Specific distribution within the Enterobacter cloacae complex of strains isolated from infected orthopedic implants.
ABSTRACT Bacteria belonging to the Enterobacter genus are frequently isolated from clinical samples but are unusual causative agents of orthopedic implant infections. Twelve genetic clusters (clusters I to XII) and one sequence crowd (sequence crowd xiii) can be distinguished within the Enterobacter cloacae nomenspecies on the basis of hsp60 sequence analysis, and until now, none of these clusters could be specifically associated with a disease. In order to investigate if specific genetic clusters would be involved in infections of orthopedic material, two series of bacterial clinical isolates identified as E. cloacae by routine phenotypic identification methods were collected either from infected orthopedic implants (n = 21) or from randomly selected samples of diverse anatomical origins (control; n = 52). Analysis of the hsp60 gene showed that genetic clusters III, VI, and VIII were the most frequent genetic clusters detected in the control group, whereas cluster III was poorly represented among the orthopedic implant isolates (P = 0.006). On the other hand, E. hormaechei (clusters VI and VIII), but not cluster III, is predominantly associated with infections of orthopedic implants and, more specifically, with infected material in the hip (P = 0.019). These results support the hypothesis that, among the isolates within the E. cloacae complex, E. hormaechei and hsp60 gene sequencing-based cluster III are involved in pathogenesis in different ways and highlight the need for more accurate routine Enterobacter identification methods.
- [show abstract] [hide abstract]
ABSTRACT: The aim of the study was to determine the impact of octenidine hydrochloride and gentamicin on bacterial survival and reduction of biofilms formed on orthopaedic metal implants. We studied metal orthopaedic components (screws, nails, fragments of wires used in Ilizarov devices) and a bone sequester. The presence and intensity of biofilm formation on the medical biomaterials was determined using the method of Richards et al. by visual evaluation of 2,3,5-triphenyl tetrazolium chloride (TTC) reduction by viable bacteria. The presence and structure of the biofilm on the components of the Ilizarov device, screws and bone sequester was also studied by electron microscopy. Bacterial survival in the biofilm following exposure to the antibiotic and antiseptic was studied by CLSI microdilution method in microtitre plates using TTC. Results. Most of the 16 strains (S. aureus, S. epidermidis, E. coli, Enterobacter) isolated from orthopaedic implants were able to form a biofilm. Established biofilms were resistant to gentamicin and octenidine hydrochloride but demonstrated greater susceptibility to octenidine. The results of the study indicate that octenidine hydrochloride is more effective than gentamicin in the treatment of infections associated with the formation of a biofilm on orthopaedic implants.Ortopedia, traumatologia, rehabilitacja 9(3):310-8.
Article: Cronobacter gen. nov., a new genus to accommodate the biogroups of Enterobacter sakazakii, and proposal of Cronobacter sakazakii gen. nov., comb. nov., Cronobacter malonaticus sp. nov., Cronobacter turicensis sp. nov., Cronobacter muytjensii sp. nov., Cronobacter dublinensis sp. nov., Cronobacter genomospecies 1, and of three subspecies, Cronobacter dublinensis subsp. dublinensis subsp. nov., Cronobacter dublinensis subsp. lausannensis subsp. nov. and Cronobacter dublinensis subsp. lactaridi sub[show abstract] [hide abstract]
ABSTRACT: [Enterobacter] sakazakii is an opportunistic pathogen that can cause infections in neonates. This study further clarifies the taxonomy of isolates described as [E.] sakazakii and completes the formal description of the proposed reclassification of these organisms as novel species and subspecies within a proposed novel genus, Cronobacter gen. nov. [E.] sakazakii was first defined in 1980, however recent polyphasic taxonomic analysis has determined that this group of organisms consists of several genomospecies. In this study, the phenotypic descriptions of the proposed novel species are expanded using Biotype 100 and Biolog Phenotype MicroArray data. Further DNA-DNA hybridization experiments showed that malonate-positive strains within the [E.] sakazakii genomospecies represent a distinct species, not a subspecies. DNA-DNA hybridizations also determined that phenotypically different strains within the proposed species, Cronobacter dublinensis sp. nov., belong to the same species and can be considered as novel subspecies. Based on these analyses, the following alternative classifications are proposed: Cronobacter sakazakii gen. nov., comb. nov. [type strain ATCC 29544(T) (=NCTC 11467(T))]; Cronobacter malonaticus sp. nov. [type strain CDC 1058-77(T) (=LMG 23826(T)=DSM 18702(T))]; Cronobacter turicensis sp. nov. [type strain z3032(T) (=LMG 23827(T)=DSM 18703(T))]; Cronobacter muytjensii sp. nov. [type strain ATCC 51329(T) (=CIP 103581(T))]; Cronobacter dublinensis sp. nov. [type strain DES187(T) (=LMG 23823(T)=DSM 18705(T))]; Cronobacter dublinensis subsp. dublinensis subsp. nov. [type strain DES187(T) (=LMG 23823(T)=DSM 18705(T))]; Cronobacter dublinensis subsp. lausannensis subsp. nov. [type strain E515(T) (=LMG 23824=DSM 18706(T))], and Cronobacter dublinensis subsp. lactaridi subsp. nov. [type strain E464(T) (=LMG 23825(T)=DSM 18707(T))].International journal of systematic and evolutionary microbiology 06/2008; 58(Pt 6):1442-7. · 2.11 Impact Factor
- Journal of Hospital Infection 06/2007; 66(1):95-7. · 2.86 Impact Factor
JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 2009, p. 2489–2495
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 47, No. 8
Specific Distribution within the Enterobacter cloacae Complex of
Strains Isolated from Infected Orthopedic Implants?
Philippe C. Morand,1,2,3* Annick Billoet,2Martin Rottman,4,5Vale ´rie Sivadon-Tardy,5,6Luc Eyrolle,7
Luc Jeanne,7Asmaa Tazi,1,2,3Philippe Anract,1,8Jean-Pierre Courpied,1,8
Claire Poyart,1,2,3and Vale ´rie Dumaine8
Faculty of Medicine, Universite ´ Paris Descartes, Paris, France1; Department of Bacteriology, Cochin Hospital (AP-HP), Paris, France2;
Institut Cochin, INSERM U567, Paris, France3; Laboratoire de Microbiologie, Ho ˆpital Raymond Poincare ´ (AP-HP),
Garches, France4; EA 3647, Faculte ´ de Me ´decine Paris-Ile de France-Ouest, Universite ´ de Versailles-Saint-Quentin en Yvelines,
Garches, France5; Department of Microbiology, Ambroise Pare ´ Hospital (AP-HP), Boulogne-Billancourt, France6;
Department of Anesthesiology, Cochin Hospital (AP-HP), Paris, France7; and Department of
Orthopedic Surgery, Cochin Hospital (AP-HP), Paris, France8
Received 9 February 2009/Returned for modification 14 April 2009/Accepted 4 June 2009
Bacteria belonging to the Enterobacter genus are frequently isolated from clinical samples but are unusual
causative agents of orthopedic implant infections. Twelve genetic clusters (clusters I to XII) and one sequence
crowd (sequence crowd xiii) can be distinguished within the Enterobacter cloacae nomenspecies on the basis of
hsp60 sequence analysis, and until now, none of these clusters could be specifically associated with a disease.
In order to investigate if specific genetic clusters would be involved in infections of orthopedic material, two
series of bacterial clinical isolates identified as E. cloacae by routine phenotypic identification methods were
collected either from infected orthopedic implants (n ? 21) or from randomly selected samples of diverse
anatomical origins (control; n ? 52). Analysis of the hsp60 gene showed that genetic clusters III, VI, and VIII
were the most frequent genetic clusters detected in the control group, whereas cluster III was poorly repre-
sented among the orthopedic implant isolates (P ? 0.006). On the other hand, E. hormaechei (clusters VI and
VIII), but not cluster III, is predominantly associated with infections of orthopedic implants and, more
specifically, with infected material in the hip (P ? 0.019). These results support the hypothesis that, among the
isolates within the E. cloacae complex, E. hormaechei and hsp60 gene sequencing-based cluster III are involved
in pathogenesis in different ways and highlight the need for more accurate routine Enterobacter identification
Prosthetic joint infection (PJI) is, after aseptic loosening, the
second most frequent complication of prosthetic joint replace-
ment. Improvements in surgical techniques and the prevention
of infection have lowered the risk of infection for primary hip
or knee replacement to less than 1 and 2%, respectively, but
the incidence of infection can increase 10-fold in the case of
surgical revision (30). The population at risk for PJI continues
to steadily increase, with an estimated 1 million arthroplasties
being carried out worldwide each year (2), and the socioeco-
nomic burden could become considerable (14–16). The aver-
age cost of combined medical and surgical treatment of an
infected joint prosthesis is estimated to be $30,000, with im-
portant discomfort and substantial economic consequences for
the patient (4). Besides joint prosthetic implants, other im-
plantable devices such as screws and internal fixation devices
are also at risk for infection in a wide range of clinical situa-
tions. Gram-positive cocci are the most frequent pathogens
encountered, but members of the Enterobacteriaceae family
can also be involved (29). Among these, Enterobacter species
are major nosocomial pathogens often found in intensive care
settings, and their role in PJI remains to be documented.
Bacteria of the Enterobacter genus are widely encountered in
nature. These microorganisms are saprophytic in the environ-
ment and commensal in the enteric flora since they are found
in soil and sewage, as well as in the human gastrointestinal
tract (17, 22). The taxonomy of the Enterobacter genus has
been iteratively updated (8–12, 19). Several species are de-
scribed in this taxon (Enterobacter aerogenes, E. amnigenus, E.
cancerogenus, E. cowanii, E. gergoviae, E. intermedius, E. pyri-
nus), as is a genetic complex, referred to as the “E. cloacae
complex,” in which other species have been identified (E. as-
buriae, E. kobei, E. ludwigii, E. hormaechei, E. nimipressuralis,
and E. cloacae). E. sakazakii strains, which belong to several
genomospecies, were recently reassigned to the newly pro-
posed genus Cronobacter (12).
Enterobacter species can act as pathogens, and the E. cloacae
complex, commonly referred to as “E. cloacae,” represents the
Enterobacter group most frequently encountered in human
clinical samples. Although identification as “E. cloacae” is rou-
tinely performed by phenotypic methods in clinical laborato-
ries, the accurate identification of isolates within this taxon is
difficult. Analysis of the 16S rRNA gene is widely used for
bacterial identification, but it is poorly discriminatory for
closely related members of the Enterobacteriaceae family and,
more specifically, for members of the Enterobacter genus (27).
* Corresponding author. Mailing address: Service de Bacte ´riologie,
Ho ˆpital Cochin AP-HP, Universite ´ Paris Descartes, 27 rue du Fau-
bourg Saint-Jacques, 75014 Paris, France. Phone: 33 (0)1 58 41 27 91.
Fax: 33 (0)1 58 41 15 48. E-mail: email@example.com.
?Published ahead of print on 10 June 2009.
Other targets for use for the molecular identification of iso-
lates within the Enterobacter genus have been described, such
as the oriC locus (23), gyrB (6), rpoB (8, 21), and hsp60 (8).
Sequence analysis of a fragment of the hsp60 gene showed that
the E. cloacae nomenspecies could be divided into 12 genetic
clusters (clusters I to XII) and one sequence crowd (sequence
crowd xiii). Specific names could be attributed to some of the
genetic clusters: E. asburiae (cluster I) (9), E. kobei (cluster II)
(9), E. ludwigii (cluster V) (11), E. nimipressuralis (cluster X)
(8), E. cloacae subsp. cloacae (cluster XI) (9), and E. cloacae
subsp. dissolvens (cluster XII) (9). Although the name E. hor-
maechei was sometimes used as a generic name for strains
belonging to different hsp60 gene sequencing-based clusters
(20), clusters VI, VII, and VIII together formally constitute the
E. hormaechei species, which has three subspecies: E. hormae-
chei subsp. oharae (cluster VI), E. hormaechei subsp. hormae-
chei (cluster VII), and E. hormaechei subsp. steigerwallti (clus-
ter VIII) (10). Species names were not attributed to clusters
III, IV, and IX and to sequence crowd xiii, although at least
cluster III is of significant clinical importance (8, 26).
The degree of genomic diversity within the E. cloacae com-
plex was recently reassessed by more global genotypic meth-
ods. Multilocus sequence analysis (MLSA) identifies seven
clusters within the E. cloacae complex, and each of these cor-
responds to one or more hsp60 gene sequencing-based genetic
clusters. Microarray-based comparative genomic hybridization
analysis (CGH) showed two genetically distinct clades (21).
Most strains associated with clinical disease belong to the
youngest CGH-based clade, to which strains of hsp60 gene
sequencing-based clusters III, VI, and VIII also belong. The
second, and older, CGH-based clade comprises heterogeneous
strains, some of which are associated with commensalism.
Single-locus-based molecular methods, as well as global ap-
proaches, such as MLSA or CGH, suggested that some genetic
clusters are more prone to cause infection, although no specific
epidemiological association could be demonstrated (8, 21).
The repeated occurrence in our clinical laboratory of orthope-
dic implant infections due to E. cloacae isolates prompted us to
further investigate these strains. We analyzed strains isolated
from infected orthopedic devices and compared them to ran-
domly selected strains isolated from clinical specimens taken
from diverse anatomical sites. Analysis of the hsp60 gene
showed that among the genetic clusters of the Enterobacter
cloacae complex, clusters VI and VIII, but not cluster III, are
predominantly associated with infections of orthopedic im-
plants and, more specifically, with hip implants. These data
support the hypothesis that genetic clusters of the E. cloacae
complex are involved in pathogenesis in different ways and
highlight the need for more accurate routine methods for the
identification of Enterobacter species.
MATERIALS AND METHODS
Bacterial strains and epidemiological data. For the study group, strains were
collected from three large academic hospitals in the Paris, France, area (Am-
broise Pare ´ Hospital, Raymond Poincare ´ Hospital, and Cochin Hospital,
AP-HP) involved in the management of osteoarticular infectious conditions. In
each hospital, all bacterial strains isolated from orthopedic device-related surgi-
cal samples are systematically and prospectively collected and stored, irrespective
of the bacterial species, the anatomical site of isolation, or the type of infection.
For the study group, the collections were submitted to identical screening criteria
(1999 to 2006 for Ambroise Pare ´ Hospital, 2000 to 2006 for Raymond Poincare ´
Hospital, 2005 to 2007 for Cochin Hospital) for the selection of strains belonging
to the E. cloacae complex that were responsible for the infection of the ortho-
pedic implants. The following criteria were applied for inclusion of the strains in
the study: (i) identification of the infecting organism as E. cloacae with routine
phenotypic identification systems (the API 20E or the Vitek 2 system; Bio-
Merieux, Marcy l’Etoile, France), (ii) clinical evidence of infection of the
orthopedic implant in an adult patient, (iii) isolation of the E. cloacae strain from
surgical samples collected at the point of contact between surrounding tissue and
the implanted material (or from the implant itself) at the time of surgical
treatment, and (iv) involvement of the E. cloacae strain as a probable causative
agent for the infection and clinical management as such. Isolation of an E.
cloacae strain from a wound, sinus tract, or drainage was not sufficient to ascer-
tain its involvement in the implant infection; and such strains were not included
in the study. One strain per patient was included, and in the case of the isolation
of multiple isolates, the most clinically relevant isolate (i.e., from the implant
itself rather than from the surrounding tissue) was used. Twenty-one strains were
selected and are referred to as strains isolated from orthopedic implanted ma-
terial (Table 1). Since different types of implants were involved, only the ana-
tomical site of infection was considered for data analysis. All selected strains
recovered from storage at ?80°C were viable for subculture. Personal data (age,
sex, site of infection, mono- or polymicrobial infection) as well as the hospital
and the date of isolation were anonymously collected for the purposes of this
For the control group (Table 2), 52 randomly selected clinical strains routinely
identified as E. cloacae in the clinical laboratory at the academic Cochin Hospital
by use of the API 20E or the Vitek 2 identification system were prospectively
collected from clinical samples taken for diagnostic purposes from adult patients
during the year 2006; environmental isolates were excluded. Thus, the control
group was representative of E. cloacae strains from an adult population routinely
identified in a clinical laboratory. One isolate was included per patient, and the
strains collected were registered on the basis of the anatomical site of isolation,
as follows: skin and soft tissue (n ? 14), upper and lower respiratory tract (n ?
15), urine (n ? 12), joint or bone (in the absence of infected material; n ? 3),
intravascular catheter (n ? 2), blood (n ? 2), and gastrointestinal suppuration
with exclusion of feces (n ? 4). Personal data (age, sex, site of isolation) were
anonymously collected for the purpose of this study.
Identification methods. Bacterial DNA from bacterial colonies grown over-
night at 37°C on 5% horse blood agar plates was prepared for PCR analysis by
using the Instagene nucleic acid purification method (Bio-Rad, Marnes la Co-
quette, France). Partial sequencing of the hsp60 gene was performed by a pre-
viously described protocol (8). Briefly, oligonucleotide primers Hsp60-F (5?-GG
TAGAAGAAGGCGTGGTTGC-3?) and Hsp60-R (5?-ATGCATTCGGTGGT
GATCATCAG-3?) were used for genomic amplification of a 341-bp fragment of
the hsp60 gene. A negative control containing all reagents except the target DNA
(which was replaced by H2O) was included in each series. PCR was performed
on a GeneAmp PCR system 9700 apparatus (Applied Biosystems) for 30 cycles
by using the following conditions: 30 s at 94°C for denaturation, 30 s at 57°C for
annealing, and 60 s at 72°C for elongation. Both strands of the purified amplified
DNA fragment were sequenced by the BigDye Terminator cycle sequencing
protocol with the same primers used for the PCR. Chromatograms of the com-
plementary strands obtained with an ABI 313 apparatus (Applied Biosystems)
were assembled by using the VectorNTi suite of programs (Invitrogen Corp.). A
272-bp fragment of the hsp60 gene was obtained for the 73 strains, and the
sequence of the fragment was compared to reference sequences from strains
previously described in taxonomic studies (8) by using the Clustal W algorithm
(www.align.genome.jp). Sequence comparisons were exported as an unrooted
neighbor-joining tree with proportional branch lengths.
Statistical analysis. Epidemiological associations were analyzed by use of the
Fisher exact test and the corresponding two-tailed P value.
Nucleotide sequence accession numbers. The sequences of the following type
strains were retrieved from the GenBank database (the information in paren-
theses is the strain designation, GenBank accession number): E. asburiae (ATCC
35953, AJ417141), E. kobei (ATCC BAA260, AJ567899), E. cloacae subsp.
dissolvens (ATCC 23373, AJ417143) (9), E. ludwigii (EN-119, AJ417114) (11),
E. hormaechei subsp. oharae (EN-314, AJ543782), E. hormaechei subsp. hor-
maechei (ATCC 49162, AJ417108), E. hormaechei subsp. steigerwaltii
(CIP108489, AJ543908) (10), E. nimipressuralis (ATCC 9912, AJ567900), E.
cancerogenus (ATCC 33241, AJ567895), E. amnigenus (ATCC 3072,
AJ567894), E. cowanii (ATCC 107300T, AJ567896), E. gergoviae (ATCC
33028, AJ567897), E. pyrinus (ATCC 49851, AJ567901), C. sakazaki (ATCC
29544, AJ567902) (12), and E. aerogenes (AB008141). For E. cloacae subsp.
cloacae, no hsp60 sequence is available for strain ATCC 13047, but one can
be found under GenBank accession number AJ417142 (strain ATCC 13049),
2490MORAND ET AL.J. CLIN. MICROBIOL.
which was used in another study (13). The GenBank accession numbers for
previously described strains (8) are listed in the Fig. 1 legend. The GenBank
accession numbers for the clinical strains described in this work are FJ595719
Distribution of strains isolated from infected orthopedic
implants within the genetic clusters of the Enterobacter cloacae
complex. Enterobacter is an uncommon causative agent of or-
thopedic implant infections. Investigation of the databases of
three large academic orthopedic surgical centers in the Paris,
France, area yielded 21 cases of orthopedic device infections
due to E. cloacae on the basis of routine phenotypic identifi-
cation methods (Table 1). The infections were found to be
polymicrobial in 9 cases (43%), whereas isolates phenotypi-
cally identified as E. cloacae were found as the sole causative
agent in 12 patients (57%). Partial sequencing of the hsp60
gene allowed the identification of all isolates as part of one of
the clusters that form the E. cloacae complex (Fig. 1). Some
genetic clusters appeared to account for most of the cases: 15
strains (71%) belonged to the E. hormaechei species (clusters
VI and VIII), 2 (9%) belonged to cluster III, and 2 (9%)
belonged to cluster V (E. ludwigii). E. cloacae subsp. cloacae
and E. asburiae (clusters XI and I, respectively) were found
only once; and clusters II (E. kobei), IV, VII (E. hormaechei
subsp. hormaechei), IX, X (E. nimipressuralis), and XII (E.
cloacae subsp. dissolvens) were absent from among the isolates
in the study group.
Predominance of E. hormaechei in hip prosthetic infections.
Various anatomical sites were involved in orthopedic implant
infections. Surprisingly, E. hormaechei (clusters VI and VIII)
was the species involved in all cases (n ? 9) of prosthetic hip
infections, whereas strains isolated from other anatomic loca-
tions also belonged to other clusters (Fig. 2). The epidemio-
logical association of E. hormaechei with hip implants was
statistically significant compared to its association with other
anatomical implant sites (9/9 cases at hip implant sites versus
6/12 cases at other anatomical implant sites; P ? 0.019), but E.
hormaechei infection of hip implants could not be linked to
other demographic or clinical factors, such as age, a history of
wound infection, or an association with other causative agents
of infection. E. hormaechei subsp. steigerwaltii (cluster VII) was
the taxon that was more frequently involved (six of nine cases)
than E. hormaechei subsp. oharae (cluster VI; three of nine
cases). The subspecies E. hormaechei subsp. hormaechei was
not found in this series.
Molecular identification of routinely identified clinical E.
cloacae isolates. In order to investigate the species distribution
among the strains isolated from routine clinical samples, we
performed sequence analysis of the hsp60 gene from the con-
trol group, which consisted of 52 randomly selected clinical
strains (Table 2). Similar to strains isolated from infected os-
teoarticular implants, hsp60 sequencing showed that all strains
TABLE 1. Characteristics of strains isolated from orthopedic implanted materiala
Strain SexAge (yr) Hospital
Form of isolation
With associated flora
With associated flora
With associated flora
With associated flora
With associated flora
With associated flora
With associated flora
With associated flora
With associated flora
aThe ratio of males (M) to females (F) was 0.9. The mean age ? standard deviation was 51 ? 20 years. CCH, Cochin Hospital; APR, Ambroise Paré Hospital; RPC,
Raymond Poincaré Hospital.
TABLE 2. Characteristics of control strainsa
No. of patients................................................................................ 52
Mean age at time of isolation ? SD (yr)...................................65 ? 18
Anatomical site of isolation (no. of isolates).............................
Skin and soft tissue.................................................................... 14
Upper and lower respiratory tract........................................... 15
Joint or bone, in the absence of infected material................
aOne isolate from each patient was studied.
bM, male; F, female.
VOL. 47, 2009E. CLOACAE COMPLEX AND ORTHOPEDIC IMPLANTS2491
phenotypically identified as E. cloacae belonged to one of the
molecular clusters of the E. cloacae complex (Fig. 1).
Without distinction by the site of isolation (Fig. 2), three
clusters (clusters III, VI, and VIII) accounted for 90% of all
strains. Similar to the implant-associated strains, genetic clus-
ters belonging to the E. hormaechei species (clusters VI and
VIII) were predominant (48% of control strains). Interest-
ingly, cluster VII (E. hormaechei subsp. hormaechei) was ab-
FIG. 1. Neighbor-joining unrooted tree resulting from analysis of the hsp60 gene sequences of 73 clinical strains and previously reported
sequences. Isolation site of clinical strains: P, infected orthopedic implant; B, blood; J, joint or bone, in the absence of implanted material; G,
gastrointestinal tract; R, respiratory tract; S, skin and soft tissue; U, urine; K, intravascular catheter. For the previously described strains,
type strains are indicated (8, 10–12). Strains labeled EN were reported previously (8) and correspond to sequences with GenBank accession
numbers AJ417125, AJ417127, AJ543819, AJ567887, AJ543876, AJ543894, AJ567893, AJ543787, AJ543803, AJ543806, AJ543829, AJ543864,
AJ543882, AJ543804, AJ543789, AJ543781, AJ543784, AJ567846, AJ543776, AJ543808, AJ543877, AJ543807, AJ543866, AJ543775, AJ543878,
AJ543881, AJ543820, AJ567878, AJ567885, AJ543857, AJ543831, AJ543821, AJ543816, AJ543798, AJ567847, AJ543777, AJ543861, AJ543768,
AJ543855, AJ543870, AJ567881, and AJ543837. EH, E. hormaechei; EC, E. cloacae. The bar indicates the number of substitutions per site. Genetic
clusters are numbered according to the description provided previously (8).
FIG. 2. Distribution of clinical strains within the genetic clusters of the E. cloacae complex. All strains could be assigned to one of the previously
reported hsp60 gene sequencing-based genetic clusters of the E. cloacae complex. (A) Strains isolated from implanted orthopedic material at different
anatomical sites (n ? 21). Hip-associated strains exclusively belonged to the E. hormaechei species (clusters VI and VIII). (B) Randomly selected clinical
strains of diverse anatomical origins (n ? 52). S, skin and soft tissue; R, upper and lower respiratory tract; U, urine; J, joint or bone, in the absence of
infected material; K, intravenous catheter; B, blood; G, gastrointestinal tract. Irrespective of the site if isolation, cluster III accounted for 42% of the
VOL. 47, 2009E. CLOACAE COMPLEX AND ORTHOPEDIC IMPLANTS2493
sent from our study, and E. hormaechei subsp. steigerwaltii
(cluster VIII, n ? 18) was predominant over E. hormaechei
subsp. oharae (cluster VI, n ? 8).
Cluster III accounted for 42% of control clinical isolates;
thus, it occurred statistically more frequently within the control
group of isolates than within the isolates from infected ortho-
pedic implants (22/52 isolates versus 2/21 isolates; P ? 0.006).
These strains were recovered from various anatomical sites
(respiratory, urinary and gastrointestinal tract, skin or soft
tissue, and blood). The three bone and joint strains obtained in
the absence of material (Fig. 2, series J [joint or bone]) were
isolated from knee synovial fluid, knee soft tissue, and hallux
valgus and belonged to clusters III, VI, and VIII, respectively;
they thus represented each of the most frequently isolated
clusters in the control group. The other clusters, cluster II (E.
kobei, n ? 1), cluster IV (n ? 1), cluster V (E. ludwigii, n ? 1),
cluster IX (n ? 1), and cluster XI (E. cloacae subsp. cloacae,
n ? 1), were poorly represented. Clusters I, VII, X, and XII
and sequence crowd xiii were not found among the isolates in
the control group. Taken together, these results show that
cluster III, together with clusters VI and VIII, accounts for
most of the strains routinely isolated from clinical specimens.
In this work, we investigated the distribution of strains in-
volved in infections of osteoarticular implanted material within
the genetic clusters of the E. cloacae complex. On the basis of
hsp60 analysis, we show that the cluster distribution for in-
fected orthopedic implant-associated strains is different from
that for randomly selected clinical strains of various anatomical
origins. Our observation that all genetic clusters are not
equally involved in pathogenesis highlights the need for more
accurate routine bacterial identification tools and for a better
understanding of the pathogenesis of the E. cloacae complex.
All strains evaluated in this study (n ? 73) could be assigned
to 1 of the 12 genetic clusters (clusters I to XII) of the E.
cloacae complex. Only one strain was found to belong to clus-
ter XI, the type strain of which is E. cloacae subsp. cloacae,
which suggests that the widely used name E. cloacae is not
representative of most clinical strains of the taxon. Preliminary
identification as E. cloacae by conventional phenotypic identi-
fication methods (performed with the API 20E or the Vitek 2
system) was a prerequisite for the inclusion of strains in either
the control or the study group. Although they might have led
to possible underrepresentation of misidentified strains that
authentically belong to the E. cloacae complex, the phenotypic
identification methods performed with the API 20E and the
Vitek 2 systems can be considered reliable tools for identifi-
cation, as long as identification as “E. cloacae” is understood as
“belonging to the E. cloacae complex.”
Although patient populations vary from one hospital to an-
other, the results of our studies of the distribution of the
control strains within genetic clusters are concordant with
those published previously, since we observed that clusters III,
VIII, and VI account for most of the clinical isolates (8).
Strains belonging to hsp60 sequence analysis-based cluster III
were shown to gather into the previously described MLSA-
based cluster 1 as well as in CGH-based clade 2, with the latter
being associated with strains that are the most frequently cul-
tured in hospitals (21). Similarly, hsp60 sequence analysis-
based cluster VI (E. hormaechei subsp. oharae) and cluster
VIII (E. hormaechei subsp. steigerwaltii) were shown to gather
in MLSA-based cluster 2 but also to belong to the clinically
relevant CGH-based clade 2. Thus, our data showing a pre-
dominance of cluster III, VI, and VIII isolates among control
clinical strains of different anatomical origins are consistent
with data presented in previous reports on the genetic di-
versity of the strains within the E. cloacae complex and
support the congruence of CGH-based clade 2 with clini-
cally relevant samples.
Some genetic clusters were absent from our study. Cluster
VII (E. hormaechei subsp. hormaechei) harbors the original
species type strain and was also poorly represented in other
studies (8). Cluster X (E. nimipressuralis) is found in potable
water reservoirs but, to our knowledge, has never been asso-
ciated with human disease (13). Cluster XII (E. cloacae subsp.
dissolvens), formerly part of the genus Erwinia, was reassigned
to the Enterobacter genus and forms a subspecies of the E.
cloacae species. It is associated with plants (maize, coffee), but
no human infections have been reported (7, 9).
Although it was the largest cluster within the group of con-
trol strains (42%), cluster III was poorly represented within the
group of orthopedic implant-associated strains (9%), and this
difference was statistically significant (P ? 0.006). Low num-
bers of cluster III isolates emphasize the large proportion of
cluster VI (E. hormaechei subsp. oharae) and cluster VIII (E.
hormaechei subsp. steigerwaltii) isolates, both of which belong
to the E. hormaechei species. Hip joint prosthesis infections
appeared to be specifically associated with E. hormaechei,
since, in our series, all cases of infections of implants at this site
were due to isolates of either cluster VIII (n ? 6) or cluster VI
(n ? 3). The third subspecies of the taxon, E. hormaechei
subsp. hormaechei (cluster VII), was not found in our analysis.
The low prevalence of cluster III isolates within the group of
isolates from infected orthopedic implants compared to their
prevalence within the group of isolates from clinical samples of
other origins suggests a specific pathogenicity and reinforces
the need for a robust and discriminatory tool for the accurate
identification of isolates within the E. cloacae complex. In this
regard, hsp60 gene sequencing-based identification appeared
to be both discriminatory and easily implementable, whereas
other sequence-based molecular methods for the identification
of Enterobacter were not as accurate. The absence of a con-
sensus for the analysis of an rpoB DNA fragment (1 kb or 500
bp) has led to contradictory results, particularly for the genetic
discrimination of isolates within hsp60 gene sequencing-based
clusters III, VI, and VIII (8, 21), as well as to the confusing use
of the species name E. hormaechei for strains that do not
belong to hsp60 gene sequencing-based clusters VI to VIII
(20). Similarly, sequence analysis of the gene encoding the
DNA gyrase subunit B (gyrB) led to the hypothesis that most
clinical isolates assigned to the E. cloacae complex by pheno-
typic semiautomated methods would belong to the E. hormae-
chei species (6). Analysis of the hsp60 gene sequence was not
included in this study, but the absence of cluster III as a specific
group within the group of clinical strains evaluated in this study
might suggest that gyrB sequencing is not as discriminatory as
hsp60 gene sequence analysis. Further investigation is needed
to elucidate the latter point.
2494MORAND ET AL.J. CLIN. MICROBIOL.
The epidemiological association between E. hormaechei and
hip prosthetic implants allows new insights into previous ob-
servations to be made. First, the E. hormaechei species was
reported in a case of prosthetic hip infection, although it was
initially misidentified as Escherichia coli (25). This strain ex-
hibited a phenotype of small-colony-variant formation, which
was shown to be associated with regulation of the hemin up-
take system (24). Although we did not systematically search for
it, at least one of the strains involved in prosthetic infection
displayed such a phenotype, with each step of subculture on
solid medium leading to the emergence of fast- and slow-
growing bacterial colonies. Second, although the study did not
specifically refer to E. hormaechei, the ability of Enterobacter
species to participate in biofilm formation on orthopedic im-
plants was reported (1). Third, the ability of E. hormaechei to
colonize implanted catheters as a biofilm and to be responsible
for systemic infection was described (3, 5). The ability to grow
as small-colony variants and the formation of biofilms are
features frequently associated with the causative agents of or-
thopedic implant infections and contribute to the increased
difficulty of diagnosis and treatment of such infections (18, 28).
Taken together, the findings from the previous reports rein-
force our observation showing the predominance of E. hormae-
chei as the cause of orthopedic implant infections. Further
work is needed in order to identify the bacterial and host
factors specifically involved in the bacterial colonization of the
implanted material and in the pathophysiology of these infec-
We thank David Biau for precious advice on the statistical analysis.
This work was supported by Universite ´ Paris Descartes, Institut
Cochin, and Assistance Publique—Ho ˆpitaux de Paris.
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