REVIEW

published: 18 May 2015

doi: 10.3389/fmicb.2015.00392

Enterobacter aerogenes and

Enterobacter cloacae; versatile
bacterial pathogens confronting
antibiotic treatment
Anne Davin-Regli * and Jean-Marie Pagès
Transporteurs Membranaires, Chimiorésistance et Drug Design, Facultés de Médecine et Pharmacie, UMR-MD1, IRBA –
Aix-Marseille Université, Marseille, France

Enterobacter aerogenes and E. cloacae have been reported as important opportunistic

and multiresistant bacterial pathogens for humans during the last three decades in
hospital wards. These Gram-negative bacteria have been largely described during
Edited by:
several outbreaks of hospital-acquired infections in Europe and particularly in France.
Marta Martins, The dissemination of Enterobacter sp. is associated with the presence of redundant
University College Dublin, Ireland
regulatory cascades that efficiently control the membrane permeability ensuring the
Reviewed by:
bacterial protection and the expression of detoxifying enzymes involved in antibiotic
Etinosa Igbinosa,
University of Benin, Nigeria degradation/inactivation. In addition, these bacterial species are able to acquire
Alessandra Polissi, numerous genetic mobile elements that strongly contribute to antibiotic resistance.
Università degli Studi
di Milano-Bicocca, Italy
Moreover, this particular fitness help them to colonize several environments and
*Correspondence:
hosts and rapidly and efficiently adapt their metabolism and physiology to external
Anne Davin-Regli, conditions and environmental stresses. Enterobacter is a versatile bacterium able to
Transporteurs Membranaires,
promptly respond to the antibiotic treatment in the colonized patient. The balance of
Chimiorésistance et Drug Design,
Facultés de Médecine et Pharmacie, the prevalence, E. aerogenes versus E. cloacae, in the reported hospital infections
UMR-MD1, IRBA – Aix-Marseille during the last period, questions about the horizontal transmission of mobile elements
Université, 27 Boulevard Jean Moulin,
13385 Marseille Cedex 05, France
containing antibiotic resistance genes, e.g., the efficacy of the exchange of resistance
anne-veronique.regli@univ-amu.fr genes Klebsiella pneumoniae to Enterobacter sp. It is also important to mention the
possible role of antibiotic use in the treatment of bacterial infectious diseases in this E.
Specialty section:
This article was submitted to aerogenes/E. cloacae evolution.
Antimicrobials, Resistance
Keywords: Enterobacter aerogenes, Enterobacter cloacae, membrane and transporters, regulation, resistance
and Chemotherapy,
mechanisms
a section of the journal
Frontiers in Microbiology

Received: 11 March 2015 Introduction

Accepted: 16 April 2015
Published: 18 May 2015 Enterobacter is a genus of a common Gram-negative, facultative anaerobic, rod-shaped, non-
Citation: spore-forming bacteria belonging to the family Enterobacteriaceae. Two of its well- known species,
Davin-Regli A and Pagès J-M (2015) Enterobacter aerogenes and E. cloacae have taken on clinical significance as opportunistic bacteria
Enterobacter aerogenes and have emerged as nosocomial pathogens from intensive care patients pathogenic, especially to
and Enterobacter cloacae; versatile
those who are on mechanical ventilation (Mezzatesta et al., 2012).
bacterial pathogens confronting
antibiotic treatment.
Enterobacter aerogenes was originally named Aerobacter aerogenes, and was later included in
Front. Microbiol. 6:392. the genus Enterobacter in 1960. In 1971, this species was proposed to be renamed Klebsiella
doi: 10.3389/fmicb.2015.00392 mobilis due to its motility conferred by peritrichous flagella and its genetic relatedness to Klebsiella

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Davin-Regli and Pagès Enterobacter sp. and antibiotic treatment

genus. It is interesting to note that phenotypic dissimilarities et al., 1997; Jalaluddin et al., 1998). Until, 2003, E. aerogenes was
between E. aerogenes and the genus Klebsiella include not only the considered as an important emerging MDR pathogen, particu-
motility but also the presence of ornithine decarboxylase (ODC) larly in ICUs (Bosi et al., 1999; Chevalier et al., 2008; Figure 1).
activity and the lack of urease activity in E. aerogenes (Farmer The situation in 1990s in Europe pointed to the dispersion
et al., 1985). However, recently, the whole genome sequenc- of an epidemic clone and, since then, it has been extensively
ing of a multidrug-resistant (MDR) clinical isolate, (including detected in European hospitals and health care facilities. The
colistin) suggested a possible reclassification of the species in event fitted in with the international spread of the ESBL TEM-24
the genus Klebsiella, under the name K. aeromobilis (Diene (blaTEM−24 ) harbored by an epidemic plasmid (Bosi et al., 1999).
et al., 2013). E. aerogenes particular phenotype can be attributed The prevalence of Enterobacter sp. infections in clinical wards
to the horizontal acquisition of additional genes from other has also increased due to the introduction of extended-spectrum
Enterobacteriaceae species and mobile elements that rapidly inte- cephalosporins and carbapenems in the antibiotic therapy (Arpin
grated and translated as easily as its own ancestral heritage (Diene et al., 1996; Anastay et al., 2013). The consequence of this antibio-
et al., 2013). For example, the flagellar genes and its assembly sys- therapy is the emergence of “pan-drug E. aerogenes isolates”
tem have been acquired in bloc from the Serratia genus. Plasmid resistant to last-line antibiotics such as carbapenems and also to
conjugation is a chimera of transposons and genetic elements colistin, for which no therapeutic option was available (Chevalier
(conjugation, integration) of various bacterial origins. E. aero- et al., 1999; Thiolas et al., 2005; Diene et al., 2013). Interestingly,
genes also contains eight rRNA operons and 87 tRNA associated the role of efflux mechanism in E. aerogenes resistance has been
with the ability to translate imported genes that use different studied within an 8 years of period. This study indicated a
codons, improving its ability to use its integrated foreign genes. noticeable increase of the prevalence of an efflux mechanism, sus-
E. aerogenes has been involved in significant European outbreak ceptible to pump inhibitor, in clinical isolates collected during
between 1993 and 2003 and is considered as the paradigm of this period (Chevalier et al., 2008). After the emergence of ESBL
opportunistic bacteria. in E. aerogenes and the characterisation of porin mutations in
Species of the E. cloacae complex are widely encountered clinical isolates, this role of efflux mechanism highlights a new
in nature, but they are also pathogens: E. cloacae and E. step in the adaptative evolution in E. aerogenes (Charrel et al.,
hormaechei are most frequently isolated from human clini- 1996; Malléa et al., 1998; Gayet et al., 2003).
cal specimens. Thus, E. cloacae is among the most common Since 2010, E. aerogenes in France is the fifth highest
Enterobacter sp. causing only nosocomial infections in the Enterobacteriaceae and the seventh highest Gram-negative
last decade and a lot has been published on the antibiotic- Bacillus responsible for notorious nosocomial infections
resistance features of these microorganisms. Despite the rele- (Carbonne et al., 2013; Figure 2). Despite its intrinsic resistance
vance of E. cloacae as a nosocomial pathogen, the pathogenic to ampicillin and constant expression of ESBL that is associated
mechanisms and factors contributing in the disease associ- with other resistance mechanisms contributing to MDR pheno-
ated with the E. cloacae complex are not understood yet; this type, its prevalence has significantly dropped (reduction factor
could be due to the scarcity and the dispersion of informa- of 20) in France (Anastay et al., 2013; Jarlier and INVS, 2014).
tion available. Its ability to form biofilms and to secrete vari- Its position was displaced in the context of hospital acquired
ous cytotoxins (enterotoxins, hemolysins, pore-forming toxins) infections, because of the dramatic rise of the E. coli pandemic
are important for its pathogenicity (Mezzatesta et al., 2012). clone O25:H4-ST131 along with K. pneumoniae and E. cloacae,
Some genotypes and species, have previously exhibited some ESBL, and/or carbapenemase producing strains. Although, E.
associations with clinical specimens, in particular urines and
sputum, when clonal outbreaks with members of the E. cloa-
cae complex were rare (Izdebski et al., 2014). Interestingly,
due to the diffusion of most frequent extended spectrum
β-lactamases (ESBL) and carbapenemases in this species, E. cloa-
cae has now become the third broad spectrum Enterobacteriaceae
species involved in nosocomial infections after Escherichia coli
and K. pneumoniae (Potron et al., 2013; Jarlier and INVS,
2014).

Epidemiology and Infections

Enterobacter aerogenes is isolated as human clinical speci-
mens from respiratory, urinary, blood, or gastrointestinal tract
(Langley et al., 2001). Epidemiology of this species has been FIGURE 1 | Schematic illustration of the timing of Enterobacter
particular in Europe: it has regularly been involved in nosoco- species during the emergence of resistance outbreaks in France
hospitals (Arpin et al., 1996; Bornet et al., 2000; Chevalier et al., 2008;
mial infections outbreaks since 1993, particularly in the Western
Lavigne et al., 2012; Mezzatesta et al., 2012; Anastay et al., 2013;
Europe (Georghiou et al., 1995; Grattard et al., 1995; Allerberger Robert et al., 2014). ESBLs, extended-spectrum β-lactamases.
et al., 1996; Arpin et al., 1996; Davin-Regli et al., 1996; De Gheldre

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Davin-Regli and Pagès Enterobacter sp. and antibiotic treatment

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 ele-
ments. The AmpC plasmid-mediated resistance is distinguished
from chromosomal enzyme production because they are not
inducible. However they represent a problem due to its increas-
ing 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
FIGURE 2 | Distribution of the main species of of β-lactamases. Fourth-generation cephalosporins retain rea-
Enterobacteriaceae-ESBL (n for 10,000 patient days): evolution sonable activity against derepressed strains, but if strains are
2002–2013 from the French national coordination of MDRB
also ESBL producers, they become resistant to this antibiotic
surveillance (Carbonne et al., 2013; Jarlier and INVS, 2014). ENT AER,
Enterobacter aerogenes; ENT CLO, E. cloacae; ESC COL, E. coli; KLE PNE, class. The prevalence of ESBL and CTX-M producers repre-
Klebsiella pneumoniae; TOT ENB, Total Enterobacteriaceae; ESBLs, sented approximatively 5% of the isolates in the recent studies
extended-spectrum β-lactamases. and ESBLs are most often plasmid-mediated. These characteris-
tics, associated with the frequent endogenous intestinal carriage
of E. cloacae, may result in abnormally high levels in the bow-
aerogenes causes septic shock more readily in patients thus els of hospitalized patients, especially those who have received
leading to a higher mortality rate (Song et al., 2010; Lavigne cephalosporins (Potron et al., 2013).
et al., 2012), 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 Enzymatic Barrier and Antibiotic
prevalent resistant genes and expressing new β-lactamases or Resistance
carbapenemases (Figure 2).
Enterobacter cloacae is ubiquitous in terrestrial and aquatic The production of β-lactamases is the prominent mechanism
environments (water, sewage, soil, and food). The species occurs responsible for β-lactam resistance in most of these species. E.
as commensal microflora in the intestinal tracts of humans aerogenes strains have a broad ability to develop antibiotics resis-
and animals and is also pathogens in plants and insects. tance mechanisms (Miro et al., 1995). They naturally express a
This diversity of habitats is mirrored by the genetic vari- chromosomal AmpC β-lactamase type cephalosporinase at low
ety of E. cloacae (Mezzatesta et al., 2012). Recently, MLST level (group 1 Bush) that induces resistance to first-generation
and PFGE epidemiological methods data revealed world cir- cephalosporins (Freney et al., 1988). Chromosomal acquired
culation of several epidemic clonal complexes (Izdebski et al., β-lactams resistance mechanisms induce the overproduction
2014). of chromosomal AmpC cephalosporinase: this results from an
It is also a well-known nosocomial pathogen contributing induction during a third-generation cephalosporin treatment or
to bacteremia, endocarditis, septic arthritis, osteomyelitis, and by a mutation in the AmpR repressor, and generates a resistance
skin/soft tissue infections, and lower respiratory tract- urinary to almost all β-lactams (Preston et al., 2000). Moreover, it has
tract and intra-abdominal infections (Fata et al., 1996). E. cloa- been described that E. aerogenes strains harboring cephalospori-
cae tends to contaminate various medical, intravenous, and other nase AmpC gene, integrated the gene of chromosomal origin
hospital devices (Dugleux et al., 1991). Nosocomial outbreaks (blaCMY-10) on a large plasmid (130 kb), contributing to a
have also been associated with the colonization of certain surgical systematic gene transmission even in the absence of antibiotic
equipment and operative cleaning solutions (Wang et al., 2000). pressure (Lee et al., 2003).
Since a decade, E. cloacae has been repeatedly reported as a noso- In 1993 appeared the first cases of nosocomial infections
comial pathogen in neonatal units and several outbreaks of infec- caused by strains with resistance to common β-lactam antibi-
tion have been reported (Fernandez-Baca et al., 2001; Pestourie otics due to ESBL (Pitout et al., 1998). The ESBL TEM- 24
et al., 2014). Today, variability among strains are less frequent associated to E. aerogenes clonal dissemination in France was
and outbreaks due to clonal E. cloacae hyper-producing AmpC constantly reported (Neuwirth et al., 1996; Bosi et al., 1999;
β-lactamase and ESBL carrier isolates are described from neonate Bertrand et al., 2003). Other ESBLs of TEM type or CTX-M
specimens, adults urines/feces samples or from environmental type (ex CTX-M-2) are often identified but TEM-24 remains
samples (Pestourie et al., 2014). associated with preferential conjugative plasmid of this species
Enterobacter cloacae has an intrinsic resistance to ampi- (Arpin et al., 2002; Dumarche et al., 2002; Biendo et al., 2008;
cillin, amoxicillin, first-generation cephalosporins, and cefoxitin Kanamori et al., 2012). Due to the well-described modification
owing to the production of constitutive AmpC β-lactamase. of porins expression and recent dissemination of plasmid bearing
It exhibits a high frequency of enzymatic resistance to broad- carbapenemases, a number of imipenem-resistant clinical strains
spectrum cephalosporins. Resistance of Enterobacter sp. to have come up (Miro et al., 1995; Bornet et al., 2000; Biendo

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Davin-Regli and Pagès Enterobacter sp. and antibiotic treatment

et al., 2008; Lavigne et al., 2012). Carbapenemases of NDM and hospital acquired E. cloacae isolates might arise via stepwise
VIM types are now, as anticipated, reported in E. aerogenes in accumulations of MDR determinants in different clones. Today,
India and those for the serine protease group as KPC or class E. cloacae is the second Enterobacteriaceae carrying carbapen-
D β-lactamases possessing carbapenemase properties as OXA-48 emase and strains co-expressing two carbapenemases has been
types are described in Europe/Asia (Khajuria et al., 2014; Torres reported (Izdebski et al., 2014).
et al., 2014). Regarding the aminoglycosides, the major mechanism of
Similarly to E. aerogenes, E. cloacae is also naturally resistant to resistance of the Enterobacteriaceae is due to aminoglycoside-
ampicillin, amoxicillin–clavulanic acid, cephalothin, and cefox- modifying enzymes that are often plasmid-encoded, but it may
itin by low production of the natural inducible cephalosporinase also be associated with transposable elements. These enzymes
of Bush group 1 (class C). They are capable of overproduc- are assigned to three groups: acetyltransferases (acetylation of
ing AmpC β-lactamases by blocking the repression of a chro- an amino group/AAC), phosphotransferases (phosphorylation
mosomal gene or by the acquisition of a transferable ampC of a hydroxyl group/APH), and adenylyltransferases (adenylyla-
gene on plasmids conferring the resistance to third-generation tion of a hydroxyl group/AAD or ANT). Plasmid exchanges and
cephalosporins (Nauciel et al., 1985; Zaher and Cimolai, 1997). disseminations of transposons facilitate the rapid acquisition of
Cefepime alone can keep its activity (Sanders and Sanders, 1997). resistance phenotypes (Mezzatesta et al., 2012).
Clinical AmpC resistance represents 50% of the isolates and fre- The resistant strain varies from 0 to 51% resistance for gen-
quently co- exists with the expression of ESBL. In 1989, appeared tamicin, and 0 to 34% for amikacin (Sanders and Sanders, 1997).
the first nosocomial isolate cases bearing plasmidic ESBL caus- In 2013, an important epidemiological study confirmed that the
ing also resistance to third generation cephalosporins except aminoglycoside-modifying genes involved in aminoglycoside-
cefamycins (De Champs et al., 1989). Together, these enzymes clinical resistance were aac(3)-IIa, aac(6 )-Ib, and ant(2 )-Ia,
are responsible for a global resistance to all β-lactams except genes that confer resistance to tobramycin, gentamicin, and
carbapenems (Pitout et al., 1997). In the last decade, E. cloa- amikacin (Miró et al., 2013). Strains have frequently more than
cae has emerged as the third most common Enterobacteriaceae one enzyme (Miró et al., 2013). This enzymatic type resistance
resistant to third generation cephalosporins with enteric E. coli is associated in 77% of clinical isolates in China to other plas-
and K. pneumoniae (Jarlier and INVS, 2014). Imipenem remains mid genes (armA, rmtB; Huang et al., 2012). Among these,
the most effective molecule for treating E. cloacae infections. the aminoglycoside AAC(6 )-Ib is the most common cause of
Since then, various ESBL of TEM, SHV, and CTX -M types amikacin resistance among members of the Enterobacteriaceae
have been characterized in E. cloacae including resistant TEM family. In a previous study, it was observed that over 40% of the
inhibitors or IRT (for inhibitor-resistant TEM; Arpin et al., 2002; E. cloacae isolates had the aac(6 )-Ib gene, although many of the
Szabo et al., 2005; Galas et al., 2008). However, among ESBL isolates with this gene were susceptible to amikacin and gentam-
producers, some sub-clones are now identified, associated with icin, which were the most active of all tested drugs (Kim et al.,
CTX-M-3 and 15 production, when other TEM or SHV (SHV-12 2009).
for example) types are also associated with epidemic-episodes- The enzymatic resistance to fluoroquinolones has been
involved isolates. Diffusion of E. cloacae producing CTX-M-15 recently described and attributed to a two-point mutation allele
ESBL is the consequence of the wide dissemination of identical of aac(6 )-Ib [named aac(6 )-Ib-cr], the aminoglycosides resis-
or related plasmids harboring the CTX-M-15 gene firstly identi- tance enzymatic determinant, which acetylates ciprofloxacin and
fied in the epidemic E. coli clone, and the CTX-M β-lactamases norfloxacin (Huang et al., 2012). A systematic molecular survey
are now the most prevalent ESBL globally (Hammami et al., reporting prevalence and characteristics of aac(6 )-Ib-cr in Korea,
2012). characterized a high prevalence of the mechanism (23%) in E.
In recent years, clinical isolates resistant by producing car- cloacae (Huang et al., 2012). Dissemination of this new enzymatic
bapenemases have been identified (Nordmann et al., 1993; Galani resistance mechanism occurs since the aac(6 )-Ib-cr is highly
et al., 2005). In 2010, CDC first reported carriage of NDM-1 associated with blaOXA−1 , ISCR1, and class 1 integron. This
in E. cloacae from patients who received medical care in India. supports the previous finding where aac(6 )-Ib-cr was located
Especially in Asia, strains harboring metallo-β-lactamases as IMP upstream of blaOXA−30 (synonymously called blaOXA−1 ) in com-
-type enzymes, NDM, GIM, VIM, and serine carbapenemase plex class 1 integron, In37 containing ISCR1 (Quiroga et al.,
type KPC have been described (Huang et al., 2012; Dai et al., 2007). A genetic linkage between aac(6 )-Ib-cr and blaCTX−M−15
2013; Hamprecht et al., 2013; Jaskulski et al., 2013). The OXA- has been demonstrated (Huang et al., 2012).
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 Membrane Barrier and Antibiotic
associated resistance (Potron et al., 2013; Torres et al., 2014). Resistance
A hike in the imipenem resistance rate in E. cloacae, from
0.4 to 8 %, has been observed (Lee et al., 2005; Poirel et al., Porin and Membrane Permeability
2007; Robert et al., 2014). An epidemic study concerning E. Carbapenems are the most powerful agents for the treat-
cloacae blood stream infections indicated a 25% production of ment of serious nosocomial infections caused by MDR
metallo-β-lactamase in corresponding strains (Khajuria et al., Enterobacteriaceae. Due to the imipenem use, it was rapidly
2014). Thus, the decreased susceptibility to carbapenems in reported a decreased penetration of β-lactams due to a change

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Davin-Regli and Pagès Enterobacter sp. and antibiotic treatment

in the expression of porins in E. aerogenes isolates. Charrel et al. 2002; Masi et al., 2005, 2006; Martins et al., 2010). Several studies
(1996) showed that MDR strains of E. aerogenes exhibited a on E. cloacae have also reported the presence of efflux pumps
characteristic phenotype associated with an altered expression belonging to RND and MATE families (Pérez et al., 2007; He
of porins and then successive studies comforted description of et al., 2011). In addition, the AcrAB–TolC and OqxAB genes
more frequent MDR strains in treated patients by β-lactams have been characterized in E. cloacae clinical resistant isolates
(Bornet et al., 2000; Fernandez-Cuenca et al., 2006). This mech- (Pérez et al., 2007, 2012; Veleba et al., 2013). In E. aerogenes and
anism of resistance is reversible upon discontinuation of treat- E. cloacae, the sequence similarities and biological activity are
ment (Bornet et al., 2000) and progressive during treatment. particularly high in AcrAB–TolC (Pradel and Pagès, 2002; Pérez
Among intermediate strains which are susceptible to imipenem et al., 2007). Moreover, various chemicals such as salicylate, chlo-
but resistant to ertapenem, there is a loss of porin Omp35 ramphenicol, and imipenem are also able to trigger the genetic
but the expression of porin Omp36 is preserved. When treat- cascade controlling the expression of Enterobacter AcrAB–TolC
ment with imipenem continues, the disappearance of two porins pump (Davin-Regli et al., 2008). The regulation seems to be asso-
and resistance to all carbapenems is noted (Lavigne et al., ciated with the internal concentration of chemicals that plays a
2013). Recently, a novel mechanism of resistance has been key role during the switch on of the cascade that provides the
observed in a clinical strain where the antibiotic cannot be effi- efflux expression (Valade et al., 2013).
ciently translocated through a mutated porin (see Mutation and MarA acts as a key regulator for the expression of porin
Antibiotic Resistance). Additionally, imipenem and carbapene- genes and tolC in Enterobacteriaceae (Levy, 2002; Piddock, 2006;
mase KPC type have been described as responsible for resistance Alekshun and Levy, 2007; Davin-Regli et al., 2008). SoxS is
to carbapenems associated to decrease in membrane permeabil- another key transcriptional regulator that is positively controlled
ity (Jaskulski et al., 2013). The conductance and selectivity of by oxidative stress and can trigger the MarA expression (Masi and
these porins, Omp35, and Omp36, correspond to the proper- Pagès, 2013). Some Enterobacteriaceae sp., such as Enterobacter,
ties obtained with OmpC and OmpF of E. coli (Bornet et al., Klebsiella, Salmonella, have an additional global regulator, RamA.
2004; James et al., 2009). Moreover, several studies have further It plays a strategic role in controlling both the porins and
described a decrease in production of these porins in resistant the efflux expression, either directly or via the MarA cascade.
isolates (Bornet et al., 2000; Yigit et al., 2002; Gayet et al., 2003; This coordinated control of influx and efflux directly and effi-
Doumith et al., 2009; Tran et al., 2009). Two major outer mem- ciently governs the intracellular accumulation of antibacterial
brane porins have been identified in E. cloacae and studied by agents. Importantly, this internal accumulation of antibacterial
liposome swelling assays (Lee et al., 1992). These porins are molecules below the threshold corresponding to the MIC can
involved in the carbapenem susceptibility (Raimondi et al., 1991; favor the emergence and acquisition of additional mechanisms
Lee et al., 1992) and exhibit important cross antigenicity with of resistance such as target mutation, production of detoxify-
the E. aerogenes porins in specific key parts, e.g., eyelet region, ing enzymes (e.g., β-lactamases, acetyltransferase, etc), and con-
membrane insertions, subunit connections (Malléa et al., 1995). tributing to the extension of MDR phenotype (Nikaido and Pagès,
In addition, their respective involvement in β-lactam and fluoro- 2012; Masi and Pagès, 2013).
quinolone uptake has been reported (Chevalier et al., 2000; James Regarding the active structure of efflux pumps involved in
et al., 2009). E. aerogenes and E. cloacae; we can hypothesize that a com-
Interestingly, the expression of porin in Enterobacteriaceae is mon structural organization is conserved, due to the high
rapidly and notably altered by various stress compounds present conserved homology between Enterobacter and E. coli. This
in the external medium (Dupont et al., 2007). During the first structural organization can be similar to the recent descrip-
hours of incubation in the presence of salicylate, novobiocin, nor- tion of the AcrAB–TolC complex in E. coli (Du et al.,
floxacin a significant increase of OmpX is observed and this over- 2014).
expression negatively controls the synthesis of porins (Dupont
et al., 2007).
Mutations and Antibiotic Resistance
Efflux and Membrane Permeability
Furthermore, an efflux mechanism that is involved in the Regarding the β-lactam antibiotics, the resistance due to tar-
expelling of molecules from the bacteria such as fluoro- get mutation occurs incidentally in Enterobacter sp. However,
quinolones, tetracycline, and chloramphenicol is active in the diverse β-lactamases identified today is the result of a series
Enterobacter sp. (Malléa et al., 1998). This mechanism is highly of mutations that have successively appeared in the original
efficient since the AcrAB–TolC efflux pump can eject about β-lactamases TEM-1/2, SHV-1, OXA-1.
80–90% of the norfloxacin during the first 10–15 min (Malléa Furthermore, strains in which AmpC cephalosporinase was
et al., 1998). Interestingly, this process is energy-dependent and derepressed have been affected by mutations affecting AmpR-
requires the membrane energy (proton motive force) as exten- promoter recognition site. Mutations that are best known and
sively described (for a recent review see Nikaido and Pagès, 2012). studied are those that affect the target of fluoroquinolones
Approximately 40% of MDR clinical strains have an active efflux and more recently those responsible for polymyxin resistance.
(Chevalier et al., 2008). The EefABC and AcrAB–TolC efflux As a matter of fact, the quinolones were widely prescribed
genes of E. aerogenes have been described and their involvement antimicrobial agents because of their proven safety, high oral
in antibiotic exportation has been studied (Pradel and Pagès, bioavailability, multiple approved indications, and bactericidal

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Davin-Regli and Pagès Enterobacter sp. and antibiotic treatment

activity. Consequently, in the microbial population, a variety of Regulation of Membrane-Associated

amino acid alterations arose from mutations within quinolone Mechanisms of Resistance
resistance-determining regions (QRDRs) of cellular target genes
gyrA and parC and conferred high-level resistance. This is one Various studies on antibiotic resistance in E. aerogenes and E.
of the most common resistance mechanisms identified among cloacae have enlightened on a group of AraC family regulators
clinical isolates of Enterobacter, despite recent characteriza- including MarA, RamA, SoxS, and RobA, which are associated
tion of plasmid-mediated quinolone resistance (PMQR) genes with a phenotype of low-level susceptibility to several antibi-
(qnrA, qnrS, aac(6 )-Ib-cr, qepA, and oqxAB; Park et al., 2009; otics and biocides by inducing the overexpression of the efflux
Kanamori et al., 2012). In E. cloacae the plasmid-borne QnrA pump (for a review see Davin-Regli et al., 2008; Davin-Regli
and QnrS resistances inducing protection from the DNA bind- and Pagès, 2012; Pérez et al., 2012). Interestingly, the role of
ing of fluoroquinolones are observed, but such mechanisms marA and ramA has been described also in the downregulation
confers low-level resistance when present alone (Corkill et al., of porins and the subsequent resistance to β-lactams in E. aero-
2005; Poirel et al., 2005; Huang et al., 2012; Kanamori et al., genes that completes the MDR phenotype of clinical resistant
2012). However, such PMRQ mechanisms have got an effi- strains (Chollet et al., 2002, 2004). Recent work has shown that
cient dissemination and are found in over 60% of the strains, the expression of another AraC- regulator, rarA, contribute to
because were found to be co-carried with various ESBLs or a multidrug-resistance phenotype, generated via the activation
AmpC-type β-lactamases on the same plasmid (Park et al., of efflux (Veleba et al., 2013). This regulator also has a role in
2009; Huang et al., 2012). Finally, associated to active efflux, the development of tigecycline resistance (Veleba et al., 2013).
target mutations are the most efficient resistance mechanisms Thus, the regulation of MDR in Enterobacter is quite complex
resulting in high MICs values, while PMRQ mechanisms confer and redundant (Davin-Regli et al., 2008; Lawler et al., 2013) and
only an additive effect on the level of fluoroquinolones resis- contributes to the rapid adaptation of the clinical isolate via the
tance. porin and efflux balance (Bornet et al., 2000, 2004). Moreover, it
Finally, pan-drug resistance is not an exceptional phenotype has been demonstrated that some two component system (TCS)
in E. aerogenes, since resistant strains to all antibiotics, includ- regulators such as OmpR-EnvZ also play a key role in the control
ing colistin pmrA substitution, were isolated and described to of porin expression in addition to OmpX and the small RNAi or
be associated with colistin resistance (Thiolas et al., 2005; Diene proteins as H-NS that govern the OmpF/OmpC balance in E. coli
et al., 2013). or efflux pump elements synthesis in E. aerogenes, respectively
Regarding the permeation pathway, it is important to men- (Stoorvogel et al., 1991; Masi et al., 2005; Dupont et al., 2007).
tion that during the last decade, we observed the emergence Interestingly, regarding the genetic control of the pump
of well-located mutation inside the pore constriction of the expression; activators MarA, RamA, and RarA, and repressors
Omp36 (OmpC like porin of E. aerogenes) that generate a strong
resistance against β-lactams (Dé et al., 2001; Thiolas et al.,
2004). 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 (Chevalier et al., 1999; Thiolas et al., 2004).
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 molec-
ular 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 (Vidal et al., 2005;
James et al., 2009; Hajjar et al., 2010a,b). These data illus-
trates the adaptive pressure that has governed the selection and
the preservation of these specific residues that filter the dif-
fusion of charged solutes. The amino acids involved represent FIGURE 3 | Schematic illustration of resistance mechanism and their
the first defense against the penetration of harmful compounds regulations. Different levels of regulation are presented: (i) Transcriptional and
and support the pioneer investigations reporting the difference translational levels controlled by various sensors [two components systems
in β-lactam susceptibility depending on porin (Pagès et al., (TCS), such as EnvZ/OmpR for porins], global regulators (RamA, MarA, SoxS
are indicated here), and local regulators (AcrR for efflux pumps and OmpX for
2008).
porins), the accumulation of trigger metabolites inside the bacterial cell can
Recently, in the context of IMI-Translocation consor- also trigger the expression via local or other regulators, MicF and MicC
tium (www.translocation.eu), the genomes of various clini- represent the small interfering RNA controlling porin mRNA stability.
cal isolates have been sequenced and the preliminary analyses (ii) Translational and final membrane assembly in a functional conformation (via
have reported several mutations in resistant strains that are chaperones and membrane factors. Porins represent Omp35, Omp36; Efflux
pumps represent the AcrAB–TolC family. IN, bacterial cytoplasm; OUT,
located in the regulators and membrane proteins (data not
external medium.
shown).

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Davin-Regli and Pagès Enterobacter sp. and antibiotic treatment

MarR, RamR, and AcrR, could be intimately associated at a global et al., 2007). This control of an outer membrane protein (OmpX)
and local level to conjointly organize the resistance in clinical on the synthesis of the outer membrane porin, in addition to
Enterobacter isolates (Davin-Regli et al., 2008). At this moment, it the major regulator of the resistance cascade that are fully active,
is also important to mention that the RamA regulator is described are present in various clinical isolates. This suggests a common
in Enterobacter, Salmonella, and Klebsiella, but not reported in evolution path and the selection of a common regulation cas-
Escherichia in contrast to the Mar regulon (Lawler et al., 2013). cade involved in the membrane adaptation to environmental
An illustration of the sophisticated regulation of the various stresses (Gayet et al., 2003). About the drug transporters, it is clear
resistance mechanisms in Enterobacter is presented in Figure 3. that AcrAB–TolC system, OqxAB, EmrE, MdfA, and MacA are
present in the two species (see data bank for a complete descrip-
tion). In addition, regarding Mar, Ram, and Sox regulators all of
Conclusion them are preserved and active in the triggering of antibiotic resis-
tance. Interestingly, the redundant global regulatory control, Mar
During the last decade, we observed the rise and the fall of sev- and Ram, are reported in E. aerogenes and E. cloacae (Veleba et al.,
eral infectious episodes due to resistant Escherichia, Enterobacter, 2013). The close species proximity is reinforced by the presence of
Klebsiella strains in French and European hospitals. Regarding similar regulators and adaptive response and support the descrip-
Enterobacter, we can note the successive waves of E. cloacae, tion of these species in the human infection and their response
followed by E. aerogenes and now again E. cloacae reported in face to antibiotic therapy.
hospital wards (Potron et al., 2013). This bacterial species is a
member of the ESKAPE group recently described as the main
contributor to the health human infection problem (Boucher Acknowledgments
et al., 2009; Rice, 2010). Consequently, it is important to exam-
ine the various ways used by E. aerogenes and E. cloacae to detect We greatly appreciate the S. Dam s’ help for his care-
and respond to the modification of environmental conditions and fully reading of the manuscript. This work was supported by
the presence of drugs in the medium. Aix-Marseille Université. The research leading to the results
Interestingly, the two Enterobacter species, aerogenes and cloa- discussed here was conduced as part of the translocation
cae, present highly preserved regulation mechanisms acting to consortium (www.translocation.eu) and has received support
modulate the expression of porins integrated into the outer mem- from the Innovative Medicines joint Undertaking under Grant
brane: for example OmpX the small outer membrane protein, Agreement no. 115525, resources which are composed of finan-
plays a role in controlling the production of the OmpF-like porin cial contribution from the European Union’s seventh frame-
(Omp35) and OmpX overproduction is reported in clinical iso- work program (FP/2007–2013) and EFPIA companies in kind
lates showing a porin failure (Stoorvogel et al., 1991; Dupont contributions.

References Bornet, C., Davin-Regli, A., Bosi, C., Pagès, J. M., and Bollet, C. (2000). Imipenem
resistance of Enterobacter aerogenes mediated by outer membrane permeability.
Alekshun, M. N., and Levy, S. B. (2007). Molecular mechanisms of antibacterial J. Clin. Microbiol. 38, 1048–1052.
multidrug resistance. Cell 128, 1037–1050. doi: 10.1016/j.cell.2007.03.004 Bornet, C., Saint, N., Fetnaci, L., Dupont, M., Davin-Régli, A., Bollet, C., et al.
Allerberger, F., Koeuth, T., Lass-Florl, C., Dierich, M. P., Putensen, C., (2004). Omp35, a new Enterobacter aerogenes porin involved in selective sus-
Schmutzhard, E., et al. (1996). Epidemiology of infections due to multiresistant ceptibility to cephalosporins. Antimicrob. Agents Chemother. 48, 2153–2158.
Enterobacter aerogenes in an university hospital. Eur. J. Clin. Microbiol. Infect. doi: 10.1128/AAC.48.6.2153-2158.2004
Dis. 15, 517–521. doi: 10.1007/BF01691323 Bosi, C., Davin-Regli, A., Bornet, C., Malléa, M., Pagès, J. M., and Bollet, C. (1999).
Anastay, M., Lagier, E., Blanc, V., and Chardon, H. (2013). Epidémiologie Most Enterobacter aerogenes strains in France belong to a prevalent clone.
des bêtalactamases à spectre étendu (BLSE) chez les entérobactéries dans J. Clin. Microbiol. 37, 2165–2169.
un hôpital du sud de la France, 1997-2007. Pathol. Biol. 61, 38–43. doi: Boucher, H. W., Talbot, G. H., Bradley, J. S., Edwards, J. E., Gilbert, D., Rice, L. B.,
10.1016/j.patbio.2012.03.001 et al. (2009). Bad bugs, no drugs: no ESKAPE! an update from the Infectious
Arpin, C., Coze, C., Rogues, A. M., Gachie, J. P., Bebear, C., and Quentin, C. (1996). Diseases Society of America. Clin. Infect. Dis. 48, 1–12. doi: 10.1086/595011
Epidemiological study of an outbreak due to multidrug-resistant Enterobacter Carbonne, A., Arnaud, I., Maugat, S., Marty, N., Dumartin, C., Bertrand, X., et al.
aerogenes in a medical intensive care unit. J. Clin. Microbiol. 34, 2163–2169. (2013). National multidrug-resistant bacteria (MDRB) surveillance in France
Arpin, C., Labia, R., Dubois, V., Noury, P., Souquet, M., and Quentin, C. through the RAISIN network: a 9 year experience. J. Antimicrob. Chemother.
(2002). TEM-80, a novel inhibitor-resistant β-lactamase in a clinical isolate 68, 954–959. doi: 10.1093/jac/dks464
of Enterobacter cloacae. Antimicrob. Agents Chemother. 46, 1183–1189. doi: Charrel, R. N., Pagès, J. M., De Micco, P., and Malléa, M. (1996). Prevalence of
10.1128/AAC.46.5.1183-1189.2002 outer membrane porin alteration in β-lactam-antibiotic-resistant Enterobacter
Bertrand, X., Hocquet, D., Boisson, K., Siebor, E., Plésiat, P., and Talon, D. (2003). aerogenes. Antimicrob. Agents Chemother. 40, 2854–2858.
Molecular epidemiology of Enterobacteriaceae producing extended-spectrum Chevalier, J., Malléa, M., and Pagès, J. M. (2000). Comparative aspects of the dif-
β-lactamase in a French university-affiliated hospital. Int. J. Antimicrob. Agents fusion of norfloxacin, cefepime and spermine through the F porin channel
22, 128–133. doi: 10.1016/S0924-8579(03)00098-0 of Enterobacter cloacae. Biochem. J. 348(Pt 1), 223–227. doi: 10.1042/0264-
Biendo, M., Canarelli, B., Thomas, D., Rousseau, F., Hamdad, F., Adjide, C., 6021:3480223
et al. (2008). Successive emergence of extended-spectrum- β-lactamase- Chevalier, J., Mulfinger, C., Garnotel, E., Nicolas, P., Davin-Régli, A., and Pagès,
producing and carbapenemase-producing Enterobacter aerogenes isolates in J. M. (2008). Identification and evolution of drug efflux pump in clinical
a university hospital. J. Clin. Microbiol. 46, 1037–1044. doi: 10.1128/JCM. Enterobacter aerogenes strains isolated in 1995 and 2003. PLoS ONE 3:e3203.
00197-07 doi: 10.1371/journal.pone.0003203

Frontiers in Microbiology | www.frontiersin.org 7 May 2015 | Volume 6 | Article 392

Davin-Regli and Pagès Enterobacter sp. and antibiotic treatment

Chevalier, J., Pagès, J. M., and Malléa, M. (1999). In vivo modification of porin Fata, F., Chittivelu, S., Tessler, S., and Kupper, Y. (1996). Gas gangrene of the
activity conferring antibiotic resistance to Enterobacter aerogenes. Biochem. arm due to Enterobacter cloacae in a neutropenic patient. South. Med. J. 89,
Biophys. Res. Commun. 266, 248–251. doi: 10.1006/bbrc.1999.1795 1095–1096. doi: 10.1097/00007611-199611000-00014
Chollet, R., Bollet, C., Chevalier, J., Malléa, M., Pagès, J. M., and Davin-Regli, A. Fernandez-Baca, V., Ballesteros, F., Hervas, J. A.,Villalon, P., and Alberti, S. (2001).
(2002). mar operon involved in multidrug resistance of Enterobacter aerogenes. Molecular epidemiological typing of Enterobacter cloacae isolates from a neona-
Antimicrob. Agents Chemother. 46, 1093–1097. doi: 10.1128/AAC.46.4.1093- tal intensive care unit: three-year prospective study. J. Hosp. Infect. 49, 173–182.
1097.2002 doi: 10.1053/jhin.2001.1053
Chollet, R., Chevalier, J., Bollet, C., Pagès, J. M., and Davin-Regli, A. (2004). Fernandez-Cuenca, F., Rodriguez-Martinez, J. M., Martinez-Martinez, J. M.
RamA is an alternate activator of the multidrug resistance cascade in and Pascual, J. M. (2006). In vivo selection of Enterobacter aerogenes
Enterobacter aerogenes. Antimicrob. Agents Chemother. 48, 2518–2523. doi: with reduced susceptibility to cefepime and carbapenems associated with
10.1128/AAC.48.7.2518-2523.2004 decreased expression of a 40 kDa outer membrane protein and hyperpro-
Corkill, J. E., Anson, J. J., and Hart, C. A. (2005). High prevalence of the plasmid- duction of AmpC β-lactamase. Int. J. Antimicrob. Agents 27, 549–552. doi:
mediated quinolone resistance determinant qnrA in multidrug-resistant 10.1016/j.ijantimicag.2006.01.005
Enterobacteriaceae from the blood cultures in Liverpool, UK. J. Antimicrob. Freney, J., Husson, M. O., Gavini, F., Madier, S., Martra, A., Izard, D., et al.
Chemother. 56, 1115–1117. doi: 10.1093/jac/dki388 (1988). Susceptibility to antibiotics and antiseptics of new species of the
Dai, W., Sun, S., Yang, P., Huang, S., Zhang, X., and Zhang, L. (2013). family Enterobacteriaceae. Antimicrob. Agents Chemother. 32, 873–876. doi:
Characterization of carbapenemases, extended spectrum β-lactamases and 10.1128/AAC.32.6.873
molecular epidemiology of carbapenem-non-susceptible Enterobacter cloa- Galani, I., Souli, M., Chryssouli, Z., Orlandou, K., and Giamarellou, H. (2005).
cae in a Chinese hospital in ChongqinG. Infect. Genet. Evol. 14, 1–7. doi: Characterization of a new integron containing, bla (VIM-1) and aac(6’)-IIc in
10.1016/j.meegid.2012.10.010 an Enterobacter cloacae clinical isolate from Greece. J. Antimicrob. Chemother.
Davin-Regli, A., Bolla, J. M., James, C. E., Lavigne, J. P., Chevalier, J., Garnotel, E., 55, 634–638. doi: 10.1093/jac/dki073
et al. (2008). Membrane permeability and regulation of drug “influx and Galas, M., Decousser, J.-W., Breton, N., Godard, T., Allouch, T., Pina, P.,
efflux” in enterobacterial pathogens. Curr. Drug Targets 9, 750–759. doi: et al. (2008). Nationwide study of the prevalence, characteristics, and
10.2174/138945008785747824 molecular epidemiology of extended-spectrum-β-lactamase-producing
Davin-Regli, A., Monnet, D., Saux, P., Bosi, C., Charrel, R. N., Barthelemy, A., et al. Enterobacteriaceae in France. Antimicrob. Agents Chemother. 52, 786–789. doi:
(1996). Molecular epidemiology of Enterobacter aerogenes acquisition: one- 10.1128/AAC.00906-07
year prospective study in two intensive care units. J. Clin. Microbiol. 34, 1474– Gayet, S., Chollet, R., Molle, G., Pagès, J. M., and Chevalier, J. (2003). Modification
1480. of outer membrane protein profile and evidence suggesting an active drug
Davin-Regli, A., and Pagès, J. M. (2012). Cross-resistance between biocides and pump in Enterobacter aerogenes clinical strains. Antimicrob. Agents Chemother.
antimicrobials: an emerging question. Rev. Sci. Tech. 31, 89–104. 47, 1555–1559. doi: 10.1128/AAC.47.5.1555-1559.2003
Dé, E., Baslé, A., Jaquinod, M., Saint, N., Malléa, M., Molle, G., et al. (2001). Georghiou, P. R., Hamill, R. J., Wright, C. E., Versalovic, J., Koeuth, T. T., and
A new mechanism of antibiotic resistance in Enterobacteriaceae induced by a Lupski, J. R. (1995). Molecular epidemiology of infections due to Enterobacter
structural modification of the major porin. Mol. Microbiol. 41, 189–198. doi: aerogenes: identification of hospital outbreak-associated strains by molecular
10.1046/j.1365-2958.2001.02501.x techniques. Clin. Infect. Dis. 20, 84–94. doi: 10.1093/clinids/20.1.84
De Champs, C., Sauvant, M. P., Chanal, C., Sirot, D., Gazuy, N., Malhuret, R., et al. Grattard, F., Pozzetto, B., Tabard, L., Petit, M., Ros, A., and Gaudin, O. G. (1995).
(1989). Prospective survey of colonization and infection caused by expanded Characterization of nosocomial strains of Enterobacter aerogenes by arbitrar-
spectrum-beta-lactamase-producing members of the family Enterobacteriaceae ily primed PCR analysis and ribotyping. Infect. Control Hosp. Epidemiol. 16,
in an intensive care unit. J. Antimicrob. Chemother. 27, 2887–2890. 224–230. doi: 10.2307/30140982
De Gheldre, Y., Maes, N., Rost, F., De Ryck, R., Clevenbergh, P., Vincent, J. L., Hajjar, E., Bessonov, A., Molitor, A., Kumar, A., Mahendran, K. R.,
et al. (1997). Molecular epidemiology of an outbreak of multidrug-resistant Winterhalter, M., et al. (2010a). Toward screening for antibiotics with
Enterobacter aerogenes infections and in vivo emergence of imipenem resis- enhanced permeation properties through bacterial porins. Biochemistry 49,
tance. J. Clin. Microbiol. 35, 152–160. 6928–6935. doi: 10.1021/bi100845x
Diene, S. M., Merhej, V., Henry, M., El Filali, A., Roux, V., Robert, C., et al. (2013). Hajjar, E., Mahendran, K. R., Kumar, A., Bessonov, A., Petrescu, M., Weingart, H.,
The rhizome of the multidrug-resistant Enterobacter aerogenes genome reveals et al. (2010b). Bridging timescales and length scales: from macroscopic flux to
how new “killer bugs” are created because of a sympatric lifestyle. Mol. Biol. the molecular mechanism of antibiotic diffusion through porins. Biophys. J. 98,
Evol. 30, 369–383. doi: 10.1093/molbev/mss236 569–575. doi: 10.1016/j.bpj.2009.10.045
Doumith, M., Ellington, M. J., Livermore, D. M., and Woodford, N. (2009). Hammami, S., Boutiba-Ben Boubaker, I., Saidani, M, Lakhal, E., Ben Hassen, A.,
Molecular mechanisms disrupting porin expression in ertapenem-resistant Kamoun, A., et al. (2012). Characterization and molecular epidemiol-
Klebsiella and Enterobacter spp. clinical isolates from the UK. J. Antimicrob. ogy of extended spectrum beta-lactamase producing Enterobacter cloacae
Chemother. 63, 659–667. doi: 10.1093/jac/dkp029 isolated from a Tunisian hospital. Microb. Drug Resist. 18, 59–65. doi:
Du, D., Wang, Z., James, N. R., Voss, J. E., Klimont, E., Ohene-Agyei, T., et al. 10.1089/mdr.2011.0074
(2014). Structure of the AcrAB-TolC multidrug efflux pump. Nature 509, Hamprecht, A., Poirel, L., Gottig, S., Seifert, H., Kaase, M., and Nordmann P.
512–515. doi: 10.1038/nature13205 (2013). Detection of the carbapenemase GIM-1 in Enterobacter cloacae in
Dugleux, G., Le Coutour, X., Hecquard, C., and Oblin, I. (1991). Septicemia caused Germany. J. Antimicrob. Chemother. 68, 558–561. doi: 10.1093/jac/dks447
by contaminated parenteral nutrition pouches: the refrigerator as an unusual He, G. X., Thorpe, C., Walsh, D., Crow, R., Chen, H., Kumar, S., et al. (2011).
cause. J. Parent. Ent. Nutr. 15, 474–475. doi: 10.1177/0148607191015004474 EmmdR, a new member of the MATE family of multidrug transporters,
Dumarche, P., De Champs, C., Sirot, D., Chanal, C., Bonnet, R., and Sirot, J. extrudes quinolones from Enterobacter cloacae. Arch. Microbiol. 193, 759–765.
(2002). TEM derivative-producing Enterobacter aerogenes strains: dissemina- doi: 10.1007/s00203-011-0738-1
tion of a prevalent clone. Antimicrob. Agents Chemother. 46, 1128–1131. doi: Huang, S., Dai, W., Sun, S., Zhang, X., and Zhang, L. (2012). Prevalence of
10.1128/AAC.46.4.1128-1131.2002 plasmid-mediated quinolone resistance and aminoglycoside resistance deter-
Dupont, M., James, C. E., Chevalier, J., and Pagès, J. M. (2007). An early response to minants among carbapeneme non-susceptible Enterobacter cloacae. PLoS ONE
environmental stress involves regulation of OmpX and OmpF, two enterobac- 7:e47636. doi: 10.1371/journal.pone.0047636
terial outer membrane pore-forming proteins. Antimicrob. Agents Chemother. Izdebski, R., Baraniak, A., Herda, M., Fiett, J., Bonten, M. J., Carmeli, Y., et al.
51, 3190–3198. doi: 10.1128/AAC.01481-06 (2014). MLST reveals potentially high-risk international clones of Enterobacter
Farmer, J. J. III, Davis, B. R., Hickman-Brenner, F. W., McWorther, A., Huntley- cloacae. J. Antimicrob. Chemother. 70, 48–56. doi: 10.1093/jac/dku359
Carter, G. P., Asbury, M. A., et al. (1985). Biochemical identification of new Jalaluddin, S., Devaster, J. M., Scheen, R., Gerard, M., and Butzler, J. P. (1998).
species and biogroups of Enterobacteriaceae isolated from clinical specimens. Molecular epidemiological study of nosocomial Enterobacter aerogenes isolates
J. Clin. Microbiol. 21, 46–76. in a Belgian hospital. J. Clin. Microbiol. 36, 1846–1852.

Frontiers in Microbiology | www.frontiersin.org 8 May 2015 | Volume 6 | Article 392

Davin-Regli and Pagès Enterobacter sp. and antibiotic treatment

James, C. E., Mahendran, K. R., Molitor, A., Bolla, J. M., Bessonov, A. N., Enterobactericeae: the OmpF/C – TolC case. Open Microbiol. J. 7, 22–33.
Winterhalter, M., et al. (2009). How beta-lactam antibiotics enter bacteria: a dia- doi: 10.2174/1874285801307010022
logue with the porins. PLoS ONE 4:e5453. doi: 10.1371/journal.pone.0005453 Masi, M., Pagès, J. M., and Pradel, E. (2006). Production of the cryptic EefABC
Jarlier, V., and INVS. (2014). Surveillance of Multidrug Resistant Bacteria in efflux pump in Enterobacter aerogenes chloramphenicol-resistant mutants.
French Healthcare Facilities BMR-Raisin Network Données 2012. Saint-Maurice: J. Antimicrob. Chemother. 57, 1223–1226. doi: 10.1093/jac/dkl139
Institut de Veille Sanitaire. Available at: http://www.invs.sante.fr Masi, M., Pagès, J. M., Villard, C., and Pradel, E. (2005). The eefABC multidrug
Jaskulski, M. R., Medeiros, B. C., Borges, J. V., Zalewsky, R., Fonseca, M. E., efflux pump operon is repressed by H-NS in Enterobacter aerogenes. J. Bacteriol.
Marinowic, D. R., et al. (2013). Assessment of extended-spectrum β-lactamase, 187, 3894–3897. doi: 10.1128/JB.187.11.3894-3897.2005
KPC carbapenemase and porin resistance mechanisms in clinical samples of Mezzatesta, M. L., Gona, F., and Stefani, S. (2012). Enterobacter cloacae complex:
Klebsiella pneumoniae and Enterobacter spp. Int. J. Antimicrob. Agents 42, clinical impact and emerging antibiotic resistance. Future Microbiol. 7, 887–902.
76–79. doi: 10.1016/j.ijantimicag.2013.03.009 doi: 10.2217/fmb.12.61
Kanamori, H., Hisakasu, Y., Yoichi, H., H Ayako, H., Kazuaki, A., Kunishima, H., Miro, E., Alonso, C., Navarro, F., Mirelis, B., and Prats, G. (1995). Resistencia
et al. (2012). Molecular characteristics of extended-spectrum β-lactamases and al imipenem en Enterobacter aerogenes. Enferm. Infecc. Microbiol. Clin. 13,
qnr determinants in Enterobacter species from Japan. PLoS ONE 7:e37967. doi: 278–282.
10.1371/journal.pone.0037967 Miró, E., Grünbaum, F., Gómez, L., Rivera, A., Mirelis, B., Coll, P., et al. (2013).
Khajuria, A., Praharaj, A. K., Kumar, M., and Grover, N. (2014). Carbapenem resis- Characterization of aminoglycoside-modifying enzymes in Enterobacteriaceae
tance among Enterobacter species in a tertiary care hospital in central India. clinical strains and characterization of the plasmids implicated in their diffu-
Chemother. Res. Pract. 2014:972646. doi: 10.1155/2014/972646 sion. Microb. Drug Resist. 19, 94–99. doi: 10.1089/mdr.2012.0125
Kim, S. Y., Park, Y. J., Yu, J. K., Kim, Y. S., and Han, K. (2009). Prevalence Nauciel, C., Philippon, A., Ronco, E., Pilliot, J., Guenounou, M., Paul, G., et al.
and characteristics of aac(6’)-Ib-cr in AmpC-producing Enterobacter (1985). Septicémies à Enterobacter cloacae et E. aerogenes: émergence de vari-
cloacae, Citrobacter freundii, and Serratia marcescens: a multicen- ants résistants. Presse Med. 14, 673–676.
ter study from Korea. Diagn. Microbiol. Infect. Dis. 63, 314–318. doi: Neuwirth, C., Siebor, E., Lopez, J., Pechinot, A., and Kazmierczak, A. (1996).
10.1016/j.diagmicrobio.2008.11.016 Outbreak of TEM-24-producing Enterobacter aerogenes in an intensive
Langley, J. M., Hanakowski, M., and Leblanc, J. C. (2001). Unique epidemiology of care unit and dissemination of the extended-spectrum β-lactamase to
nosocomial urinary tract infection in children. Am. J. Infect. Control 29, 94–98. other members of the family Enterobacteriacae. J. Clin. Microbiol. 34,
doi: 10.1067/mic.2001.111537 76–79.
Lavigne, J. P., Sotto, A., Nicolas-Chanoine, M. H., Bouziges, N., Bourg, G., Nikaido, H., and Pagès, J. M. (2012). Broad-specificity efflux pumps and their role
Davin-Regli, A., et al. (2012). Membrane permeability, a pivotal function in multidrug resistance of Gram-negative bacteria. FEMS Microbiol. Rev. 36,
involved in antibiotic resistance and virulence in Enterobacter aerogenes clin- 340–363. doi: 10.1111/j.1574-6976.2011.00290.x
ical isolates. Clin. Microbiol. Infect. 18, 539–545. doi: 10.1111/j.1469-0691.2011. Nordmann, P., Mariotte, S., Naas, T., Labia, R., and Nicolas, M. H. (1993).
03607.x Biochemical properties of a carbapenem-hydrolyzing beta-lactamase from
Lavigne, J. P., Sotto, A., Nicolas-Chanoine, M. H., Bouziges, N., Pagès, J. M., Enterobacter cloacae and cloning of the gene into Escherichia coli. Antimicrob.
and Davin-Regli, A. (2013). An adaptive response of Enterobacter aerogenes to Agents Chemother. 37, 939–946. doi: 10.1128/AAC.37.5.939
imipenem: regulation of porin balance in clinical isolates. Int. J. Antimicrob. Pagès, J. M., James, C. E., and Winterhalter, M. (2008). The porin and the permeat-
Agents 41, 130–136. doi: 10.1016/j.ijantimicag.2012.10.010 ing antibiotic: a selective diffusion barrier in Gram-negative bacteria. Nat. Rev.
Lawler, A. J., Ricci, V., Busby, S. J., and Piddock, L. J. (2013). Genetic inactiva- Microbiol. 6, 893–903. doi: 10.1038/nrmicro1994
tion of acrAB or inhibition of efflux induces expression of ramA. J. Antimicrob. Park, Y.-J., Yu, J.-K., Kim, S.-I., Lee, K., and Arakawa, Y. (2009). Accumulation
Chemother. 68, 1551–1557. doi: 10.1093/jac/dkt069 of plasmid-mediated fluoroquinolone resistance genes, qepA and qnrS1, in
Lee, E. H., Collatz, E., Trias, J., and Gutmann, L. (1992). Diffusion of β-lactam Enterobacter aerogenes co-producing RmtB and Class A β-lactamase LAP-1.
antibiotics into proteoliposomes reconstituted with outer membranes of iso- Ann. Clin. Lab. Sci. 39, 55–59.
genic imipenem-susceptible and -resistant strains of Enterobacter cloacae. Pérez, A., Canle, D., Latasa, C., Poza, M., Beceiro, A., Tomás Mdel, M., et al. (2007).
J. Gen. Microbiol. 138, 2347–2351. doi: 10.1099/00221287-138-11-2347 Cloning, nucleotide sequencing, and analysis of the AcrAB-TolC efflux pump
Lee, H. K., Park, Y. J., Kim, J. Y., Chang, E., Cho, S. G., Chae, H. S., of Enterobacter cloacae and determination of its involvement in antibiotic resis-
et al. (2005). Prevalence of decreased susceptibility to carbapenems among tance in a clinical isolate. Antimicrob. Agents Chemother. 51, 3247–3253. doi:
Serratia marcescens, Enterobacter cloacae, and Citrobacter freundii and inves- 10.1128/AAC.00072-07
tigation of carbapenemases. Diagn. Microbiol. Infect. Dis. 52, 331–336. doi: Pérez, A., Poza, M., Aranda, J., Latasa, C., Medrano, F. J., Tomás, M., et al. (2012).
10.1016/j.diagmicrobio.2005.04.012 Effect of transcriptional activators SoxS, RobA, and RamA on expression of
Lee, S. H., Jeong, S. H., and Park, Y. M. (2003). Characterization of blaCMY-10 multidrug efflux pump AcrAB-TolC in Enterobacter cloacae. Antimicrob. Agents
a novel, plasmid-encoded AmpC -type β-lactamase gene in a clinical isolate Chemother. 56, 6256–6266. doi: 10.1128/AAC.01085-12
of Enterobacter aerogenes. J. Appl. Microbiol. 95, 744–752. doi: 10.1046/j.1365- Pestourie, N., Garnier, F., Barraud, O., Bedu, A., Ploy, M. C., and Mounier, M.
2672.2003.02040.x (2014). Outbreak of AmpC β-lactamase-hyper-producing Enterobacter cloacae
Levy, S. B. (2002). Active efflux, a common mechanism for biocide and antibiotic in a neonatal intensive care unit in a French teaching hospital. Am. J. Infect.
resistance. Symp. Ser. Soc. Appl. Microbiol. 92, 65S–71S. doi: 10.1046/j.1365- Control 42, 456–458. doi: 10.1016/j.ajic.2013.11.005
2672.92.5s1.4.x Piddock, L. J. (2006). Clinically relevant chromosomally encoded multidrug
Malléa, M., Chevalier, J., Bornet, C. E., Eyraud, A., Davin-Regli, A., Bollet, C., resistance efflux pumps in bacteria. Clin. Microbiol. Rev. 19, 382–402. doi:
et al. (1998). Porin alteration and active efflux: two in vivo drug resistance 10.1128/CMR.19.2.382-402.2006
strategies used by Enterobacter aerogenes. Microbiology 144, 3003–3009. doi: Pitout, J. D., Sanders, C. C., and Sanders, W. E. Jr. (1997). Antimicrobial resistance
10.1099/00221287-144-11-3003 with focus on beta-lactam resistance in gram-negative bacilli. Am. J. Med. 103,
Malléa, M., Simonet, V., Lee, E. H., Gervier, R., Collatz, E., Gutmann, L., et al. 51–59. doi: 10.1016/S0002-9343(97)00044-2
(1995). Biological and immunological comparisons of Enterobacter cloacae and Pitout, J. D. D., Thomson, K. S., Hanson, N. D., Ehrhardt, A. F., Coudron, P.,
Escherichia coli porins. FEMS Microbiol. Lett. 129, 273–279. doi: 10.1016/0378- and Sanders, C. C. (1998). Plasmid-mediated resistance to expanded-spectrum
1097(95)00171-Z cephalosporins among Enterobacter aerogenes strains. Antimicrob. Agents
Martins, A., Spengler, G., Martins, M., Rodrigues, L., Viveiros, M., Davin-Regli, A., Chemother. 42, 596–600.
et al. (2010). Physiological characterisation of the efflux pump system of Poirel, L., Pitout, J. D., and Nordmann, P. (2007). Carbapenemases: molecu-
antibiotic-susceptible and multidrug-resistant Enterobacter aerogenes. Int. J. lar diversity and clinical consequences. Future Microbiol. 2, 501–512. doi:
Antimicrob. Agents 36, 313–318. doi: 10.1016/j.ijantimicag.2010.06.036 10.2217/17460913.2.5.501
Masi, M., and Pagès, J. M. (2013). Structure, function and regula- Poirel, L., Van De Loo, M., Mammeri, H., and Nordmann, P. (2005).
tion of outer membrane proteins involved in drug transport in Association of plasmid-mediated quinolone resistance with extended-spectrum

Frontiers in Microbiology | www.frontiersin.org 9 May 2015 | Volume 6 | Article 392

Davin-Regli and Pagès Enterobacter sp. and antibiotic treatment

beta-lactamase VEB-1. Antimicrob. Agents Chemother. 49, 3091–3094. doi: Thiolas, A., Bornet, C., Davin-Regli, A., Pagès, J. M., and Bollet, C. (2004).
10.1128/AAC.49.7.3091-3094.2005 Resistance to imipenem, cefepime, and cefpirome associated with mutation in
Potron, A., Poirel, L., Rondinaud, E., and Nordmann, P. (2013). Intercontinental Omp36 osmoporin of Enterobacter aerogenes. Biochem. Biophys. Res. Commun.
spread of OXA-48 β-lactamase-producing Enterobacteriaceae over a 11- 317, 851–856. doi: 10.1016/j.bbrc.2004.03.130
year period, 2001 to 2011. Euro. Surveill. 18:20549. doi: 10.2807/1560- Torres, E., López-Cerero, L., Del Toro, M. D., and Pascual, A. (2014). First
7917.ES2013.18.31.20549 detection and characterization of an OXA-48-producing Enterobacter
Pradel, E., and Pagès, J. M. (2002). The AcrAB-TolC efflux pump contributes aerogenes isolate. Enferm. Infecc. Microbiol. Clin. 32, 469–470. doi:
to multidrug resistance in the nosocomial pathogen Enterobacter aerogenes. 10.1016/j.eimc.2013.10.008
Antimicrob. Agents Chemother. 46, 2640–2643. doi: 10.1128/AAC.46.8.2640- Tran, Q. T., Dupont, M., Lavigne, J. P., Chevalier, J., Pagès, J. M., Sotto, A., et al.
2643.2002 (2009). Occurrence of efflux mechanism and cephalosporinase variant in a
Preston, K. E., Radomski, C. C. A., and Venezia, R. A. (2000). Nucleotide sequence population of Enterobacter aerogenes and Klebsiella pneumoniae isolates pro-
of the chromosomal ampC gene of Enterobacter aerogenes. Antimicrob. Agents ducing extended-spectrum beta-lactamases. Antimicrob. Agents Chemother. 53,
Chemother. 44, 3158–3162. doi: 10.1128/AAC.44.11.3158-3162.2000 1652–1656. doi: 10.1128/AAC.00822-08
Quiroga, M. P., Andres, P., Petroni, A., Soler Bistué, A. J., Guerriero, L., Vargas, Valade, E., Davin-Regli, A., Bolla, J.-M., and Pagès, J.-M. (2013). “Bacterial mem-
L. J., et al. (2007). Complex class 1 integrons with diverse variable regions, brane, a key for controlling drug influx and efflux,” in Antibiotics: Targets,
including aac(6’)-Ib-cr, and a novel allele, qnrB10, associated with ISCR1 in Mechanisms and Resistance, eds C. O. Gualerzi, L. Brandi, A. Fabbretti, and
clinical enterobacterial isolates from Argentina. Antimicrob. Agents Chemother. C. L. Pon (Weinheim: Wiley-VCH Verlag GmbH & Co. KGaA), 217–240. doi:
51, 4466–4470. doi: 10.1128/AAC.00726-07 10.1002/9783527659685.ch9
Raimondi, A., Traverso, A., and Nikaido, H. (1991). Imipenem- and meropenem- Veleba, M., De Majumdar, S., Hornsey, M., Woodford, N., and Schneiders, T.
resistant mutants of Enterobacter cloacae and Proteus rettgeri lack porins. (2013). Genetic characterization of tigecycline resistance in clinical isolates of
Antimicrob. Agents Chemother. 35, 1174–1180. doi: 10.1128/AAC.35.6.1174 Enterobacter cloacae and Enterobacter aerogenes. J. Antimicrob. Chemother. 68,
Rice, L. B. (2010). Progress, and challenges in implementing the research on 1011–1018. doi: 10.1093/jac/dks530
ESKAPE pathogens. Infect. Control. Hosp. Epidemiol. Suppl. 1, S7–S10. doi: Vidal, S., Bredin, J., Pagès, J.-M., and Barbe, J. (2005). β-lactam screening by
10.1086/655995 specific residues of the OmpF eyelet. J. Med. Chem. 48, 1395–400. doi:
Robert, J., Pantel, A., Mérens, A., Lavigne, J. P., Nicolas-Chanoine, M. H., and 10.1021/jm049652e
ONERBA’s Carbapenem Resistance Study Group. (2014). Incidence rates of Wang, S. A., Tokars, J. I., Bianchine, P. J., Carson, L. A., Arduino, M. J., Smith, A. L.,
carbapenemase-producing Enterobacteriaceae clinical isolates in France: a et al. (2000). Enterobacter cloacae bloodstream infections traced to contamined
prospective nationwide study in 2011-12. J. Antimicrob. Chemother. 69, 2706– human albumine. Clin. Infect. Dis. 30, 35–40. doi: 10.1086/313585
2712. doi: 10.1093/jac/dku208 Yigit, H., Anderson, G. J., Biddle, J. W., Steward, C. D., Rasheed, J. K.,
Sanders, W. E., and Sanders, C. C. (1997). Enterobacter spp.: pathogens poised to Valera, L. L., et al. (2002). Carbapenem resistance in a clinical isolate of
flourish at the turn of the century. Clin. Microbiol. Rev. 10, 220–241. Enterobacter aerogenes is associated with decreased expression of OmpF and
Song, E.-H., Park, K.-H., Jang, E.-Y., Lee, E.-J., Chong, Y.-P., Cho, O.-H., et al. OmpC porin analogs. Antimicrob. Agents Chemother. 46, 3817–3822. doi:
(2010). Comparison of the clinical and microbiologic characteristics of patients 10.1128/AAC.46.12.3817-3822.2002
with Enterobacter cloacae and Enterobacter aerogenes bacteremia: a prospec- Zaher, A., and Cimolai, N. (1997). ERIC-PCR typing profiles of Enterobacter
tive observation study. Diagnos. Microbiol. Infect. Dis. 66, 436–440. doi: cloacae are stable after development of advanced cephalosporin resis-
10.1016/j.diagmicrobio.2009.11.007 tance. Int. J. Antimicrob. Agents 9, 165–167. doi: 10.1016/S0924-8579(97)
Stoorvogel, J., van Bussel, M. J., Tommassen, J., and van de Klundert, J. A. (1991). 00046-0
Molecular characterization of an Enterobacter cloacae outer membrane protein
(OmpX). J. Bacteriol. 173, 156–160. Conflict of Interest Statement: The authors declare that the research was con-
Szabo, D., Melan, M. A., Hujer, A. M., Bonomo, R. A. Hujer, K. M., Bethel, C. R., ducted in the absence of any commercial or financial relationships that could be
et al. (2005). Molecular analysis of the simultaneous production of two SHV- construed as a potential conflict of interest.
type extended-spectrum beta-lactamases in a clinical isolate of Enterobacter
cloacae by using single-nucleotide polymorphism genotyping. Antimicrob. Copyright © 2015 Davin-Regli and Pagès. This is an open-access article distributed
Agents Chemother. 49, 4716–4720. doi: 10.1128/AAC.49.11.4716-4720.2005 under the terms of the Creative Commons Attribution License (CC BY). The use,
Thiolas, A., Bollet, C., La Scola, B., Raoult, D., and Pagès, J. M. (2005). distribution or reproduction in other forums is permitted, provided the original
Successive emergence of Enterobacter aerogenes strains resistant to imipenem author(s) or licensor are credited and that the original publication in this jour-
and colistin in a patient. Antimicrob. Agents Chemother. 49, 1354–1358. doi: nal is cited, in accordance with accepted academic practice. No use, distribution or
10.1128/AAC.49.4.1354-1358.2005 reproduction is permitted which does not comply with these terms.

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