Enterobacter aerogenes and Enterobacter cloacae; versatile bacterial pathogens confronting antibiotic treatment

1Enterobacter is a genus of a common Gram-negative, facultative anaerobic, rod-shaped, non-spore-forming bacteria belonging to the family Enterobacteriaceae. Two of its well- known species, Enterobacter aerogenes and E. cloacae have taken on clinical significance as opportunistic bacteria and have emerged as nosocomial pathogens from intensive care patients pathogenic, especially to those who are on mechanical ventilation.

2Enterobacter aerogenes was originally named Aerobacter aerogenes, and was later included in the genus Enterobacter in 1960. In 1971, this species was proposed to be renamed Klebsiella mobilis due to its motility conferred by peritrichous flagella and its genetic relatedness to Klebsiella genus. It is interesting to note that phenotypic dissimilarities between E. aerogenes and the genus Klebsiella include not only the motility but also the presence of ornithine decarboxylase (ODC) activity and the lack of urease activity in E. aerogenes. However, recently, the whole genome sequencing of a multidrug-resistant (MDR) clinical isolate, (including colistin) suggested a possible reclassification of the species in the genus Klebsiella, under the name K. aeromobilis. E. aerogenes particular phenotype can be attributed to the horizontal acquisition of additional genes from other Enterobacteriaceae species and mobile elements that rapidly integrated and translated as easily as its own ancestral heritage. For example, the flagellar genes and its assembly system have been acquired in bloc from the Serratia genus. Plasmid conjugation is a chimera of transposons and genetic elements (conjugation, integration) of various bacterial origins. E. aerogenes also contains eight rRNA operons and 87 tRNA associated with the ability to translate imported genes that use different codons, improving its ability to use its integrated foreign genes. E. aerogenes has been involved in significant European outbreak between 1993 and 2003 and is considered as the paradigm of opportunistic bacteria.

3Species of the E. cloacae complex are widely encountered in nature, but they are also pathogens: E. cloacae and E. hormaechei are most frequently isolated from human clinical specimens. Thus, E. cloacae is among the most common Enterobacter sp. causing only nosocomial infections in the last decade and a lot has been published on the antibiotic-resistance features of these microorganisms. Despite the relevance of E. cloacae as a nosocomial pathogen, the pathogenic mechanisms and factors contributing in the disease associated with the E. cloacae complex are not understood yet; this could be due to the scarcity and the dispersion of information available. Its ability to form biofilms and to secrete various cytotoxins (enterotoxins, hemolysins, pore-forming toxins) are important for its pathogenicity. Some genotypes and species, have previously exhibited some associations with clinical specimens, in particular urines and sputum, when clonal outbreaks with members of the E. cloacae complex were rare. Interestingly, due to the diffusion of most frequent extended spectrum β-lactamases (ESBL) and carbapenemases in this species, E. cloacae has now become the third broad spectrum Enterobacteriaceae species involved in nosocomial infections after Escherichia coli and K. pneumoniae.

4Enterobacter aerogenes is isolated as human clinical specimens from respiratory, urinary, blood, or gastrointestinal tract. Epidemiology of this species has been particular in Europe: it has regularly been involved in nosocomial infections outbreaks since 1993, particularly in the Western Europe. Until, 2003, E. aerogenes was considered as an important emerging MDR pathogen, particularly in ICUs (;; Figure 1). The situation in 1990s in Europe pointed to the dispersion of an epidemic clone and, since then, it has been extensively detected in European hospitals and health care facilities. The event fitted in with the international spread of the ESBL TEM-24 (blaTEM-24) harbored by an epidemic plasmid. The prevalence of Enterobacter sp. infections in clinical wards has also increased due to the introduction of extended-spectrum cephalosporins and carbapenems in the antibiotic therapy. The consequence of this antibiotherapy is the emergence of “pan-drug E. aerogenes isolates” resistant to last-line antibiotics such as carbapenems and also to colistin, for which no therapeutic option was available. Interestingly, the role of efflux mechanism in E. aerogenes resistance has been studied within an 8 years of period. This study indicated a noticeable increase of the prevalence of an efflux mechanism, susceptible to pump inhibitor, in clinical isolates collected during this period. After the emergence of ESBL in E. aerogenes and the characterisation of porin mutations in clinical isolates, this role of efflux mechanism highlights a new step in the adaptative evolution in E. aerogenes.

5Since 2010, E. aerogenes in France is the fifth highest Enterobacteriaceae and the seventh highest Gram-negative Bacillus responsible for notorious nosocomial infections (; Figure 2). Despite its intrinsic resistance to ampicillin and constant expression of ESBL that is associated with other resistance mechanisms contributing to MDR phenotype, its prevalence has significantly dropped (reduction factor of 20) in France. Its position was displaced in the context of hospital acquired infections, because of the dramatic rise of the E. coli pandemic clone O25:H4-ST131 along with K. pneumoniae and E. cloacae, ESBL, and/or carbapenemase producing strains. Although, E. aerogenes causes septic shock more readily in patients thus leading to a higher mortality rate, E. cloacae is now the most frequently observed clinical isolate among Enterobacter sp. It can be associated with the dissemination of actual epidemic plasmids bearing most prevalent resistant genes and expressing new β-lactamases or carbapenemases (Figure 2).

6Enterobacter cloacae is ubiquitous in terrestrial and aquatic environments (water, sewage, soil, and food). The species occurs as commensal microflora in the intestinal tracts of humans and animals and is also pathogens in plants and insects. This diversity of habitats is mirrored by the genetic variety of E. cloacae. Recently, MLST and PFGE epidemiological methods data revealed world circulation of several epidemic clonal complexes.

7It is also a well-known nosocomial pathogen contributing to bacteremia, endocarditis, septic arthritis, osteomyelitis, and skin/soft tissue infections, and lower respiratory tract- urinary tract and intra-abdominal infections. E. cloacae tends to contaminate various medical, intravenous, and other hospital devices. Nosocomial outbreaks have also been associated with the colonization of certain surgical equipment and operative cleaning solutions. Since a decade, E. cloacae has been repeatedly reported as a nosocomial pathogen in neonatal units and several outbreaks of infection have been reported. Today, variability among strains are less frequent and outbreaks due to clonal E. cloacae hyper-producing AmpC β-lactamase and ESBL carrier isolates are described from neonate specimens, adults urines/feces samples or from environmental samples.

8Enterobacter cloacae has an intrinsic resistance to ampicillin, amoxicillin, first-generation cephalosporins, and cefoxitin owing to the production of constitutive AmpC β-lactamase. It exhibits a high frequency of enzymatic resistance to broad-spectrum cephalosporins. Resistance of Enterobacter sp. to third-generation cephalosporins is most typically caused by overproduction of AmpC β-lactamases, and thus treatment with third-generation cephalosporins may select for AmpC-overproducing mutants. AmpC overproduction is due to the derepression of a chromosomal gene or by the acquisition of a transferable ampC gene from plasmids or other mobile elements. The AmpC plasmid-mediated resistance is distinguished from chromosomal enzyme production because they are not inducible. However they represent a problem due to its increasing prevalence among clinical isolates. The enzyme confers a resistance to third-generation cephalosporins and ureido- and carboxy-penicillins and is not inhibited by common inhibitors of β-lactamases. Fourth-generation cephalosporins retain reasonable activity against derepressed strains, but if strains are also ESBL producers, they become resistant to this antibiotic class. The prevalence of ESBL and CTX-M producers represented approximatively 5% of the isolates in the recent studies and ESBLs are most often plasmid-mediated. These characteristics, associated with the frequent endogenous intestinal carriage of E. cloacae, may result in abnormally high levels in the bowels of hospitalized patients, especially those who have received cephalosporins.

9The production of β-lactamases is the prominent mechanism responsible for β-lactam resistance in most of these species. E. aerogenes strains have a broad ability to develop antibiotics resistance mechanisms. They naturally express a chromosomal AmpC β-lactamase type cephalosporinase at low level (group 1 Bush) that induces resistance to first-generation cephalosporins. Chromosomal acquired β-lactams resistance mechanisms induce the overproduction of chromosomal AmpC cephalosporinase: this results from an induction during a third-generation cephalosporin treatment or by a mutation in the AmpR repressor, and generates a resistance to almost all β-lactams. Moreover, it has been described that E. aerogenes strains harboring cephalosporinase AmpC gene, integrated the gene of chromosomal origin (blaCMY-10) on a large plasmid (130 kb), contributing to a systematic gene transmission even in the absence of antibiotic pressure.

10In 1993 appeared the first cases of nosocomial infections caused by strains with resistance to common β-lactam antibiotics due to ESBL. The ESBL TEM- 24 associated to E. aerogenes clonal dissemination in France was constantly reported. Other ESBLs of TEM type or CTX-M type (ex CTX-M-2) are often identified but TEM-24 remains associated with preferential conjugative plasmid of this species. Due to the well-described modification of porins expression and recent dissemination of plasmid bearing carbapenemases, a number of imipenem-resistant clinical strains have come up. Carbapenemases of NDM and VIM types are now, as anticipated, reported in E. aerogenes in India and those for the serine protease group as KPC or class D β-lactamases possessing carbapenemase properties as OXA-48 types are described in Europe/Asia.

11Similarly to E. aerogenes, E. cloacae is also naturally resistant to ampicillin, amoxicillin–clavulanic acid, cephalothin, and cefoxitin by low production of the natural inducible cephalosporinase of Bush group 1 (class C). They are capable of overproducing AmpC β-lactamases by blocking the repression of a chromosomal gene or by the acquisition of a transferable ampC gene on plasmids conferring the resistance to third-generation cephalosporins. Cefepime alone can keep its activity. Clinical AmpC resistance represents 50% of the isolates and frequently co- exists with the expression of ESBL. In 1989, appeared the first nosocomial isolate cases bearing plasmidic ESBL causing also resistance to third generation cephalosporins except cefamycins. Together, these enzymes are responsible for a global resistance to all β-lactams except carbapenems. In the last decade, E. cloacae has emerged as the third most common Enterobacteriaceae resistant to third generation cephalosporins with enteric E. coli and K. pneumoniae. Imipenem remains the most effective molecule for treating E. cloacae infections. Since then, various ESBL of TEM, SHV, and CTX -M types have been characterized in E. cloacae including resistant TEM inhibitors or IRT (for inhibitor-resistant TEM;;; ). However, among ESBL producers, some sub-clones are now identified, associated with CTX-M-3 and 15 production, when other TEM or SHV (SHV-12 for example) types are also associated with epidemic-episodes-involved isolates. Diffusion of E. cloacae producing CTX-M-15 ESBL is the consequence of the wide dissemination of identical or related plasmids harboring the CTX-M-15 gene firstly identified in the epidemic E. coli clone, and the CTX-M β-lactamases are now the most prevalent ESBL globally.

12In recent years, clinical isolates resistant by producing carbapenemases have been identified. In 2010, CDC first reported carriage of NDM-1 in E. cloacae from patients who received medical care in India. Especially in Asia, strains harboring metallo-β-lactamases as IMP -type enzymes, NDM, GIM, VIM, and serine carbapenemase type KPC have been described. The OXA-48 type serine carbapenemase is the most prevalent because its gene is located on a plasmid, associated to the bla-CTX-M-15 gene coding ESBL, thus explaining its spread and the associated resistance. A hike in the imipenem resistance rate in E. cloacae, from 0.4 to 8 %, has been observed. An epidemic study concerning E. cloacae blood stream infections indicated a 25% production of metallo-β-lactamase in corresponding strains. Thus, the decreased susceptibility to carbapenems in hospital acquired E. cloacae isolates might arise via stepwise accumulations of MDR determinants in different clones. Today, E. cloacae is the second Enterobacteriaceae carrying carbapenemase and strains co-expressing two carbapenemases has been reported.

13Regarding the aminoglycosides, the major mechanism of resistance of the Enterobacteriaceae is due to aminoglycoside-modifying enzymes that are often plasmid-encoded, but it may also be associated with transposable elements. These enzymes are assigned to three groups: acetyltransferases (acetylation of an amino group/AAC), phosphotransferases (phosphorylation of a hydroxyl group/APH), and adenylyltransferases (adenylylation of a hydroxyl group/AAD or ANT). Plasmid exchanges and disseminations of transposons facilitate the rapid acquisition of resistance phenotypes.

14The resistant strain varies from 0 to 51% resistance for gentamicin, and 0 to 34% for amikacin. In 2013, an important epidemiological study confirmed that the aminoglycoside-modifying genes involved in aminoglycoside-clinical resistance were aac(3)-IIa, aac(6′)-Ib, and ant(2′′)-Ia, genes that confer resistance to tobramycin, gentamicin, and amikacin. Strains have frequently more than one enzyme. This enzymatic type resistance is associated in 77% of clinical isolates in China to other plasmid genes (armA, rmtB; ). Among these, the aminoglycoside AAC(6′)-Ib is the most common cause of amikacin resistance among members of the Enterobacteriaceae family. In a previous study, it was observed that over 40% of the E. cloacae isolates had the aac(6′)-Ib gene, although many of the isolates with this gene were susceptible to amikacin and gentamicin, which were the most active of all tested drugs.

15The enzymatic resistance to fluoroquinolones has been recently described and attributed to a two-point mutation allele of aac(6′)-Ib [named aac(6′)-Ib-cr], the aminoglycosides resistance enzymatic determinant, which acetylates ciprofloxacin and norfloxacin. A systematic molecular survey reporting prevalence and characteristics of aac(6′)-Ib-cr in Korea, characterized a high prevalence of the mechanism (23%) in E. cloacae. Dissemination of this new enzymatic resistance mechanism occurs since the aac(6′)-Ib-cr is highly associated with blaOXA-1, ISCR1, and class 1 integron. This supports the previous finding where aac(6′)-Ib-cr was located upstream of blaOXA-30 (synonymously called blaOXA-1) in complex class 1 integron, In37 containing ISCR1. A genetic linkage between aac(6′)-Ib-cr and blaCTX-M-15 has been demonstrated.

16Carbapenems are the most powerful agents for the treatment of serious nosocomial infections caused by MDR Enterobacteriaceae. Due to the imipenem use, it was rapidly reported a decreased penetration of β-lactams due to a change in the expression of porins in E. aerogenes isolates. showed that MDR strains of E. aerogenes exhibited a characteristic phenotype associated with an altered expression of porins and then successive studies comforted description of more frequent MDR strains in treated patients by β-lactams. This mechanism of resistance is reversible upon discontinuation of treatment and progressive during treatment. Among intermediate strains which are susceptible to imipenem but resistant to ertapenem, there is a loss of porin Omp35 but the expression of porin Omp36 is preserved. When treatment with imipenem continues, the disappearance of two porins and resistance to all carbapenems is noted. Recently, a novel mechanism of resistance has been observed in a clinical strain where the antibiotic cannot be efficiently translocated through a mutated porin (see Mutation and Antibiotic Resistance). Additionally, imipenem and carbapenemase KPC type have been described as responsible for resistance to carbapenems associated to decrease in membrane permeability. The conductance and selectivity of these porins, Omp35, and Omp36, correspond to the properties obtained with OmpC and OmpF of E. coli. Moreover, several studies have further described a decrease in production of these porins in resistant isolates. Two major outer membrane porins have been identified in E. cloacae and studied by liposome swelling assays. These porins are involved in the carbapenem susceptibility and exhibit important cross antigenicity with the E. aerogenes porins in specific key parts, e.g., eyelet region, membrane insertions, subunit connections. In addition, their respective involvement in β-lactam and fluoroquinolone uptake has been reported.

17Interestingly, the expression of porin in Enterobacteriaceae is rapidly and notably altered by various stress compounds present in the external medium. During the first hours of incubation in the presence of salicylate, novobiocin, norfloxacin a significant increase of OmpX is observed and this overexpression negatively controls the synthesis of porins.

18Furthermore, an efflux mechanism that is involved in the expelling of molecules from the bacteria such as fluoroquinolones, tetracycline, and chloramphenicol is active in Enterobacter sp.. This mechanism is highly efficient since the AcrAB–TolC efflux pump can eject about 80–90% of the norfloxacin during the first 10–15 min. Interestingly, this process is energy-dependent and requires the membrane energy (proton motive force) as extensively described (for a recent review see ). Approximately 40% of MDR clinical strains have an active efflux. The EefABC and AcrAB–TolC efflux genes of E. aerogenes have been described and their involvement in antibiotic exportation has been studied. Several studies on E. cloacae have also reported the presence of efflux pumps belonging to RND and MATE families. In addition, the AcrAB–TolC and OqxAB genes have been characterized in E. cloacae clinical resistant isolates. In E. aerogenes and E. cloacae, the sequence similarities and biological activity are particularly high in AcrAB–TolC. Moreover, various chemicals such as salicylate, chloramphenicol, and imipenem are also able to trigger the genetic cascade controlling the expression of Enterobacter AcrAB–TolC pump. The regulation seems to be associated with the internal concentration of chemicals that plays a key role during the switch on of the cascade that provides the efflux expression.

19MarA acts as a key regulator for the expression of porin genes and tolC in Enterobacteriaceae. SoxS is another key transcriptional regulator that is positively controlled by oxidative stress and can trigger the MarA expression. Some Enterobacteriaceae sp., such as Enterobacter, Klebsiella, Salmonella, have an additional global regulator, RamA. It plays a strategic role in controlling both the porins and the efflux expression, either directly or via the MarA cascade. This coordinated control of influx and efflux directly and efficiently governs the intracellular accumulation of antibacterial agents. Importantly, this internal accumulation of antibacterial molecules below the threshold corresponding to the MIC can favor the emergence and acquisition of additional mechanisms of resistance such as target mutation, production of detoxifying enzymes (e.g., β-lactamases, acetyltransferase, etc), and contributing to the extension of MDR phenotype.

20Regarding the active structure of efflux pumps involved in E. aerogenes and E. cloacae; we can hypothesize that a common structural organization is conserved, due to the high conserved homology between Enterobacter and E. coli. This structural organization can be similar to the recent description of the AcrAB–TolC complex in E. coli.

21Regarding the β-lactam antibiotics, the resistance due to target mutation occurs incidentally in Enterobacter sp. However, the diverse β-lactamases identified today is the result of a series of mutations that have successively appeared in the original β-lactamases TEM-1/2, SHV-1, OXA-1.

22Furthermore, strains in which AmpC cephalosporinase was derepressed have been affected by mutations affecting AmpR-promoter recognition site. Mutations that are best known and studied are those that affect the target of fluoroquinolones and more recently those responsible for polymyxin resistance. As a matter of fact, the quinolones were widely prescribed antimicrobial agents because of their proven safety, high oral bioavailability, multiple approved indications, and bactericidal activity. Consequently, in the microbial population, a variety of amino acid alterations arose from mutations within quinolone resistance-determining regions (QRDRs) of cellular target genes gyrA and parC and conferred high-level resistance. This is one of the most common resistance mechanisms identified among clinical isolates of Enterobacter, despite recent characterization of plasmid-mediated quinolone resistance (PMQR) genes (qnrA, qnrS, aac(6′)-Ib-cr, qepA, and oqxAB;; ). In E. cloacae the plasmid-borne QnrA and QnrS resistances inducing protection from the DNA binding of fluoroquinolones are observed, but such mechanisms confers low-level resistance when present alone. However, such PMRQ mechanisms have got an efficient dissemination and are found in over 60% of the strains, because were found to be co-carried with various ESBLs or AmpC-type β-lactamases on the same plasmid. Finally, associated to active efflux, target mutations are the most efficient resistance mechanisms resulting in high MICs values, while PMRQ mechanisms confer only an additive effect on the level of fluoroquinolones resistance.

23Finally, pan-drug resistance is not an exceptional phenotype in E. aerogenes, since resistant strains to all antibiotics, including colistin pmrA substitution, were isolated and described to be associated with colistin resistance.

24Regarding the permeation pathway, it is important to mention that during the last decade, we observed the emergence of well-located mutation inside the pore constriction of the Omp36 (OmpC like porin of E. aerogenes) that generate a strong resistance against β-lactams. This specific mutation altering the pore characteristics impairs the diffusion of all β-lactams including cephalosporins and carbapenems, represents the first type of an adaptative mutation of bacterial porin in a resistant clinical isolates of Enterobacteriaceae. Interestingly, the intensity of MIC modification conferred by the specific residues depends on the structure and charge of the antibiotic molecules. A recent study reports the molecular simulations and dynamics of β-lactams inside the wild type and mutated channel during the travel of the molecule from outside to the periplasmic space. These data illustrates the adaptive pressure that has governed the selection and the preservation of these specific residues that filter the diffusion of charged solutes. The amino acids involved represent the first defense against the penetration of harmful compounds and support the pioneer investigations reporting the difference in β-lactam susceptibility depending on porin.

25Recently, in the context of IMI-Translocation consortium (www.translocation.eu), the genomes of various clinical isolates have been sequenced and the preliminary analyses have reported several mutations in resistant strains that are located in the regulators and membrane proteins (data not shown).

26Various studies on antibiotic resistance in E. aerogenes and E. cloacae have enlightened on a group of AraC family regulators including MarA, RamA, SoxS, and RobA, which are associated with a phenotype of low-level susceptibility to several antibiotics and biocides by inducing the overexpression of the efflux pump (for a review see;; ). Interestingly, the role of marA and ramA has been described also in the downregulation of porins and the subsequent resistance to β-lactams in E. aerogenes that completes the MDR phenotype of clinical resistant strains. Recent work has shown that the expression of another AraC- regulator, rarA, contribute to a multidrug-resistance phenotype, generated via the activation of efflux. This regulator also has a role in the development of tigecycline resistance. Thus, the regulation of MDR in Enterobacter is quite complex and redundant and contributes to the rapid adaptation of the clinical isolate via the porin and efflux balance. Moreover, it has been demonstrated that some two component system (TCS) regulators such as OmpR-EnvZ also play a key role in the control of porin expression in addition to OmpX and the small RNAi or proteins as H-NS that govern the OmpF/OmpC balance in E. coli or efflux pump elements synthesis in E. aerogenes, respectively.

27Interestingly, regarding the genetic control of the pump expression; activators MarA, RamA, and RarA, and repressors MarR, RamR, and AcrR, could be intimately associated at a global and local level to conjointly organize the resistance in clinical Enterobacter isolates. At this moment, it is also important to mention that the RamA regulator is described in Enterobacter, Salmonella, and Klebsiella, but not reported in Escherichia in contrast to the Mar regulon.

28An illustration of the sophisticated regulation of the various resistance mechanisms in Enterobacter is presented in Figure 3.

29During the last decade, we observed the rise and the fall of several infectious episodes due to resistant Escherichia, Enterobacter, Klebsiella strains in French and European hospitals. Regarding Enterobacter, we can note the successive waves of E. cloacae, followed by E. aerogenes and now again E. cloacae reported in hospital wards. This bacterial species is a member of the ESKAPE group recently described as the main contributor to the health human infection problem. Consequently, it is important to examine the various ways used by E. aerogenes and E. cloacae to detect and respond to the modification of environmental conditions and the presence of drugs in the medium.

30Interestingly, the two Enterobacter species, aerogenes and cloacae, present highly preserved regulation mechanisms acting to modulate the expression of porins integrated into the outer membrane: for example OmpX the small outer membrane protein, plays a role in controlling the production of the OmpF-like porin (Omp35) and OmpX overproduction is reported in clinical isolates showing a porin failure. This control of an outer membrane protein (OmpX) on the synthesis of the outer membrane porin, in addition to the major regulator of the resistance cascade that are fully active, are present in various clinical isolates. This suggests a common evolution path and the selection of a common regulation cascade involved in the membrane adaptation to environmental stresses. About the drug transporters, it is clear that AcrAB–TolC system, OqxAB, EmrE, MdfA, and MacA are present in the two species (see data bank for a complete description). In addition, regarding Mar, Ram, and Sox regulators all of them are preserved and active in the triggering of antibiotic resistance. Interestingly, the redundant global regulatory control, Mar and Ram, are reported in E. aerogenes and E. cloacae. The close species proximity is reinforced by the presence of similar regulators and adaptive response and support the description of these species in the human infection and their response face to antibiotic therapy.

31The 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.