Clinical Isolates of Antimicrobial-Resistant Enterobacter Species Can Persist in Human Macrophages Without Replication and Overt Cellular Cytotoxicity.

Abstract

Enterobacter species are opportunistic, multidrug resistant gram-negative bacteria associated with morbidity and mortality worldwide. Because very little is known about the infection biology of Enterobacter spp, we investigated the intracellular trafficking of a subset of Enterobacter clinical isolates, including colistin-resistant strains, within human macrophages and determined the macrophage response to the intracellular infection. Phagocytosis of 11 clinical isolates representing Enterobacter cloacae, Enterobacter bugandensis, Enterobacter kobei, Enterobacter xiangfangensis, Enterobacter roggenkampii, Enterobacter hoffmannii, and Enterobacter ludwigii was investigated in primary human macrophages. Intracellular bacterial trafficking was followed by confocal fluorescence microscopy, intracellular bacterial replication was assessed by bacterial enumeration, and a fluorescence dilution approach was used to follow bacterial cell division over time. Macrophage cell cytotoxicity was investigated by quantifying the release of lactate dehydrogenase during infection and by determining cleavage of the proinflammatory markers caspase-1, gasdermin D, and prointerleukin-1β. Enterobacter isolates did not replicate in human macrophages, exhibiting long-term survival (up to 44 hours) within a modified late phagolysosome compartment. Survival did not correlate with colistin resistance, lipopolysaccharide modifications, or bacterial pathogenicity in the Galleria mellonella infection model. Intracellular bacteria induced low levels of macrophage cytotoxicity that correlated with absence of cleavage of proinflammatory markers in infected macrophages. Enterobacter spp clinical isolates can persist without replication inside human macrophages with minimal effects on cell integrity and inflammation. These observations could have implications for clinical outcome of patients that cannot readily clear Enterobacter infections, which can potentially lead to prolonged intracellular survival and infection relapse.