Exploring Cluster-Dependent Antibacterial Activities and Resistance Pathways of NOSO-502 and Colistin against Enterobacter cloacae Complex Species

The Enterobacter cloacae complex (ECC) is a group of diverse bacterial species of clinical and environmental relevance (1). These facultative anaerobic bacteria belonging to the Gram-negative Enterobacteriales family are widely present in nature and are part of the gut commensal microbiota of animal and human populations (2). ECC species were clustered by Hoffmann and Roggenkamp in 12 groups on the basis of DNA sequence of their hsp60 genes (designated C-I to C-XII, Table S1) (3). Some of them, frequently belonging to C-III, C-VI, and C-VIII, are associated with a variety of human infections and have emerged as one of the leading causes of nosocomial infections worldwide, accounting for up to 5% of hospital-acquired pneumonia and bacteremia, 4% of nosocomial urinary tract infections, and 10% of postsurgical peritonitis (4). The intrinsic resistance of ECC to several antibiotics and their ability to acquire resistance to many others, including the last resort antibiotics such as carbapenems and colistin (CST), makes some infections caused by ECC difficult to treat (5, 6). Indeed, these species are intrinsically resistant to aminopenicillins and first- and second-generation cephalosporins due to the presence of a chromosomal inducible AmpC β-lactamase (7). Moreover, the acquisition of plasmids carrying extended-spectrum β-lactamases (ESBL)-encoding genes confers resistance to most clinically relevant β-lactams (8). Most worrisome, many studies have also reported the global emergence of carbapenem-resistant E. cloacae (CREC), suggesting accelerating resistance acquisition in this organism (9–11). Carbapenem resistance in ECC results from either the constitutive overexpression of AmpC/ESBL combined with decreased permeability or the acquisition of plasmid-encoded carbapenemase genes (e.g., bla_KPC, bla_NDM, or bla_OXA-48-like) (7). In 2013, the need to develop new antibiotics active against carbapenem-resistant Enterobacterales (including CREC strains) was classified as urgent by the U.S. Centers for Disease Control and Prevention (CDC) (12). The status of CST is different from that of other antibiotics. This drug is a member of the polymyxin family and was used for the treatment of various Gram-negative infections. Its use by intravenous route was abandoned in the early 1980s because of significant side effects and the introduction into clinical practice of less toxic antibiotics (13). With the increasing prevalence of infections caused by multidrug-resistant (MDR) Gram-negative bacteria and the failure to develop new effective antibiotics, CST has reemerged as therapeutic option for many infections, including those due to ECC (14). Currently, many programs that aim at designing novel polymyxin derivatives are under clinical development (15). Mechanistically, CST is a cyclic cationic lipopeptide that binds to the lipid A part of the LPS, inducing an outer membrane permeabilization and an inner membrane disruption, leading to cell lysis. Consequently, LPS modification by addition of positively charged moieties is the main mechanism of resistance to CST. In Enterobacterales, lipid A modification mainly occurs by the addition of phosphoethanolamine (PEtN) or 4-amino-4-deoxy-l-arabinose (L-Ara4N) with regulation by the two-component systems (TCS) PmrAB and/or PhoPQ in response to environmental signals such as the presence of cationic antimicrobial peptides (CAMPs), low magnesium, or acidic pH. PmrAB can activate both arnBCADTEF operon expression encoding enzymes responsible for the synthesis and transfer of the L-Ara4N to lipid A but also the pEtN transferase PmrC. Depending on the species, PhoPQ can either directly activate the arnBCADTEF operon or indirectly activate by cross-activation of PmrAB via the connector protein PmrD (16). In ECC, CST resistance is regularly associated with a cluster-dependent hetero-resistance phenotype, a phenomenon observed in different Gram-negative and Gram-positive species, in which the major population of susceptible cells is killed in the presence of a given antibiotic whereas a preexisting subpopulation of resistant cells can rapidly multiply (17). It has been postulated that it may cause antibiotic treatment failure and be induced by the host immune system (18, 19). As expected, CST-resistant clinical ECC have also emerged with a high prevalence in some studies (20). In the race against time to develop new antibiotics, the odilorhabdins (ODLs) have emerged as a promising new family. ODL members are cationic peptides that specifically inhibit the bacterial translation by interacting with the 30S subunit of the bacterial ribosome (21, 22). NOSO-502 is the first preclinical candidate of this novel antibiotic class (23). In a recent publication, Racine et al. reported that NOSO-502 exhibits potent activity against MDR colistin-resistant, and carbapenemase-producing Enterobacterales, including ECC isolates from different European hospitals (KPC-2, NDM-1, OXA-48) (23). NOSO-502 and CST coresistant K. pneumoniae mutants bearing mutations in crrB gene were identified (23). CrrB is a signal-transducing histidine kinase and CrrA is an adjacent response regulator belonging to a TCS named CrrAB. It was established that mutations in the CrrAB TCS induce resistance to NOSO-502 by an upregulation of the efflux pump component KexD when it generates CST resistance by addition of L-Ara4N or pEtN to LPS regulated by the PmrAB or PhoPQ TCS, and via the protein CrrC (24). KexD is predicted to be an energy-dependent efflux pump subunit belonging to the resistance nodulation division (RND) family and CrrC a modulator that interacts with PmrAB to alter arn operon expression (25).

The purpose of this study was first to evaluate the activity of NOSO-502 against a large panel of ECC clinical isolates, from different Hoffmann’s clusters, and then to investigate the associated resistance mechanisms. Indeed, understanding and anticipating the emergence of resistance is an essential step in the development of new antibiotics. Our preliminary results confirmed the potent antibacterial activity of NOSO-502 against the most problematic ECC strains, but also surprisingly highlighted two specific clusters were less susceptible to both NOSO-502 and CST, leading us to analyses the associated mechanisms of resistance. A new essential TCS mediating hetero-resistance to both antibiotics by different molecular mechanisms that have never been investigated among ECC members was identified.

NOSO-502 is the first preclinical candidate of a novel antibiotic class, the odilorhabdins (ODLs). This cationic peptide inhibits bacterial protein synthesis by targeting the ribosome and it is currently developed to treat infections caused by Enterobacteriales such as E. coli, K. pneumoniae, or ECC. In this study, we focused on the characterization of NOSO-502 antibacterial activity against ECC strains that pose a serious threat to human health worldwide due both to the emergence and spread of carbapenem-resistant isolates but also because several of them exhibit hetero-resistance to CST. NOSO-502 demonstrated improved potency, based on MIC values, against a recent and representative population of ECC hospital isolates belonging to all ECC clusters. Intriguingly, only strains from ECC C-XI and C-XII exhibited higher MICs for NOSO-502. To date, isolates of clusters C-XI and C-XII represent only a few cases of ECC isolated from patients. E. cloacae (C-III), E. hormaechei subsp. oharae (C-VI), and E. hormaechei subsp. steigerwaltii (C-VIII), including carbapenemase-producing strains, are the most frequent species isolated from intensive care unit (ICU) patients and have previously been identified in a wide variety of nosocomial infections, including cerebral abscess, pneumonia, meningitis, bacteremia, wound, and urinary tract and abdominal cavity/intestinal infections (1, 28). A recent publication reported that in a Spanish hospital between 2005 and 2018, 80% of isolated carbapenemase-producing ECC strains were members of C-III, C-IV, C-VI, or C-VIII, while only 0.5% belong to C-XII. No strains from C-XI were identified (29). Other reports confirm that isolates from cluster XI are rare among carbapenemase-producing ECC isolates (30, 31). Nevertheless, some outbreaks were reported in Togo and the French island of Mayotte (32, 33).

We clearly demonstrated that the TCS ECL_01761/ECL_01762 plays a pivotal role in the cluster-dependent resistance to NOSO-502 or CST of ECC isolates. In E. cloacae subsp. cloacae ATCC 13047 (C-XI), the TCS ECL_01761/ECL_01762 responds directly to the presence of NOSO-502 or CST leading to resistant phenotypes, to NOSO-502 via an upregulation of the ECL_01758 efflux pump component but also to CST via the overexpression of the transporter protein ECL_01760 (Fig. 6). Nevertheless, the role of the histidine kinase ECL_01762 seems limited in the CST-resistance pathway while that of the response regulator ECL_01761 is preponderant. Many bacteria possess considerable numbers of TCS and the high similarity between some systems raises the possibility of cross talk between a histidine kinase and a noncognate response regulator (34). In this case, another unidentified histidine kinase seems to work with ECL_01761. Increased drug efflux driven by TCS was found across many species of MDR bacteria (35). ECL_01758 complements the hypersusceptibility phenotype of the ΔacrB ECL13047 mutant to restore wild-type susceptibility to diverse antibiotics classes such as fluoroquinolones, aminoglycosides, or tetracyclines (4). However, we noted that the inactivation of AcrB increased the resistance of ECL13047 to NOSO-502 while the deletion of tolC or acrA had the opposite effect, confirming a possible functional competition between ECL_01758 and AcrB to work with AcrA and TolC. In K. pneumoniae, Ogawa and collaborators have already showed that KexD, an ortholog gene of ECL_01758, functions with AcrA and TolC (27). Guérin and collaborators have also identified that the inactivation of ECL_01758 increased the virulence of ECL13047 in a Galleria mellonella model compared with the wild-type strain, while the acrB deletion mutant was avirulent (4). This is an important point because it means that cationic peptides like NOSO-502, polymyxin, or AMPs of innate immunity could downregulate the virulence of E. cloacae by induction of ECL_01761/ECL_01762 and by overexpression of ECL_01758. Although only present in clusters I, II, IV, and IX, this tripartite system seems to be solely functional in isolates from C-XI and to a lesser degree from C-XII. It could explain the very low prevalence of C-XI bacterial isolates in human infections. As previously observed in K. pneumoniae, we cannot exclude that mutation in the ECL_01762 gene induces overexpression of the ECL_01758 efflux pump component leading to NOSO-502 resistance in strains belonging to these clusters.

As previously mentioned, the TCS ECL_01761/ECL_01762 led to CST resistance by overexpressing the transporter protein ECL_01760. The presence of CST activates both ECL_01761/ECL_01762 and PhoPQ (Fig. 6). Previous works described CST hetero-resistance in ECL13047 was due to a modification of the LPS, achieved by the addition of L-Ara4N mediated by enzymes of the arnBCADTEF operon under the control of the PhoPQ two-component system (16). However, we observed that deletion of ECL_01760 restores full susceptibility to CST, implying that this gene also plays a role in this resistance phenotype. ECL_01760 codes for a small integral membrane protein of 128 amino acids, predicted to be a transporter with four transmembrane domains. It has already been reported that some small proteins connect different TCSs to adapt cells to environmental stress. In Salmonella, the PhoP activated PmrD protein binds to the phosphorylated form of PmrA resulting in the expression of PmrA-regulated LPS modification genes (36). In E. coli, SafA formerly B1500, is a 65 amino-acid membrane protein connecting both TCSs EvgAS and PhoPQ. Its expression is regulated by the EvgAS and this protein directly interacts with PhoQ to activate the PhoPQ system (37, 38). In our study, ECL_01760 acts downstream of the arnBCADTEF operon expression and, therefore, of the PhoPQ system. Indeed, similar upregulations of phoPQ and arnB expression were observed in CST-treated cultures of the CST-susceptible ECL13047 ΔECL_01760 mutant and of the CST-resistant wild-type strain. ArnBCAD enzymes were involved in the biosynthesis of the undecaprenyl phosphate-α-l-Ara4N while ArnEF ensure its transport across the inner membrane. These steps precede periplasmic modification of lipid A with l-Ara4N by the undecaprenyl phosphate-alpha-4-amino-4-deoxy-l-arabinose arabinosyl transferase ArnT. ECL_01760 could, therefore, act on the inner membrane transport of the undecaprenyl phosphate-α-l-Ara4N cytoplasmic precursor (Fig. 6). These results seem different from those observed in K. pneumoniae by Cheng et al. who demonstrated that CrrC, an ortholog gene of ECL_01760, regulated the pmrHFIJKLM operon, also called arnBCADTEF, and pmrC through the PmrAB TCS (25). Interestingly, in K. pneumoniae, the small protein DkcA, displays similarity to membrane transporter and is required for CST resistance in a lipid A-dependent and-independent manner (39). DkcA is required for lipid A modification with Ara4N by a yet unidentified mechanism, but authors hypothesized that it would play a role in the arn pathway or by maintaining an inner membrane potential necessary for the CST antibacterial activity.

This work was partially performed within the framework of IMI’s GNA-NOW Program. We thank Anne Lanois (INRAE) from DGIMI laboratory (UM-INRAE) and Philippe Clair from the Montpellier GenomiX qPCR HD facility for expert technical assistance with real-time PCR.