formation, mice were intravenously infected with 10⁶ colony forming units (CFU) of either hv<i>Kp</i> isolates (six strains) or non-hv<i>Kp</i> isolates (seven strains). Intracellular bacterial replication and neutrophil influx in liver and spleen was quantified by fluorescence microscopy of sliced cryopreserved organs of mice collected 30 min, 6 h, and 24 h after infection with the aim to provide data of bacterial association to Kupffer cells in the liver and to the different tissue macrophages in the spleen. Microbiological and microscopy analysis of an ex-vivo model of pig liver and spleen infection were used to confirm within-macrophage replication. Pig organs were perfused with heparinised, autologous pig's blood and injected with 6·5 × 10⁷ CFU of hv<i>Kp</i> K2 sequence type 25 strain GMR151. Blood and tissue biopsies collected before infection and 30 min, 1 h, 2 h, 3 h, 4 h, and 5 h after infection were used to measure bacterial counts and to identify the subcellular localisation of bacteria by immunohistochemistry analysis. We show that hv<i>Kp</i> resisted phagocyte-mediated clearance and replicated in mouse liver macrophages to form clusters 6 h after infection, with a mean of 7·0 bacteria per Kupffer cell (SD 6·2); however, non-hv<i>Kp</i> were efficiently cleared (mean 1·5 bacteria per cell [SD 1·1]). Hv<i>Kp</i> infection promoted neutrophil recruitment to sites of infection, which in the liver resulted in histopathological signs of abscess formation as early as 24 h post-infection. Experiments in pig organs which share a high functional and anatomical resemblance to human organs, provided strong evidence for the propensity of hv<i>Kp</i> to replicate within the hepatic macrophages. These findings show subversion of innate immune processes in the liver by <i>K pneumoniae</i> and resistance to Kupffer cell mediated clearance as an explanation for the propensity of hv<i>Kp</i> strains to cause hepatic abscesses. University of Oxford and a Royal Society Wolfson grant funded biosafety facility.