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Enterobacter cloacae Complex: Clinical Impact and Emerging Antibiotic

Resistance

Article in Future Microbiology · July 2012

DOI: 10.2217/fmb.12.61 · Source: PubMed

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Enterobacter cloacae complex:

Review
Future Microbiology
clinical impact and emerging
antibiotic resistance
Maria Lina Mezzatesta*1, Floriana Gona1 & Stefania Stefani1
1
Department of Bio-Medical Sciences, Section of Microbiology, University of Catania, Via Androne 81,
95124 Catania, Italy
*Author for correspondence: Tel.: +39 095 2504733 n Fax: +39 095 2504733 n [email protected]

Species of the Enterobacter cloacae complex are widely encountered in nature,

but they can act as pathogens. The biochemical and molecular studies on
E. cloacae have shown genomic heterogeneity, comprising six species :
Enterobacter cloacae, Enterobacter asburiae, Enterobacter hormaechei,
Enterobacter kobei, Enterobacter ludwigii and Enterobacter nimipressuralis,
E. cloacae and E. hormaechei are the most frequently isolated in human clinical
specimens. Phenotypic identification of all species belonging to this taxon is
usually difficult and not always reliable; therefore, molecular methods are often
used. Although the E. cloacae complex strains are among the most common
Enterobacter spp. causing nosocomial bloodstream infections in the last decade,
little is known about their virulence-associated properties. By contrast, much has
been published on the antibiotic-resistance features of these microorganisms.
In fact, they are capable of overproducing AmpC b-lactamases by derepression
of a chromosomal gene or by the acquisition of a transferable ampC gene on
plasmids conferring the antibiotic resistance. Many other resistance determinants
that are able to render ineffective almost all antibiotic families have been
recently acquired. Most studies on antimicrobial susceptibility are focused on
E. cloacae, E. hormaechei and E. asburiae; these studies reported small variations
between the species, and the only significant differences had no discriminating
features.

Bacteria of the Enterobacter genus are faculta- and Enterobacter nimipressuralis. Its most impor-
tive anaerobic Gram-negative strains belong- tant representative, E. cloacae, has emerged as
ing to the family of Enterobacteriaceae and a troublesome pathogen for healthcare institu-
widely found in nature. These microorganisms tions globally. E. cloacae accounts for up to 5%
are sapro­phytic in the environment, as they are of hospital-acquired sepsis, 5% of nosocomial
found in soil and sewage, and are also part of the pneumonias, 4% of nosocomial urinary tract
commensal enteric flora of the human GI tract. infections and 10% of postsurgical peritonitis
Over recent decades, the Enterobacter spp. cases [5,6] . Their clinical significance, especially
have taken on clinical significance and have over the last 15 years, has been reported in many
emerged as nosocomial pathogens from inten- publications, demonstrating their remarkable
sive care patients [1] . The National Nosocomial ability to upregulate or acquire resistance deter-
Infections Surveillance System reported data on minants and making them some of the most
noso­comial bacteremia from 1976 to 1989 in worrying microorganisms of the current anti-
the USA, and the National Healthcare Safety biotic era. The last review of Enterobacter spp.
Network (2008) reported that Enterobacter was written in 1997 by Sanders and Sanders [1] ,
spp. account for approximately 5% of noso- and since then, there have been many changes
comial bacteremia cases; these data have not in the taxonomy of this genus in which the spe- Keywords
varied [1–3] . Another multicenter study focused cies of the E. cloacae complex, due to common n antibiotic resistance
on 24,179 cases of nosocomial bloodstream characteristics, have emerged as nosocomial n E. cloacae complex
n Enterobacter asburiae
infections between 1995 and 2002, showing pathogens. Moreover, there have also been many
n Enterobacter cloacae
Enterobacter spp. to be among the ten most developments in clinical diagnostic methods and n Enterobacter hormaechei
commonly isolated noso­comial pathogens with a in nosocomial surveillance of these species that n Enterobacter kobei

greater incidence in intensive care unit wards [4] . today use new molecular methods. n Enterobacter ludwigii
n Enterobacter nimipressuralis
Currently, six species have been assigned to the For these reasons, we propose, with this first
Enterobacter cloacae complex, including E. cloa- review of the E. cloacae complex, to describe
cae, Enterobacter asburiae, Enterobacter hormae- the advances, and also provide a comprehen-
chei, Enterobacter kobei, Enterobacter ludwigii sive appraisal, of the relevant microbiological part of

10.2217/FMB.12.61 © 2012 Future Medicine Ltd Future Microbiol. (2012) 7(7), 887–902 ISSN 1746-0913 887
 Review Mezzatesta, Gona & Stefani

identification methods, as well as their clinical which is able to discriminate 12 genetic clusters
impact. We also report emerging antibiotic- (clusters I–XII) as reported by Hoffmann and
resistance trends of the six species belonging to Roggenkamp [6] .
this complex. Specific names have been attributed to some
of the genetic clusters; nine of the clusters corre-
Taxonomy & identification spond to species: E. asburiae (cluster I), E. kobei
The genus Enterobacter was first described by (cluster II), E. ludwigii (cluster V), E. hormae-
Hormaeche and Edwards in 1960 [7] and has chei subsp. oharae (cluster VI), E. hormaechei
undergone significant taxonomic modification subsp. hormaechei (cluster VII), E. hormaechei
over the last 50 years: Enterobacter agglomerans subsp. steigerwallti (cluster VIII), E. nimipres-
has been transferred to the genus Pantoea, and suralis (cluster X), E. cloacae subsp. cloacae
Enterobacter sakazakii was recently reassigned to (cluster XI) and E. cloacae subsp. dissolvens (clus-
the newly proposed genus Cronobacter [8] . ter XII); while three clusters do not have specific
Currently, there are 22 species in the genus names and are referred to as E. cloacae cluster III,
Enterobacter [201] : Enterobacter aerogenes, amnige- E. cloacae cluster IV and E. cloacae cluster IX [8] .
nus, arachidis, asburiae, cancerogenus, cloacae, The second method consists of the sequencing of
cowanii, dissolvens, gergoviae, helveticus, hormae- rpoB, and could be a valid alternative for strain
chei, kobei, ludwigii, mori, nimipressuralis, ory- identification because of the high resolution for
zae, pulveris, pyrinus, radicincitans, soli, taylorae differentiating between closely related species.
and turicensis [9] . Six of these species – E. cloa- The degree of genomic diversity within the
cae, asburiae, hormaechei, kobei, ludwigii and E. cloacae complex was recently reassessed by
nimipressuralis – are combined in the so-called other genotypic methods. Two of these deserve
E. cloacae complex, because most of them share some attention and are the multilocus sequence
a DNA relatedness with E. cloacae ranging from ana­lysis and the comparative genomic hybrid-
61 to 67% [6] . The taxonomy of the E. cloacae ization. Multilocus sequence ana­lysis identifies
complex is mainly based on whole-genome seven clusters within the E. cloacae complex,
DNA–DNA hybridizations and phenotypic and each of these corresponds to one or more
characteristics. hsp60 gene sequencing-based genetic cluster.
Brief ly, the E. cloacae complex exhib- The microarray-based comparative genomic
its the general characteristics of the genus hybridization ana­lysis is a powerful method for
Enterobacter: they are catalase-positive, oxi- performing genome-wide studies on different
dase- and DNAase-negative, fermentative and bacteria [5] .
nonpigmented. Their identification is routinely Recently, in 2012, the results on the use the
performed by using phenotypic methods: com- matrix-assisted laser desorption ionization time-
mercial systems for Enterobacteriaceae, API® of-flight mass spectrometry (MALDI-TOF MS)
20E or Vitek® 2 (BioMerieux, France), are able method for fast identification within the E. cloa-
to discriminate only E. cloacae and E. asburiae, cae complex have demonstrated that it can be
while for the discrimination of the other species, used for distinguishing E. cloacae and E. nimi-
Biotype 100™ (BioMérieux) system and other pressuralis; however, it is inadequate for distin-
conventional tests are necessary [10,11] . guishing E. asburiae, E. hormaechei, E. kobei and
Table 1 summarizes some of the key tests for E. ludwigii from E. cloacae. Instead, the combi-
the phenotypic differentiation of the E. cloa- nation of MALDI-TOF MS with the E. cloacae-
cae complex species; for example, the Voges– specific duplex real-time PCR is an appropriate
Proskauer test is the only one able to differenti- method for identification of the six species of the
ate E. kobei from E. cloacae, while E. ludwigii E. cloacae complex [15] .
identification is mainly possible by its ability The identification of the species within the
to grow on 3-0-methyl-d-gluco-pyranose, E. cloacae complex, with respect to E. cloacae, in
while E. nimipressuralis is the only nonmotile clinical specimens is important because it gives
species [12,13] . more information on their clinical relevance
Enterobacter spp. are unambiguously iden- that otherwise could remain underestimated.
tified by molecular methods [14] by using the An example is a case report of nosocomial uro-
16S rRNA gene, the oriC locus and gyrB [8] . sepsis caused by E. kobei that was previously
Recently, hsp60- and rpoB-genotyping appeared identified as E. cloacae [16] . From the antibiotic
to be promising novel methods. In particular, therapy point of view, the identification is less
the first method uses genotypic identifica- important because these species possess similar
tion, via sequencing, of a fragment of hsp60, susceptibility patterns.

888 Future Microbiol. (2012) 7(7) future science group

 Enterobacter cloacae complex: clinical impact & emerging antibiotic resistance Review

Finally, the identification by only phenotypic

Putrescine
methods may lead to the misidentification of
E. hormaechei as Cronobacter sakazakii, which
prevents the full disclosure of sources, infections

-/+
and of its virulence [17] .

±
+

-
d -sorbitol
Epidemiology & molecular typing
An increasing number of clonal outbreaks
caused by members of the E. cloacae complex

+/-
+

-
is being reported. In recent years, genotypic

Dulcitol
methods have been developed to increase the
discriminatory power of epidemiological inves-
tigations. Sensitive and reproducible molecular

±
+
-

-
markers, including those used in ribotyping [18] ,

gluco-pyranose
small-fragment restriction endonuclease ana­lysis

a- d -melibiose Sucrose 3-hydroxy- 3-methyl- d -

and pulsed-field gel electrophoresis (PFGE) [19]
have been applied with success to the E. cloacae
complex. PFGE is considered the gold standard,
but PCR-based methods, including enterobacte-

+
-

-
rial repetitive intergenic consensus (ERIC) PCR,
repetitive extragenic palindromic (REP) PCR

butyrate
and arbitrarily primed PCR [20] belong to a new

Table 1. Key reactions for biochemical differentiation of the Enterobacter cloacae complex.
generation of typing methods in which a single

+/-
+

+
-

-
short primer with arbitrarily chosen nucleotide
sequences is used in PCR to amplify genomic
DNA, and these are cheaper, easier to perform
and provide faster results.

-
When comparing DNA macrorestriction ana­
lysis by PFGE with PCR-based DNA finger-
printing techniques, the discriminatory power
of PFGE was found to be higher than those of +/-

-/+
PCR-based techniques [19] .
+

+
In the recent study by Stumpf et al., the
a- d -methyl-
glycosidase

E. cloacae complex isolates were assigned to

their respective genetic cluster by partial hsp60
sequencing; all strains were selected to evalu-
ate the specificity of ERIC and REP PCR for
+

+
the identification of clonal isolates belonging to
Esculin

the E. cloacae complex [21] . Comparing ERIC

-: 0–10%; -/+: 10–20%; ±: 20–80%; +/-: 80–90%; +: 90–100%.

PCR with PFGE, a specificity of 14%, con-
+

+
±

sidering the detection of ‘clonal’ isolates, was

-

calculated. REP PCR performed much better,

Motility

yielding a specificity of 90%. ERIC PCR pat-

terns allowed an accurate classification of the
+/-

+/-

+/-
+

isolates with their respective genomovars, sug-

gesting that ERIC PCR differentiates between
genomovars rather than between strains. By
Proskauer

contrast, REP PCR differentiates better at the

Voges–

strain level. A proposed diagnostic system for

+/-

the detection of outbreak strains of the E. cloa-

+

+
-

cae complex is based on an initial REP PCR,

which should be confirmed by PFGE in cases
nimipressuralis
Enterobacter

Enterobacter

Enterobacter

Enterobacter

Enterobacter

Enterobacter
hormaechei

of identical patterns, whereas ERIC PCR does

Species

asburiae

not seem to be useful for the detection of out-

ludwigii
cloacae

kobei

break strains when dealing with isolates of the

E. cloacae complex. Therefore, both PFGE- and

future science group www.futuremedicine.com 889

 Review Mezzatesta, Gona & Stefani

PCR-based fingerprinting are useful for typing curli fimbriae and the genotypic detection of the
of the E. cloacae complex. However, PFGE can curli gene, suggesting the importance of these
detect minor mutations among outbreak strains, fimbriae in the formation and morphology of
and this is important for epidemiological studies biofilms in E. cloacae [28] .
of these species in a complex endemic setting [21] .
Species of the E. cloacae complex
Mechanisms of pathogenicity E. cloacae
Little is known about the factors impacting the E. cloacae was described for the first time
pathogenicity and virulence of Enterobacter spp. in 1890 by Jordan [201] as ‘Bacillus cloacae’,
As Gram-negative pathogens, they possess endo- and then underwent numerous taxonomical
toxins and thus have all of the pathogenetic prop- changes, becoming ‘Bacterium cloacae’ in 1896
erties imparted to an organism by this virulence (Lehmann and Neumann), ‘Cloaca cloacae’ in
factor [1] . The possible pathogenetic mechanism 1919 (Castellani and Chalmers), it was identi-
is complex and multifactorial, with the involve- fied as ‘Aerobacter cloacae’ in 1923 (Bergey et al.),
ment of a number of putative virulence factors, ‘Aerobacter cloacae’ in 1958 (Hormaeche and
whose role in the development of the disease is Edwards) and E. cloacae in 1960 (Hormaeche
still not clear. Barnes et al. report that after adhe- and Edwards), by which it is still known
sion to the epithelial cells, in vitro Enterobacter today [7] . E. cloacae is ubiquitous in terrestrial
spp. strains produce many potential virulence and aquatic environments (water, sewage, soil
factors, including enterotoxins, a-hemolysin and and food). These strains occur as commensal
thiol-activated pore-forming cytotoxins similar microflora in the intestinal tracts of humans
to Shiga-like toxin II, which require treatment and animals [1] and play an important role as
with 2-mercaptoethanol [22] . In several Gram- pathogens in plants and insects. This diversity of
negative bacteria, the type III secretion system habitats is mirrored by the genetic variety of the
(TTSS) has been shown to be an important nomenspecies E. cloacae [6] . E. cloacae is also an
factor that plays a crucial role in host–patho- important nosocomial pathogen responsible for
gen interactions. The TTSS consists of several bacteremia and lower respiratory tract, urinary
proteins assembled in a system that delivers tract and intra-abdominal infections, as well as
virulence factors, usually toxins, directly into endocarditis, septic arthritis, osteomyelitis and
the host cells. Hence, the presence of the TTSS skin and soft tissue infections. The skin and the
can be used as a general indicator of bacterial GI tract are the most common sites through
virulence [23] . In a study by Krzyminska et al., which E. cloacae can be contracted [1,29] .
TTSS genes were present in 27% of the E. cloa- E. cloacae tends to contaminate various medi-
cae isolated from clinical specimens, indicating cal, intravenous and other hospital devices.
that these bacteria may destroy phagocytes and Nosocomial outbreaks have also been associ-
epithelial cells, leading to their spread within the ated with colonization of certain surgical equip-
host [24] . The results of this study demonstrate ment and operative cleaning solutions. Another
that cytotoxic enterotoxins activated by 2-mer- potential reservoir for nosocomial bacteremia
captoethanol and the TTSS may contribute to is the heparin solution used to irrigate certain
E. cloacae pathogenesis. In a recent study, it has intravascular devices continually. This fluid
been demonstrated that the E. cloacae complex had been implicated as a reservoir for out-
strains may induce apoptosis of HEp-2 cells [25] . breaks of device-associated bacteremia in several
This process may be a primary strategy of the instances [30] .
strains, resulting in tissue destruction, bacterial In recent years, E. cloacae has emerged as one
spreading and, as a consequence, invasive disease of the most commonly found nosocomial patho-
or systemic infection. These mechanisms may gen in neonatal units, with several outbreaks of
be considered as contributing factors in disease infection being reported [31] . In 1998, van Nierop
development. Finally, some studies have found et al. reported an outbreak in a neonatal inten-
that the biofilm formation on and the coloni- sive care unit with nine deaths [32] , and in 2003,
zation of inert surfaces in Enterobacteriaceae Kuboyama et al. reported three outbreaks with
are due to a class of proteinaceous extracellular 42 systemic infections and a mortality of 34%
fibers, defined ‘curli fimbriae’, which mediate [33] . This microorganism may be transmitted
binding to a variety of host proteins and also to neonates through contaminated intra­venous
mediate host cell adhesion and invasion [26,27] . fluids, total parenteral nutrition solutions and
Recently, Kim et al. have demonstrated a good medical equipment. Many single-clone out-
correlation between the phenotypic detection of breaks, probably caused by cross-transmission

890 Future Microbiol. (2012) 7(7) future science group

 Enterobacter cloacae complex: clinical impact & emerging antibiotic resistance Review

via healthcare workers, have been described, sug- (with PR Edwards) proposed and defined the
gesting that inpatients can also act as a reservoir genus Enterobacter [7] . The name E. hormaechei
[31] . The type strains of the species are E. cloacae was formerly called enteric group 75, which con-
ATCC 49162 and 13047. This latter strain is the tained 11 strains that were sent to the CDC for
first complete genome sequence of the E. cloacae identification between 1973 and 1984. Twelve
species and the type strain is E. cloacae subsp. additional strains were received from 1985 to
cloacae. 1987, three of which were blood isolates. E. hor-
The complete E. cloacae subsp. cloacae ATCC maechei was first described on the basis of 23 iso-
13047 genome contains a single circular chromo- lates sent to the CDC for identification. At that
some of 5,314,588 bp and two circular plasmids, time, they could not be assigned to a species
pECL_A and pECL_B, of 200,370 and 85,650 since they were negative in the d-sorbitol and
bp (GenBank accession numbers CP001918, melibiose tests and did not fit the biochemical
CP001919 and CP001920, respectively) [34] . profile of any established Enterobacter species.
The other genomes of E. cloacae that have The species E. hormaechei was proposed to be
been sequenced are deposited in GenBank under lactose-, d-sorbitol-, raffinose-, melibiose- and
accession numbers CP002272, CP002886, esculin-negative and 87% dulcitol-­p ositive.
FP929040 and AGSY00000000. These species were originally defined by
O’Hara et al. when a large hybridization group
E. asburiae of enteric organisms was isolated and found to
E. asburiae is named after Mary Alyce Fife- be associated with bloodstream infections [10] .
Asbury, an American bacteriologist who made The type strain of E. hormaechei is ATCC
many important contributions to the classifi- 49162 and was isolated from the sputum of
cation of Enterobacteriaceae, particularly in a man in California in 1977 [10] . The whole-
describing new Klebsiella and Salmonella sero- genome shotgun sequencing project was submit-
types [35–37] , new genera and new species [38–42] . ted in 2011 to the Human Genome Sequencing
E. asburiae sp. nov. was described in 1986 based Center (TX, USA; GenBank accession number
on the enteric group 17 [43] . This group was AFHR00000000).
defined in 1978 as a group of biochemically E. hormaechei consists of three different sub-
similar strains isolated from different human species: E. hormaechei subsp. oharae, E. hormae-
specimens [44] and sent to the CDC. Before the chei subsp. hormaechei and E. hormaechei subsp.
designation of ‘enteric group 17’, these strains steigerwaltii, which corresponds to genetic clus-
had been reported as unidentified or atypical ters VI, VII and VIII, respectively [8] . The dif-
Citrobacter or Enterobacter strains [44] . After ferentiation of these subspecies is based on their
several studies, it was shown that these strains particular properties and biochemical tests [11] .
represent a single new species in the genus E. hormaechei is commonly isolated as a noso-
Enterobacter, which was named E. asburiae. comial pathogen of clinical significance [45,46] ;
E. asburiae strains have been isolated from the it has been reported in several outbreaks of sep-
soil and implicated in the mobilization of phos- sis in neonatal intensive care units in the USA
phate for plant nutrition from calcium phos- [47] and in Brazil, where the outbreak originated
phate, but most E. asburiae species have been from contaminated parenteral nutrition [48] .
isolated from human sources. The type strain
of the species E. asburiae is ATCC 35953 and E. kobei
was isolated from lochia exudates of a 22‑year- E. kobei is named after Kobe City (Japan), where
old woman in the USA [43] . The only sequenced the type strain of this species was isolated. E. kobei
strain of E. asburiae is LF7a, which contains a was first described by Kosako et al. based on a
circular DNA (4,812,833 bp) and two circu- collection of 23 strains with the general traits of
lar plasmids, pENTAS01 (166,725 bp) and E. cloacae and the common phenotypic difference
pENTAS02 (32,574 bp), which were submitted of being Voges–Proskauer-negative [49] . The name
by Lucas et al. in 2011 to the US DOE Joint E. kobei is proposed for a group of organisms
Genome Institute (CA, USA; GenBank acces- referred to as NIH group 21 at the NIH, Tokyo. It
sion numbers CP003026.1, CP003027.1 and was later found that NIH group 21 also resembled
CP003028.1, respectively). the CDC enteric group 69 [50] , and E. kobei was
compared with the latter. On the basis of DNA
E. hormaechei relatedness, both organisms could be included in
E. hormaechei is named after Estenio a single taxon. However, the CDC enteric group
Hormaeche, a Uruguayan microbiologist who 69 was described as positive in Voges–Proskauer

future science group www.futuremedicine.com 891

 Review Mezzatesta, Gona & Stefani

and yellow pigmentation [50] , whereas all strains in 2010, a pseudobacteremia was reported to
of E. kobei were Voges–Proskauer- and pigmen- be caused by contaminated saline cotton at
tation-negative. These findings suggest that the the venipuncture sites where blood was drawn
relationship of both organisms is at the sub­species [54] . There have also been a limited number of
or biogroup level. The type strain of E. kobei is reports regarding E. kobei infections of clinical
NIH 1485-1479 and was isolated by blood culture significance; a case of nosocomial urosepsis has
of a diabetic patient. been described as being caused by an E. kobei
strain that was Voges–Proskauer test-positive and
E. ludwigii resistant to b-lactams, ciprofloxacin, gentamicin
E. ludwigii, named after Wolfgang Ludwig, a and cotrimoxazole [16] . The literature on E. lud-
microbiologist working in bacterial systematics wigii reports type strain EN-119T, as described
[51] and who developed the ARB databases as above, and an imipenem-resistant E. ludwigii
well as making them public [52] . This descrip- producing VIM-2 located on a plasmid. This
tion is based on the phylogenetic analyses of microorganism was isolated from sewage water
partial hsp60 sequence data collected in a pop- in a hospital of Mallorca (Spain), suggesting that
ulation genetic study [6] , as well as on DNA– environmental bacteria represent a reservoir for
DNA hybridization assays and phenotypic the dissemination of metallo-b-lactamase (MBL)
characterizations. genes [55] .
The type strain EN-119T was isolated from Most isolates of the E. cloacae complex are sus-
midstream urine of an 18-year-old male patient ceptible to fluoroquinolones, trimethoprim/sulfa-
with a nosocomial urinary tract infection methoxazole, chloramphenicol, amino­glycosides,
while he was hospitalized at the Grosshadern tetracyclines, piperacillin–tazobactam and carba­
University-Hospital Munich, Germany. The penems, while they are intrinsically resistant to
GenBank accession number of the 16S rDNA ampicillin, amoxicillin, amoxicillin–clavulanate,
of strain EN-119T is AJ853891 [12] . first-generation cephalosporins and cefoxitin
owing to the production of constitutive AmpC
E. nimipressuralis b-lactamase. In particular, fosfomycin seems
The species E. nimipressuralis was originally to have a different activity against all species,
defined by Brenner et al. and was formerly called because E. cloacae and E. asburiae are both natu-
Erwinia nimipressuralis, which was isolated from rally susceptible and resistant while E. hormaechei
nonclinical sources (e.g., elm trees with a disease is only naturally sensitive [53] .
called wet wood) [43] . Erwinia nimipressuralis The production of b-lactamases is the most
was inserted in the Approved Lists of Bacterial important mechanism responsible for b-lactam
Names in 1980. This microorganism is bio- resistance in most of these species. This pheno­
chemically similar to E. cloacae, but it is different typic group of enzymes is a heterogeneous
for acid production from sucrose and raffinose, mixture of b-lactamases belonging to the
whereas E. cloacae is positive in these tests. The four molecular Ambler classes: class A (peni-
type strain of E. nimipressuralis is ATCC 9912 cillinases), class B (metalloenzymes), class C
and isolated from the elm Ulmus spp. in the USA (cephalosporinases) and class D (oxacillinases).
(GenBank accession number AJ567900). All kinds of b-lactamases are found in E. cloa-
cae and, to a lesser extent, in E. hormaechei and
Antibiotic resistance of the E. cloacae E. asburiae.
complex These microorganisms are capable of over­
The in vitro activity of a variety of antimicro- producing AmpC b-lactamases by derepression
bial agents against the E. cloacae complex has of a chromosomal gene, or by the acquisition of a
been examined by numerous investigators, but transferable ampC gene on plasmids or on other
the majority of studies are focused on E. cloa- mobile elements, commonly named as being
cae, E. hormaechei and E. asburiae [53] , which are AmpC plasmid-mediated. The AmpC plasmid-
the species most frequently isolated from clinical mediated strains are distinguished from chromo-
sources. somal strains because, with only a few exceptions,
Table 2 shows the resistance mechanisms of they are not inducible. The AmpC plasmid-medi-
species of the E. cloacae complex to each of the ated strains represent a problem because the dere-
major classes of antibiotics in published reports. pression of this enzyme is increasingly frequent
E. nimipressuralis is a plant pathogen and among clinical isolates and confers resistance to
has not been associated with human diseases. third-generation cephalo­sporins and ureido- and
There are very few reports about this species; carboxy-penicillins, and they are not inhibited

892 Future Microbiol. (2012) 7(7) future science group

 Enterobacter cloacae complex: clinical impact & emerging antibiotic resistance Review

Table 2. Mechanisms of antibiotic resistance in the Enterobacter cloacae complex.

Antibiotic Resistance type Resistance Enzyme type Species Ref.
class mechanism
b-lactams Enzymatic degradation Ambler class A TEM, SHV, CTX-M Enterobacter cloacae, [14,16,63,64]
(b-lactamases) Enterobacter hormaechei
VEB, GES/IBC, KPC E. cloacae [62,65,80,81,83,84,116]

NMC-A, IMI-1, IMI-2 E. cloacae, [74–79,82]

Enterobacter asburiae
Ambler class B VIM E. cloacae, [55,89,92]
Enterobacter ludwigii
IMP, NDM-1 E. cloacae [86–88,90,91,96,97]

Ambler class C Amp-C, ACT-1 E. cloacae, E. asburiae, [57,113]

E. hormaechei,
Enterobacter kobei
Ambler class D OXA-48 E. cloacae [98,101]

Fluoroquinolones Plasmid-mediated Acetyltransferase AAC(6´)-Ib-cr E. cloacae [108,109,111,116]

quinolone resistance
Qnr-mediated Qnr (A, B, S, C, D) E. cloacae, E. hormaechei [46,115]
topoisom protection
Efflux-pump QepA E. cloacae [107]

Aminoglycosides Enzymatic Acetyltransferase AAC(6´)-Ib E. cloacae [118,119]

modification
Tigecycline Target overproduction Efflux pump AcrAB E. cloacae, E. hormaechei [122,124]

by common inhibitors of b-lactamases, such as b-lactamases may not be critical, since the thera-
clavulanate, but by boronic acid and/or cloxacillin peutic options for infections caused by organisms
instead. Fourth-generation cephalosporins retain that possess any of these mechanisms of resistance
reasonable activity against derepressed strains, but are similarly limited. Nevertheless, the detection
if they are also extended-spectrum b-lactamase of such ‘hidden’ ESBLs is still of epidemiologi-
(ESBL) producers, they become resistant to this cal importance for the hospital environment [59] .
class of antibiotics [56] . ESBLs and AmpC-type The detection of ESBLs by methods based on the
cephalo­sporinases that can hydrolyze third- inhibitory effects of clavulanic acid (conventional
generation cephalosporins such as cefotaxime or double-disk synergy test [DDST] and Vitek
ceftazidime confer resistance more frequently to ESBL detection test) is expected to be difficult
E. cloacae strains isolated in patients who previ- and is dependent on the level of chromosomal
ously received these antibiotics [29] . E. hormae- enzyme production. Studies by Tzelepi et al. sug-
chei is also an AmpC producer, and it has been gested that the ESBL detection methods used are
recently demonstrated that the plasmid–mediated inadequate in cases of overproduction of Bush
AmpC blaACT-1 gene originated from the chro- group 1 b-lactamases [60,61] .
mosome of E. hormaechei, which was previously In a recent report, the Vitek ESBL detec-
believed to derive from E. cloacae [57] . tion test, as well as the conventional DDST,
ESBLs are of molecular class A, in the classifi- were unable to detect the SHV-5 b-lactamase
cation scheme of Ambler. They are b-lactamases in a Klebsiella pneumoniae strain that produced
capable of hydrolyzing penicillins, first-, second- a plasmid-borne AmpC-type b-lactamase; the
and third-generation cephalosporins and aztreo- ESBL present in that strain had been successfully
nam (but not cephamycins or carbapenems), and detected by a DDST that combined amoxicil-
are inhibited by b-lactamase inhibitors such as lin/clavulanate with cefepime. In accordance
clavulanic acid. with this observation, the use of cefepime
Moreover, recent studies suggest a high increased the sensitivity of the DDST for the
prevalence of ESBL production in these species, detection of ESBLs in Enterobacter from 16 to
thus limiting the choice of antimicrobial agents 61% when the disks were applied at the stan-
capable of treating invasive infections [58] . dard distance of 30 mm from clavulanate, and
From a clinical point of view, the discrimina- from 71 to 90% with closer application of the
tion between ESBLs and overproduced class C disks [61] .

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 Review Mezzatesta, Gona & Stefani

The findings of this study indicate that the fre- as KPC and GES, or both, such as IMI. NMC-A
quency of ESBLs can easily be underestimated in (not metallo­enzyme carba­penemase) was detected
Enterobacter spp. characterized by a high preva- for the first time in 1990 in Paris (France) and
lence of derepressed variants. For such situations, appeared in Argentina in 2000 and in the USA
the application of DDSTs that combine amoxicil- in 2003 [74–76] . The chromosome-­borne IMI-1
lin/clavulanate with cefepime may increase the (imipenem-­hydrolyzing b-lactamase) enzyme
possibility of ESBL detection because they are appeared for the first time in E. cloacae strains in
important factors to be considered in the man- 1984 in California and the most recently in 2011
agement of antibiotic-resistant E. cloacae infec- in France [77] , while plasmid-encoded IMI-2 was
tions, and their spread in the community is a isolated in 2001 in China [78,79] . The IMI and
general concern. NMC-A enzymes have been detected in rare clini-
E. cloacae may express ESBLs that are not cal isolates of E. cloacae in the USA, France and
closely related to the TEM or SHV type, includ- Argentina [76,80,81] . NMC-A and IMI-1 have 97%
ing CTX‑M- and VEB-type ESBLs [62–64] . VEB- amino acid identity [82] . The IMI-2 enzymes were
producing Enterobacteriaceae have been pre- also found on plasmids in 22 imipenem-resistant
dominantly found in Vietnam and Thailand; an E. asburiae that naturally produce a cephalospo-
E. cloacae outbreak was recently documented in rinase, but not carba­penemase. In these strains,
China [65] . Another important factor that concerns which were isolated from US rivers sampled from
ESBLs is that they are typically plasmid-mediated, 1999 to 2001, an upstream LysR-type regula-
rather than chromosomal-mediated b-lactamases; tor gene was identified; this regulation can be
this is of great concern given their easy spread. responsible for the inducibility of IMI-2 expres-
In 2005, a study by Ho reported bloodstream sion. b-lactamase IMI-2 is therefore the first
infections by E. hormaechei isolates producing inducible and plasmid-encoded carba­penemase.
CTX-M-, SHV- and TEM-related enzymes [66] . The E. asburiae isolates were clonally related,
Of the E. hormaechei (n.539) isolates, 17.9% were belonging to a single pulse-field type, although
ESBL-positive, which was frequently attributed to they were obtained from distantly related mid-
blaCTX‑M genes [66] . In a neonatal unit of an African western rivers; the identification of clonally related
hospital, CTX-M 15 E. hormaechei strains were E. asburiae isolates from distant rivers indicates
also detected from powdered milk [14] . an environmental and enterobacterial reservoir for
The E. cloacae complex produces not only carbapenemase genes [82] .
ESBLs, but also b-lactamases with carbapen- The GES/IBC family of b-lactamases is an
emase activity that have a broader range activ- infrequently encountered family of enzymes that
ity and may be grouped as either serine carba­ was first described in 2000 with reports of IBC‑1,
penemases (classes A and D) or MBLs (class B) a novel class A ESBL that was encoded by a class 1
according to Ambler classification [67,68] . integron-associated gene (integron-borne cepha-
For the screening of carbapenemase-produc- losporinase) from an E. cloacae isolate in Greece
ing Enterobacteriaceae, CHROMagar™ KPC [83] . This integron was located in a multidrug-
(Hy-Labs, Israel) commercial medium could be resistant (MDR) transferable plasmid found in
used, where Enterobacter spp. appears as metallic an E. cloacae clinical strain.
blue colonies [69] . The phenotypic tests for the con- The most recently reported class A enzyme
firmation of carbapenemases production are mod- with carbapenemase activity is KPC, which is
ified Hodge test (classes A and D), the combined found mostly on plasmids in K. pneumoniae;
disk of meropenem ± boronic acid (class A) and these enzymes also hydrolyze the cephalo­
the combined disk of meropenem ± di­picolinic sporins, such as cefotaxime. Although the
acid or meropenem/imipenem ± ethylene diamine KPC b-lactamases are predominantly found in
tetra-acetic acid (class B) [70,71] . K. pneumoniae, there are few reports of the pres-
Carbapenemase-producing E. cloacae are cur- ence of these enzymes in E. cloacae. KPC-2 and
rently relatively rare, but there are concerns about KPC-3 determinants were found in 2001–2003
their emergence and spread. The class A carba­ in New York from a patient with renal failure and
penemases appeared sporadically in clinical isolates continuous bacteremia [81,84] .
since their first discovery over 20 years ago [72,73] . Another mechanism of carbapenem resistance
They have the ability to hydrolyze a broad variety observed in E. cloacae is MBL production. These
of b-lactams, including carba­penems, and are usu- enzymes are capable of hydrolyzing all b-lactams,
ally inhibited by clavulanate and tazobactam. They except monobactams, and are not susceptible
can be chromosome-encoded, such as NMC-A, to therapeutic b-lactamase inhibitors such as
SME, SFC-1 and BIC-1, plasmid-­encoded, such clavulanate, sulbactam and tazobactam [85] .

894 Future Microbiol. (2012) 7(7) future science group

 Enterobacter cloacae complex: clinical impact & emerging antibiotic resistance Review

MBLs can be either chromosomally encoded or was first identified in a K. pneumoniae isolate from
plasmid-mediated, and most of them are inserted Turkey with a worldwide spread. In addition to
in integrons. The most commonly acquired K. pneumoniae, E. coli, Citrobacter freundii and
MBL enzymes include IMP, VIM, SPM, GIM, Providencia rettgeri, OXA-48 has been identified
SIM and NDM; IMP and VIM are common in in E. cloacae in different countries [98–101] .
Pseudomonas aeruginosa and other nonferment- Finally, the resistance to carbapenems may
ing Gram-negative bacteria. The first appearance be due to the hyperproduction of an AmpC-
of MBLs in E. cloacae were reported in E. cloa- type cephalosporinase or ESBL combined with
cae clinical isolates from various geographical decreased drug permeability through the outer
areas, with IMP-1 from Turkey [86] , IMP-8 from membrane, and carbapenem-hydrolyzing b-lac-
Taiwan [87] , VIM-2 from Korea [88] , VIM 1–4 tamases [102] . The hyperproduction of chromo­
from Italy [89–91] and VIM-12 from Greece [92] . somal cephalosporinase combined with porin
Even if these papers describe the presence alterations confers imipenem resistance to clinical
of the enzymes, alone or associated with other isolates of E. cloacae [103,104] .
determinants, they are probably under-reported The E. cloacae complex has also acquired resis-
due to the difficulties related to their recogni- tance to another widely used class of antibiotics
tion by standard susceptibility tests; in some – fluoroquinolones – which are extensively used
cases, only ertapenem showed a reduced sus- in clinical therapy of sustained Enterobacteriaceae
ceptibility phenotype. These findings are con- infections. These antibiotics act by interacting
cordant with some reports that analyzed the with type II topoisomerases (DNA gyrase and
microbiological features of VIM-1-producing topoisomerase IV). Related to this mechanism
Enterobacteriaceae, in which ertapenem was of action, bacteria have developed resistance
shown to be a better indicator for phenotypic mechanisms consisting of target mutations
carbapenemase detection [93,94] . In an Italian GyrA/GyrB for DNA gyrase and ParC/ParE
study, most of the VIM-1-producing E. cloacae for topoisomerase IV, or in reduced access to the
had imipenem and meropenem MICs within the target itself, by either decreased permeability or
range of susceptibility, while ertapenem MICs augmenting expression of efflux pumps, such as
were between 0.25 and 8 mg/l. The patients AcrAB and MexAB [105,106] . The genes encoding
included in this study, who were treated with these mechanisms are of chromosomal origin,
carbapenem, had therapy failure, which led to but since 1998, plasmid-mediated quinolone-
the investigation of their mechanisms of resis- resistance genes have been reported in clinical
tance. All strains were VIM-1 producers, and the Enterobacteriaceae isolates. These genes are qnr
discrepancy with phenotypic tests was explained (qnrA, qnrB, qnrS, qnrC and qnrD), aac(6´)‑Ib-cr
by low expression levels of the bla VIM-1 gene that and a plasmid-mediated efflux pump qepA [107] ,
is related to an inactivated P2 promoter and to which confer low-level resistance to quinolones
a weak activity of another promoter, P1, which [108–110] and have an additive effect on the level of
drives the expression of the gene cassette inserted resistance caused by other mechanisms. The lit-
into the class 1 integron, which was detected in erature reports quinolone resistance in E. cloacae
all E. cloacae isolates carrying VIM-1 [91] . and E. hormaechei (Table 2) .
The latest MBL detected is New Delhi MBL From the epidemiological point of view, in the
found in K. pneumoniae and Escherichia coli last few years, quinolone resistance has increased
isolates from a patient coming from India in E. cloacae isolates worldwide [108,109,111] . The
and hospitalized in Sweden in 2008 [95] . This qnr genes were found to be cocarried with vari-
enzyme hydrolyzes all b-lactam antibiotics ous ESBLs or AmpC-type b-lactamases on the
except for aztreonam, which is usually inacti- same plasmid [112–114] , as well as with MBLs [115] .
vated by coproduced ESBLs or AmpC b-lac- Unfortunately, increasing in vitro resistance of
tamases. The rapid spread of this gene, which ESBL producers to fluoroquinolones is limiting
has already been described in several species of the role of these important classes of antibiotics
Enterobacteriaceae, has also been detected in for the future. In a recent report, the associa-
E. cloacae worldwide [96,97] . tion of qnrB2 with blaKPC-2 carbapenemase was
Acquired class D b-lactamases possessin­g described on a single plasmid in two genetically
carba­penemase properties were previously identi- unrelated clones of E. cloacae [116] and this report
fied mainly in Acinetobacter spp. and occasionally highlights a very worrying problem for future
in Enterobacteriaceae. The chromosome-­encoded treatment of these infections.
oxacillinase OXA-23 was previously described for A nationwide outbreak in The Netherlands
Proteus mirabilis, and the oxacillinase OXA‑48 was caused by E. hormaechei strains carrying a

future science group www.futuremedicine.com 895

 Review Mezzatesta, Gona & Stefani

conjugative plasmid pQC, with several complex Asia–Pacific rim and Latin America. Middle
integrons containing qnrA1, aadB, blaCTX-M-9 and East and Africa were excluded because of the
tetA genes [46] . low numbers of isolates.
With regard to another class of antibiotics – Although tigecycline has good in vitro activ-
the aminoglycosides – the major mechanism ity against E. cloacae, a few clinical strains of
of resistance of the Enterobacteriaceae is usu- E. cloacae with decreased susceptibility to tige-
ally attributable to aminoglycoside-modifying cycline have been recently described, resulting
enzymes that are often plasmid-encoded, but may from their RamA-mediated overexpression of
also be associated with transposable elements. the AcrAB efflux pump [122,123] .
Plasmid exchange and dissemination of transpo- A recent case report describes a tigecycline-
sons facilitate the rapid acquisition of resistance resistance induction under tigecycline therapy
phenotypes [117] . These enzymes are assigned in an E. hormaechei strain isolated from a patient
to three groups: acetyltransferases (acetylation after liver transplantation [124] .
of an amino group/AAC), phosphotransferases Over the last few years, an old antibiotic, colis-
(phosphorylation of a hydroxyl group/APH) and tin, has been reintroduced in clinical therapy
adenylyltransferases (adenylylation of a hydroxyl because it is active against several MDR aerobic
group/AAD or ANT). Gram-negative bacilli, including Enterobacter
Among these, the aminoglycoside AAC(6´)-Ib spp. [125] . The limited usage of colistin in the
is the most common cause of amikacin resistance 1980s was due to reports of a high incidence of
among members of the Enterobacteriaceae fam- toxicity [126,127] , which may have been due to
ily [118] . In a previous study, it was observed inappropriate patient selection and monitoring
that over 40% of the E. cloacae isolates had the [128] . Early clinical papers report the results of
aac(6´)-Ib gene, although many of the isolates pharmokinetic studies to reduce and optimize
with this gene were susceptible to amikacin the doses of colistin, which remains a valid alter-
and gentamicin, which were the most active of native for the treatment of infections caused by
all drugs tested [119] . The association between MDR enterobacteria [129] .
aac(6´)-IIc and bla VIM-1 has been reported in the The data on the mechanisms of resistance to
same integron previously described in P. aerugi- polimixins in Enterobacteriaceae suggest that
nosa and E. coli, suggesting extensive spread of they are due to the changes in negatively charged
resistance genes between various Gram-negative surface lipopolysaccharides induced by the regu-
species [120] . latory loci pmrA and phoP, generating resistance
A valid alternative to carbapenems and fluoro­ to these antibiotics [130] . Previously published
quinolones is tigecycline, especially against data report a case of heteroresistance to colistin
clinical isolates exhibiting MDR phenotypes. in E. cloacae isolates and two colistin-resistant,
Tigecycline, a novel broad-spectrum glycyl­ VIM-1-producing E. cloacae strains isolated
cycline antibiotic derivative of minocycline, from a patient with a complicated urinary tract
has the ability to evade common mechanisms infection [91,131] .
of resistance to tetracyclines expressed in
Gram-negative and Gram-positive bacteria. Future perspective
The vast majority of studies evaluating the Species of the E. cloacae complex, especially
in vitro susceptibility of tigecycline against E. cloacae, E. asburiae and E. hormaechei, are
species belonging to the E. cloacae complex are causing severe infections in community and
focused on E. cloacae, while there are no data nosocomial outbreaks; therefore it is important
on other species of the E. cloacae complex. A to identify them correctly, because incorrect
recent paper reporting results from studies on identification could underestimate the infections
23,918 Gram-negative isolates from intensive caused by these microorganisms.
care units worldwide between 2004 and 2009 Most clinical laboratories in developed
demonstrated excellent activity of tigecycline countries routinely identify Enterobacter spp.
against 3789 E. cloacae isolates collected, with by phenotypic methods employing commer-
a susceptibility >90% for all regions, while the cially available kits or semiautomated systems
largest variation in susceptibility was seen for that are limited to E. cloacae and E. asburiae,
minocycline (from 79.2% in North America while for further identification and discrimi-
to 59.0% in the Middle East) [121] . Resistance nation of the other species in this genus, bio-
to minocycline increased from 2004 to 2009 chemical tests or molecular methods such as
between 24 and 30.8% among isolates of E. clo- 16S rRNA, rpoB and hsp60 gene sequencing
acae collected from North America, Europe, the should be used.

896 Future Microbiol. (2012) 7(7) future science group

 Enterobacter cloacae complex: clinical impact & emerging antibiotic resistance Review

A high risk of antibiotic failure in treating be inappropriate; therefore, colistin, although

infections sustained by these microorganisms nephrotoxic, alone or in combination with other
could exist among patients treated with carba­ antibiotics (cephalosporins, quinolones, amino-
penem therapy because these species could be glycosides or carbapenems), may represent the
VIM producers. For the phenotypic detection only effective therapeutic option against MDR
of carbapenemase, MIC screening for mero­ Enterobacter spp. [132] .
penem could be set at ≥0.5 mg/l. For sporadic Members of the E. cloacae complex are part
carbapenemase producers with low MIC values of the endogenous flora of humans and conse-
for meropenem (<0.5 mg/l), carbapenemase is quently of patients who have become chronically
not detected; therefore, ertapenem, even if less colonized, making it difficult to apply generalized
specific, remains a valid marker [70,91,202] . strategies of infection control for these micro­
MBL-producing E. cloacae are unusual causes organisms that are able to acquire resistance deter-
of severe infections in compromised hosts, minants and so becoming MDR [133,134] . Prompt
and empiric therapy with carbapenems may attention should be given to rapid detection of

Executive summary
Taxonomy & identification
„„The Enterobacter cloacae complex includes six species: Enterobacter cloacae, Enterobacter asburiae, Enterobacter hormaechei,

Enterobacter kobei, Enterobacter ludwigii and Enterobacter nimipressuralis, which are all closely related to E. cloacae.
„„The E. cloacae complex exhibits the same phenotypic characteristics of the genus Enterobacter.

„„Only E. cloacae and E. asburiae may be identified by conventional methods.

„„For identification of all species of this taxon, additional biochemical and molecular methods such as 16S rRNA, hsp60 and rpoB genes

are needed.
Mechanisms of pathogenicity
„„Enterobacter spp. strains produce enterotoxins, a-hemolysin and thiol-activated pore-forming cytotoxins similar to Shiga-like toxin II.

„„The type III secretion system delivers virulence factors, usually toxins, into the host cells.

„„Type III secretion system genes were found in E. cloacae strains, indicating that this system may contribute to its pathogenesis.

„„Curli fimbriae seem to be involved in the formation of biofilms in E. cloacae.

Epidemiology & molecular typing

„„The clonal outbreaks of species belonging to the E. cloacae complex are being increasingly reported.

„„Pulsed-field gel electrophoresis and PCR-based fingerprinting are useful for typing of the E. cloacae complex; pulsed-field gel

electrophoresis has the ability to detect minor mutations among outbreak strains.
Species of the E. cloacae complex
„„E. cloacae is the most important within this taxon because it is responsible for bacteremia and lower respiratory, urinary, skin and

intra-abdominal nosocomial infections.

„„E. asburiae was originally isolated from the soil, but some strains have been isolated from human sources.

„„E. hormaechei consists of three different subspecies: E. hormaechei subsp. oharae, E. hormaechei subsp. hormaechei and

E. hormaechei subsp. steigerwalti. E. hormaechei is an important nosocomial pathogen also isolated in neonatal intensive care units.
„„E. kobei differs from E. cloacae because it is Voges–Proskauer-negative. There very few clinical reports about this species.

„„E. ludwigii is an imipenem-resistant strain was isolated from sewage water in a hospital.

„„E. nimipressuralis was isolated from nonclinical sources.

Antibiotic resistance of the E. cloacae complex

„„The E. cloacae complex is naturally susceptible to fluoroquinolones, trimethoprim/sulfamethoxazole, chloramphenicol, aminoglycosides,

tetracyclines, piperacillin–tazobactam and carbapenems.

„„The E. cloacae complex is intrinsically resistant to ampicillin, amoxicillin, amoxicillin–clavulanate, first-generation cephalosporins and

cefoxitin for constitutive AmpC b-lactamase production.

„„Resistance to b-lactams is due to extended-spectrum b-lactamase production, such as TEM-, SHV-, CTX-M- and VEB-type enzymes,

which are only found in E. cloacae and E. hormaechei.

„„Carbapenemase-producing E. cloacae are currently relatively rare. However, in the last decade, they have been increasingly isolated

from clinical sources.

„„The qnr, aac(6´)-Ib-cr and qepA genes confer low resistance to fluoroquinolones. Published studies report resistance in E. cloacae and

in E. hormaechei.
„„Aminoglycoside resistance is due to the enzymatic modification by AAC(6´)-Ib acetyltransferase found in E. cloacae.

„„Tigecycline has good activity against E. cloacae strains.

„„Colistin resistance is rare in E. cloacae complex species.

future science group www.futuremedicine.com 897

 Review Mezzatesta, Gona & Stefani

antibiotic-resistant nosocomial pathogens, espe- conclusion that we need urgent action to slow
cially carbapenem-resistant Enterobacteriaceae. down and control the worldwide epidemic
The CDC recently proposed a protocol for the spread of these bacteria, especially of the emerg-
detection of carbapenemases in Klebsiella spp. ing carbapenemase-producing E. cloacae com-
and E. coli in stool samples [133] . Some authors plex strains because they are transmitted by
indicate the use of CHROMagar KPC or mobile genetic elements.
McConkey agar with carbapenem disks or PCR
for blaKPC for KPC-producing Enterobacteriaceae Acknowledgements
from rectal swabs. Of these three methods, PCR The authors would like to thank A Bridgewood for the
has a higher sensitivity and specificity but is the language revision of this manuscript.
most expensive; CHROMagar and McConkey
agar have the same sensitivity and specificity, but Financial & competing interests disclosure
McConkey agar is cheaper [69,135] . The authors have no relevant affiliations or financial
The pathogenetic mechanisms and contrib- involvement with any organization or entity with a
uting factors in disease development in the financial interest in or financial conflict with the sub-
E. cloacae complex are still not well understood; ject matter or materials discussed in the manuscript.
this could be due to the scarcity of information This includes employment, consultancies, honoraria,
available. stock ownership or options, expert testimony, grants or
All these observations, together with the patents received or pending, or royalties.
awareness of the limited therapeutic options to No writing assistance was utilized in the production
treat these infections, bring us to the alarming of this manuscript.

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