Clinical isolates ofEnterobacterspecies can persist in human macrophages without bacterial replication and minimal cellular cytotoxicity
Authors: Parau Georgiana , Parks Hannah J. , Anderson Amy J. G. , Bisaro Fabiana , García-Romero Inmaculada , Gilmore Michael C. , Korankye Samuel O. , Marshall Helina , Valvano Miguel A. 
Published: February 2025

SummaryBackgroundTheEnterobacter cloacaecomplex (Ecc) encompasses opportunistic Gram-negative bacteria demonstrating considerable phenotypic and genotypic diversity. Bloodstream, respiratory and urinary tract infections by Ecc bacteria are associated with morbidity and mortality worldwide. These infections are often difficult to treat since Ecc bacteria are resistant to penicillins, quinolones, aminoglycosides, and third-generation cephalosporins. Resistance also extends to carbapenems, leaving only polymyxins, such as colistin, as a last resort antibiotic for treatment. However, colistin resistance in Ecc isolates is also unexpectedly frequent. Despite extensive information on antibiotic resistance byEnterobacterspecies, much less is known about their infection biology. There are few reports on the survival and persistence of selectedEnterobacterspecies in macrophages and epithelial cells, but howEnterobacterisolates interact with innate immune host cells upon engulfment remains unexplored. In this study, we have investigated the intracellular trafficking of a subset of antimicrobial resistant Ecc clinical isolates, including colistin-resistant strains, within human macrophages, and determined the macrophage response to the intracellular infection.MethodsPhagocytosis of 11 clinical Ecc isolates, includingE. cloacae, E. bugandensis, E. kobei, E. xiangfangensis, E. roggenkampii, E. hoffmannii, andE. ludwigiiwas investigated in THP-1 and human monocyte derived macrophages (HMDMs). Confocal fluorescence microscopy was used to ascertain intracellular trafficking via co-localisation of cell markers with fluorescent bacteria. Intracellular bacterial replication was assessed by bacterial enumeration in cell lysates after killing extracellular bacteria and by a fluorescence dilution approach to follow the synthesis of the bacterial cell wall over time. Macrophage cell cytotoxicity was investigated by quantifying the release of lactate dehydrogenase during infection with all isolates. Two prototypic isolates, theE. cloacaeATCC13047 type strain and theE. bugandensis104107, were used to explore in more detail the response of macrophages to the intracellular infection by determining cleavage of the proinflammatory markers caspase-1, gasdermin D and pro-interleukin-1β.FindingsWe found that Ecc isolates do not replicate in human macrophages but survive within a modified late phagolysosome compartment. Survival occurred in all species investigated and did not correlate with colistin resistance, lipopolysaccharide modifications, or bacterial pathogenicity in theGalleria mellonellainfection model. All isolates induced macrophage cell cytotoxicity at significantly lower levels than controls treated with lipopolysaccharide and nigericin treatment (to induce a proinflammatory response). Low cytotoxicity also correlated with absence of cleavage of proinflammatory markers in infected macrophages.InterpretationEcc species can survive without replication inside human macrophages with minimal effects on cell viability and inflammation. These observations could have implications in the clinical outcome of patients that cannot readily clear infecting Ecc bacteria. This can potentially lead to prolonged intracellular survival and infection relapse.FundingBiotechnology and Biological Sciences Research Council grants BB/T005807/1 and BB/S006281/1.Research In contextEvidence before this studyWe searched PubMed with the terms “Enterobacter” AND “macrophage”, “Enterobacter” AND “monocytes”, and “Enterobacter” AND “intracellular” for original articles published in English up to July 9, 2024. The search excluded terms “sakazakii” and “aerogenes” sinceE. aerogenesandE. sakazakiihave been moved to the genusKlebsiellaandCronobacter, respectively. Of the 55, 15, and 181 studies we identified, respectively, only one reported testingEnterobacter cloacaephagocytosis. Another study reported intracellular bacterial communities in uroepithelial cells, which serve as a reservoir during urinary tract infection. One other study reported the isolation ofE. hormaecheifrom human atherosclerotic tissue and described studies in THP-1 monocytic macrophages. A few earlier studies also reportedEnterobactercytotoxins affecting immune cells, and anE. cloacaepolysaccharide capable of inducing apoptosis in epithelial cells. These studies did not investigate mechanisms and there have been no more recent follow-ups; importantly, it remains unclear if the strains employed in these studies were properly identified asEnterobacterspecies. Therefore, despite isolated observations of survival ofEnterobacterisolates surviving in immune and epithelial cells, there is an overall knowledge gap in our understanding of these pathogens concerning intracellular survival compartments, kinetics of survival, and induction of macrophage cytotoxicity and inflammatory responses.Added value of this studyOur study is the first to investigate in detail how clinical isolates of variousEnterobacterspecies can survive intracellularly in human macrophages. All isolates display multidrug antimicrobial resistance, including some with colistin resistance, and can survive intracellularly in human macrophages. Our data demonstrate that intracellularEnterobacterresides in vacuoles for up to 44 hours without replication. Colocalization experiments with various fluid phase and membrane cellular markers revealed that bacteria-containing vacuoles are modified late phagolysosomes, which do not accumulate the autophagosome marker LC3B. Intracellular bacterial survival did not associate to anyEnterobacterspecies tested, the presence of colistin resistance, lipopolysaccharide modifications, or virulence in theGalleria mellonellainfection model. Moreover, intracellular infection caused minimal cytotoxicity in macrophages without evidence of macrophage proinflammatory cell death by pyroptosis.Implications of all the available evidenceOur findings underscore the capacity ofEnterobacterspecies, traditionally viewed as extracellular bacteria, to hideout in macrophages without inducing a significant inflammatory response. These properties may further complicate the treatment of antibiotic-resistantEnterobacterinfections in susceptible populations such as the elderly and neonates. These findings open a door to the development of host-directed therapeutics to enhance bacterial clearance by macrophage-mediated killing.