Colistin heteroresistance in Enterobacter cloacae is mediated by PmrAB-independent 4-amino-4-deoxy-L-arabinose addition to lipid A

The Enterobacter cloacae complex (ECC) consists of closely-related, but genetically distinct bacteria commonly associated with the human microbiota. ECC have been increasingly isolated from healthcare-associated infections, demonstrating that these Enterobacteriaceae are emerging nosocomial pathogens. ECC strains can rapidly acquire multidrug resistance to conventional antibiotics. Cationic antimicrobial peptides (CAMPs) have served as therapeutic alternatives because they target the highly conserved lipid A component of the Gram-negative outer membrane to lyse the bacterial cell. Many Gram-negative Enterobacteriaceae fortify their outer membrane with cationic amine-containing moieties to protect from CAMP-inflicted lysis. The PmrAB two-component system (TCS) transcriptionally activates 4-amino-4-deoxy- L -arabinose ( L -Ara4N) biosynthesis to result in amine moiety addition to lipid A in many Enterobacteriaceae such as E. coli and Salmonella . In contrast, PmrAB in E. cloacae is dispensable for CAMP resistance. Instead, fitness against CAMPs presents as heteroresistance, or a subpopulation of cells that exhibit clinically significant increases in resistance levels compared to the majority population. We demonstrate that E. cloacae lipid A is modified with L -Ara4N to induce CAMP heteroresistance and that the regulatory mechanism is independent of the PmrAB Ecl TCS. We show that the response regulator, PhoP Ecl , directly binds to the arnB Ecl promoter to induce expression of L -Ara4N biosynthesis and PmrAB-independent addition to the lipid A disaccharolipid. Therefore, we have identified a mechanism of ECC colistin heteroresistance that directly involves the PhoPQ system. Importance Members of the Enterobacter cloacae complex (ECC) are Gram-negative nosocomial pathogens that have emerged within healthcare facilities around the world. ECC infections are associated with immunocompromised patients and infections are often life threatening. The cationic antimicrobial peptide, colistin (polymyxin E), is a last-line treatment option to combat Gram-negative multidrug resistant infections. However, many ECC intrinsically encode a colistin heteroresistance mechanism. Our analysis to characterize colistin heteroresistance in E. cloacae revealed that 4-amino-4-deoxy- L -arabinose is conjugated to the lipid A disaccharolipid to protect from colistin-mediated lysis. Additionally, this mechanism is directly regulated by the PhoPQ Ecl two-component system. Elucidation of outer membrane antimicrobial resistance modifications and their regulatory pathways in E. cloacae isolates will advance our understanding of CAMP heteroresistance.