Multidrug-Resistant Enterobacter cloacae Complex Emerging as a Global, Diversifying Threat

1Enterobacter spp., the second most common carbapenem-resistant Enterobacteriaceae (CRE) in the United States, increasingly contribute to the spread of carbapenem-resistant infections. In particular, Enterobacter cloacae complex (ECC) are common nosocomial pathogens capable of producing a wide variety of infections, such as pneumonia, urinary tract infections, and septicemia. The emergence of multidrug resistance (MDR), including resistance to the last-resort carbapenems meropenem, imipenem, and ertapenem, has led to an increased interest in these organisms.

2Molecular analyses based on multilocus sequence typing (MLST) and heat-shock protein (hsp) typing have led to the re-definition of members within this complex. Whole-genome sequencing (WGS), moreover, has allowed for reproducible population-level analyses to determine clonal structure and diversity in ECC and CREC collections ranging from localized, regional outbreaks to global studies. These methods have facilitated analyses of phylogenetic structure and evolutionary history on a global scale.

3Importantly, clinical and genomic studies have revealed a striking facility for ECC to acquire genes encoding broad-spectrum antibiotic resistance, including a variety of carbapenemase genes, superimposed on intrinsic β-lactam resistance conferred by chromosomal ampC genes. Here, we address recent advances in the molecular epidemiology, resistance mechanisms, global spread, and genomics of MDR ECC, focusing on CREC.

4The E. cloacae complex is polyphyletic based on the traditionally employed 16S rRNA gene typing. Phenotypic methods and antibiotic susceptibility patterns were insufficient to resolve this genetically diverse species cluster. Molecular and genomic advances have enabled more refined species designations of ECC based on single amplicon (hsp60 or rpoB) genotyping, multilocus sequence analysis (MLSA), comparative genomic hybridization (CGH), pulsed-field gel electrophoresis (PFGE), and more recently, MLST and WGS. Based on hsp60 allelic variation, ECC was previously classified into thirteen genovars (clusters I-XIII). These encompass Enterobacter asburiae (cluster I), Enterobacter kobei (cluster II), Enterobacter ludwigii (cluster V), Enterobacter hormaechei subsp. oharae (cluster VI), subsp. hormaechei (cluster VII), and subsp. steigerwaltii (cluster VIII), Enterobacter nimipressuralis (cluster X), E. cloacae subsp. cloacae (cluster XI) and subsp. dissolvens (cluster XII), unnamed E. cloacae Hoffmann clusters III, IV, and IX, and an unstable E. cloacae sequence crowd (cluster XIII). However, using hsp60 or rpoB alone led to significant discrepancies in identification of subspecies.

5Multilocus sequence analysis based on 6 housekeeping genes (rpoB, fusA, gyrB, leuS, pyrG, and rplB) suggested the emergence of two distinct ECC clades: a recent clade including the three E. hormaechei subspecies and a heterogeneous older clade including multiple ECC clusters. The observed recombination:mutation ratio of 1.04 (95% confidence interval 0.72–1.45) across ancestral clades also indicates potential recombination events in the early evolution of ECC, likely accounting for discrepancies between single amplicon methods. Based on MLSA, Enterobacter mori, Enterobacter xiangfangensis (cluster VI), and Enterobacter cancerogenus were recently classified. The remarkable genomic heterogeneity within ECC has even been used to suggest broad re-classification of the complex into five distinct genera based on MLSA. Despite ongoing debate regarding nomenclature within ECC, E. cloacae and E. hormaechei and related subspecies remain the most clinically relevant. In 2013, dnaA was added to the six genes of MLSA to develop an MLST scheme, which has emerged as a more robust tool for identifying closely related ECC isolates. To date, 1069 sequence types (STs) have been reported.1

6Comparison of the entire genome through WGS provides the opportunity to explore the genetic relationships between genomes at even higher resolution, and has further refined ECC classification into 18 clusters (A-R). These encompass the 12 Hoffmann clusters, E. mori, and five novel clusters (K, L, N, O, and P) ( and Supplementary Figure S1). Thus, the advent of WGS has greatly improved the ability to identify, investigate, and compare the emergence of ECC in diverse settings with high resolution, despite its polyphyletic and genomic diversity.

7A variety of intrinsic and acquired antimicrobial resistance mechanisms have diminished the arsenal of effective therapeutics for treatment of ECC infections. ECC is intrinsically resistant to penicillins and first- and second-generation cephalosporins due to low-level expression of chromosomal ampC genes encoding an inducible AmpC-type Bush group 1 (class C) cephalosporinase. Resistance to third-generation cephalosporins and aztreonam can result from mutations, usually in ampD, leading to constitutive hyperproduction (derepression) of AmpC.

8Extended-spectrum β-lactamase (ESBL) genes confer resistance to most β-lactam antibiotics, including extended spectrum (i.e., second and third-generation) cephalosporins (ESCs) and monobactams (i.e., aztreonam). These genes are typically plasmid-encoded and were first identified in ECC in 1989. Since then, ESBL-encoding ECC have increased in prevalence, particularly in nosocomial settings and among patients with previous antibiotic exposure. ESBL- and AmpC-mediated resistance now commonly coincide, leading to near-pan-resistance to β-lactams.

9Carbapenem-resistance in ECC is conferred through either constitutive overexpression of AmpC combined with disrupted membrane permeability, or more commonly through the acquisition of plasmid-encoded carbapenemase genes. Two major categories of carbapenemases have been identified in CREC, carbapenem-hydrolyzing serine β-lactamases (Ambler class A and D) and metallo-β-lactamases (MBLs; Ambler class B) (Supplementary Table S1). The Klebsiella pneumoniae carbapenemase (KPC), a class A β-lactamase which predominates in the United States, and the New Delhi metallo-β-lactamase-1 (NDM-1) have been most frequently described in ECC, although substantial regional variation has been reported. Rarely, ECC may also harbor chromosomally encoded carbapenemase genes.

10In addition to β-lactam resistance, ECC harbor a variety of multi-class antibiotic resistance genes. This includes aminoglycoside resistance primarily due to the acquisition of plasmids or mobile genetic cassettes encoding aminoglycoside 6’-N-acetyltransferase type I [AAC(6’)-I]. Mutations in DNA gyrase, DNA topoisomerase, or efflux pump genes have been associated with resistance to fluoroquinolones. Notably, ESBL and carbapenemase genes are often collocated with aminoglycoside-resistance genes on plasmids, engendering multi-class antibiotic resistance phenotypes.

11An AAC(6′)-I variant produced by aac(6′)-Ib-cr, or the presence of plasmid-borne qnr or qep genes, can confer low-level quinolone resistance in ECC. In addition, specific substitutions in chromosomal fluoroquinolone resistance-determining regions (QRDRs), such as the previously characterized double-serine/threonine substitutions in gyrA and parC, have been associated with improved fitness in major STs of other Enterobacteriaceae, including ESBL-producing Escherichia coli and K. pneumoniae. This fitness advantage has been hypothesized to contribute to the spread of high-risk international STs while selecting against minor STs. QRDR mutations have been detected in ECC and appear to be widespread in CREC. However, their contribution to the spread of specific ECC and CREC clones has yet to be determined.

12E. cloacae complex was one of the first KPC-producing organisms identified, and has recently demonstrated an increase in prevalence and regional distribution. Current literature indicates that the emergence and spread of CREC is due to high diversity of clonal lineages and carbapenemases. A recent study leveraging two global surveillance programs demonstrated the remarkable dissemination and variety of carbapenemase genes in ECC.

13We found 61 publicly available English-language publications identifying carbapenemase alleles in ECC with a corresponding geographic location (Supplementary Table S2). These encompassed 36 carbapenemase alleles (IMP-1,4,8,11,13,14,26,34; IMI-1,2,3,4,5,6,7,9; KPC-2,3,4,5,18; NDM-1,5,6,7; NMC-A; OXA-48; VIM-1,2,4,5,11,23,31; FRI-1,2; GES-7) in ECC from 44 countries, including single isolates and single or multi-institutional outbreak collections (Figure 1 and Supplementary Table S2). In the United States and Canada, blaKPC-positive ECC have been mostly encountered, with rare reports of IMI- and NMC-A-encoding organisms. Isolates harboring blaKPC have also been detected in Europe and South America. While blaNDM-1 is endemic in the Indian subcontinent, multiple blaNDM alleles were detected in hospitals throughout Eastern China. IMP-encoding genes have been reported widely in Southeast Asia, including China, Japan, Korea, the Philippines, Taiwan, and Australia, and are thought to be endemic to this area. On the other hand, VIM variants are more prevalent across Europe with rare reports from South America and Southeast Asia. OXA-48-like carbapenemases, thought to originate in Turkey, have spread into the Middle East, North Africa, and Europe.

14Previous multinational surveillance studies employing MLST found substantial clonal diversity of both ESBL-producing ECC and CREC, with evidence for several potential high-risk clones. The most widespread ESBL-producing ECC were ST66, ST78, ST108, and ST114, each having at least 10 isolates from three to five countries. Several epidemic clonal complexes (CC), such as CC74 (including ST78) or CC114 (including ST66) were identified, including specific ST66, ST78, and ST114 pulsotypes associated with carriage of CTX-M-15 β-lactamase. Likewise, ST114, (E. xiangfangensis), ST93 and ST90 (E. hormaechei subsp. steigerwaltii), and ST78 (E. cloacae cluster III) were widespread among global CREC isolates from 37 countries, while ST105 (E. xiangfangensis) and ST108 were also identified in multiple countries.

15While carbapenem-resistant K. pneumoniae (CRKP) appears to be declining in high-prevalence areas such as the Northeastern United States, multiple sites across the United States have reported increasing prevalence of CREC. By 2015, over 4% of ECC clinical isolates collected in the United States Veteran’s Health Administration (VHA) nationwide were carbapenem non-susceptible, with especially high rates along the West Coast and Southwestern United States. Most recently, New York City, Boston, Western Pennsylvania, North Carolina, and Minnesota/North Dakota have reported significant increases in CREC infections.

16Limited information is available regarding specific genomic features of ECC potentiating its transmission and recent epidemiological success. However, the few available genomic studies suggest that establishment of successful clones as well as acquisition of MDR phenotypes by diverse lineages may have been substantial contributors.

17ST171 has been identified as a major CREC clone with epidemic potential in the United States. We previously found phylogenomic evidence that all ST171 with publicly available sequences formed two major clades which diverged and spread in parallel from the Northeastern to the Mid-Atlantic and Midwestern United States. Our analysis estimated that these clades diverged prior to 1962, roughly two decades before the widespread use of carbapenems and fluoroquinolones, suggesting antibiotic pressure as a key factor in the proliferation of ST171.

18ST171 is primarily associated with blaKPC-3, although a handful of blaKPC-2- and blaKPC-4-containing isolates have been identified. In the Northeast, CREC ST171 primarily contained a blaKPC-3 gene located on IncFIA plasmids (e.g., p34978, pNR3024). These plasmids were nearly identical to pBK30683, a ∼70 kb IncFIA plasmid which was widespread among blaKPC-producing K. pneumoniae in New York and New Jersey hospitals. Interestingly, a different study reported ST171 isolates from Minnesota and North Dakota which contained blaKPC-3 on a truncated (∼120 kb) IncFIA plasmid pMNCRE44. The truncated pMNCRE44 shared key regions with other ST171 IncFIA plasmids, but lacked genes encoding conjugation machinery. A small cluster of ST171 isolates from Boston instead contained blaKPC-4 on an unrelated IncHI2 plasmid. A duodenoscope-mediated outbreak of CREC in a Michigan hospital also found likely patient-to-patient transmission of blaKPC-positive ST171 (KPC allele unreported). However, the hospital collection included diverse clones in which carbapenem-resistance was driven primarily by chromosomal mutations rather than carbapenemase genes. ST171 was rare in global surveys of both primarily carbapenem-susceptible and carbapenemase-producing ECC, harboring three different carbapenemase genes presumably on different plasmid backbones. This suggests that stable uptake of the IncFIA plasmid by ST171 largely enabled its successful proliferation throughout the Northeastern United States, while isolates lacking this plasmid remain uncommon.

19In contrast, ST78 was identified as a high-risk clone among both ESBL-producing ECC and CREC. CREC ST78 has largely been isolated in the Northeastern United States, with multiple sporadic uptake events of blaKPC-containing plasmids, and has not exhibited the same rapid clonal proliferation as ST171. ST78 has been associated with various KPC-types on IncN plasmids, even within the New York City area. Global carbapenemase-producing ST78 isolates have also been associated with a variety of plasmid backbones, highlighting its unique ability to acquire MDR plasmids. demonstrated 4 different carbapenemases (blaV IM-1, blaIMP-4, blaIMP-8, blaOXA-48) on multiple different genetic backbones in ST78, although the carbapenemase-harboring plasmid could not be determined using short-read sequencing. In Japan, ST78 isolates harbored blaIMP-1 on class 1 integrons encoded on multiple different plasmids including IncHI2, IncW, and IncFIB.

20Other CREC STs have been associated with diverse KPC subtypes on IncN, IncX7, IncL/M, IncA/C, pKpQIL, and pKPC_UVA01-like plasmids, and plasmids with unknown replicon types. However, few molecular studies include complete plasmid analyses, particularly for non-blaKPC carbapenemases. Notably, although region-specific associations between carbapenemase genes and specific genetic backbones have been reported, shuffling of these genetic structures among different ECC clones appears to occur commonly including in geographically diverse areas.

21Since the mid-1990s, when KPC was first described, the spread of CRKP has largely been attributed to the stable association between blaKPC and the successful CRKP clone ST258 (; Figure 2). Although isolated instances of CREC were reported around the same time, the diversification of both blaKPC and plasmid backbones harboring these genes may have enabled uptake into diverse ECC. In contrast to CRKP, the spread of CREC can be attributed to not only stable blaKPC-clone associations, as in the case of ST171 and blaKPC-3-encoding IncFIA plasmids, but also the sporadic uptake of diverse plasmids by heterogeneous clones. This includes clones with epidemic potential capable of harboring diverse blaKPC-containing plasmid backbones, such as ST78.

22In addition to the presence of blaKPC genes, other genomic factors linked to carbapenem- or other MDR may have aided in the rapid proliferation of CREC. Several lineage-specific genomic islands in both ST171 and ST78, encode for toxin-antitoxin and cell stress response systems. Genes for toxin-antitoxin systems and heavy metal resistance have been found on MDR plasmids in CREC isolates. These factors may further contribute to the success of this organism, particularly in nosocomial settings, although their specific impact on virulence and fitness has yet to be determined.

23Virulence of CREC compared to carbapenem-susceptible ECC has not been extensively assessed. However, murine macrophage cytotoxicity assays did reveal significantly reduced cell killing of CREC vs. ESBL isolates and, more specifically, reduced toxicity of CREC ST171 vs. ESBL ST78 isolates. Of note, no significant differences in cytotoxicity by site of collection or KPC-subtype were observed. Although previous studies reported detection of Shiga-like toxins in ECC, candidate genes were not detected in genomic analysis of these clones. Thus, CREC may be a low-virulence pathogen with specific adaptations that enable success in nosocomial environments. In particular, cross-class antibiotic resistance and the acquisition of carbapenem- and fluoroquinolone-resistance determinants prior to the widespread use of these drugs point to the role of antibiotic pressure in hospital settings, rather than increased virulence, in the spread of CREC ST171 in the United States. However, as previously suggested, potential fitness advantages conferred by QRDR mutations may play a role in the spread of major CREC STs, including ST171 and ST78, and should be evaluated further.

24Analogous to the pan-genome, the concept of a “pan-metabolome” has also been applied to ECC. Several metabolite targets were identified, which discriminated between CREC and carbapenem-susceptible ECC, indicating a distinct metabolomic signature for each phenotype, beyond the presence of a single carbapenemase gene. The use of metabolomics and transcriptomics in future studies will be important to fully understand the complex relationships between genomic background, acquired carbapenemase resistance and virulence factors, and variable resistance phenotypes.

25Several gaps remain in our understanding of CREC. The notable diversity of CREC clones, carbapenemase genes, and plasmid backbones harboring MDR genes have thus far led to uncertainty regarding a clear timeline and evolutionary history of these organisms. Virulence, fitness, or other genomic factors potentiating the spread of CREC have not been completely defined or assessed in vitro. Moreover, despite recent advancements potentiated by WGS and comparative genomics, transcriptomics and/or metabolomics approaches may be useful in future studies to define the metabolic activity of CREC under different conditions. Lastly, the underreporting of CREC remains a possibility, and may influence findings regarding both population-level diversity and genomic mechanisms of resistance.

26Regardless, the unique diversity of CREC, even compared to other CRE such as CRKP, necessitates a tailored approach to preventing its transmission and further diversification. The establishment of high-risk global CREC clones, coupled with the apparent high frequency of plasmid uptake into diverse ECC, suggests that vigilant tracking of both localized outbreaks and the potential for horizontal plasmid transfer is required.

27A-CU initiated the review. AG-S performed a literature search. MKA wrote the first draft. All authors edited and reviewed the manuscript draft.

28A-CU has received research funding from Merck, GSK and Allergan, unrelated to the current study. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.