Abstract
Six species and six additional genovars are combined within the so-called Enterobacter cloacae complex, with one of them being the species Enterobacter hormaechei. In a recent population genetic study, two genetic clusters were found in close phylogenetic proximity to the genetic cluster of E. hormaechei. In order to prove the hypothesis that these three genetic clusters belong to the same species, we performed cross-hybridization experiments in microplates with DNAs of representatives of each genetic cluster. The close phylogenetic relationship among the clusters was reflected by their relatively low ΔTm values, ranging from 0.3 to 4.8, confirming the hypothesis that the clusters are parts of the same species. These clusters can be distinguished from the other species of the E. cloacae complex, which have ΔTm values of 5.6 to 10.3. Forty-eight E. hormaechei strains from the different genetic clusters were phenotypically characterized with 129 biochemical tests. In this way, E. hormaechei could be differentiated from the other species of the E. cloacae complex because it tests negative in the 3-hydroxy-butyrate test. The three genetic clusters of E. hormaechei could also be differentiated from each other by using phenotypic tests. Hence, we propose three new subspecies of E. hormaechei corresponding to genetic clusters VI, VII, and VIII of the E. cloacae complex. E. hormaechei subsp. hormaechei comb. nov. corresponds to the original species description, as it gives negative results for the adonitol, d-arabitol, d-sorbitol, and d-melibiose tests and a positive result for the dulcitol test. E. hormaechei subsp. oharae subsp. nov. gives negative results for the dulcitol, adonitol, and d-arabitol tests and positive results for the d-sorbitol and d-melibiose tests. E. hormaechei subsp. steigerwaltii subsp. nov. gives a negative result for the dulcitol test and positive results for the adonitol, d-arabitol, d-sorbitol, and d-melibiose tests. Among the members of the E. cloacae complex, E. hormaechei seems to be the species most frequently recovered from clinical specimens.
The genus Enterobacter was first described by Hormaeche and Edwards (9). Since the transfer of Enterobacter agglomerans to the genus Pantoea (5), 14 species are included in the genus (1), including Enterobacter aerogenes, which is considered a homotypic synonym of Klebsiella mobilis because it has the same type strain (13). Around Enterobacter cloacae, six genetically related and phenotypically similar species have been combined within the so-called E. cloacae complex (7), i.e., E. cloacae, Enterobacter asburiae, Enterobacter dissolvens, Enterobacter hormaechei, Enterobacter kobei, and Enterobacter nimipressuralis. In addition to these species, at least six genetic clusters are phylogenetically delineated within the complex (7). Two of the most prominent clusters (clusters VI and VIII) are closely related to the species E. hormaechei, together forming the so-called E. hormaechei metacluster. They displayed 98 to 99% sequence identity in an analysis of three housekeeping genes and showed nearly identical restriction patterns of their ampC genes, which chromosomally code for a Bush class 1 beta-lactamase (7).
E. hormaechei was first described on the basis of 23 isolates sent to the Centers for Disease Control and Prevention (Atlanta, Ga.) for identification. At that time, they could not be assigned to a species, since they were negative in d-sorbitol and melibiose tests and did not fit the biochemical profile of any established Enterobacter species. Later, this preselected set of isolates turned out to be genetically closely related to each other. The species E. hormaechei was proposed to be lactose, d-sorbitol, raffinose, melibiose, and esculin negative and 87% dulcitol positive (12). Subsequently, a clinical outbreak was observed that was caused by a different biotype of E. hormaechei, challenging the original species description (4). In a hybridization study of clinical isolates of the E. cloacae complex performed by Grimont and Grimont (6), E. hormaechei represented the most prominent proportion of all isolates studied (33%). Similarly, the E. hormaechei metacluster comprised 44% of all strains in our recent population genetic study (7). Because of its apparently highly underestimated clinical relevance (6, 7), we studied the E. hormaechei metacluster further, concluding that it consists of three different subspecies, for which we propose the names E. hormaechei subsp. oharae, E. hormaechei subsp. hormaechei, and E. hormaechei subsp. steigerwaltii.
MATERIALS AND METHODS
Forty-eight strains and seven type strains were included in this study (Table 1). They were assigned to their respective genetic clusters of the E. cloacae complex by partial sequence comparisons of their hsp60 genes as previously described (7). Type strains were purchased from the American Type Culture Collection (ATCC) or the Collection de l'Institut Pasteur (CIP).
TABLE 1.
Clustera | Strain | Speciesb | Source materialc | Origin |
---|---|---|---|---|
VI | EN-18 | ENOH | Stool | Munich, Germany |
EN-190 | ENOH | Trachea | Munich, Germany | |
EN-210 | ENOH | BAL | Munich, Germany | |
EN-218 | ENOH | Ear | Munich, Germany | |
EN-232 | ENOH | Throat | Munich, Germany | |
EN-248 | ENOH | Sputum | Munich, Germany | |
EN-260 | ENOH | Trachea | Munich, Germany | |
EN-276 | ENOH | Blood | Munich, Germany | |
EN-277 | ENOH | Blood | Munich, Germany | |
EN-312 | ENOH | NS | Hannover, Germany | |
EN-314T | ENOH | Mouth (2-yr-old infant) | Hannover, Germany | |
EN-332 | ENOH | BAL | Gelsenkirchen, Germany | |
EN-334 | ENOH | Urine | Gelsenkirchen, Germany | |
EN-351 | ENOH | Throat | Jena, Germany | |
EN-366 | ENOH | Abscess | Lausanne, Switzerland | |
VII | ATCC 49162T | ENHO | Sputum | California |
EN-280 | ENHO | Sputum | Munich, Germany | |
EN-291 | ENHO | Throat | Regensburg, Germany | |
EN-449 | ENHO | Blood | Munich, Germany | |
EN-670 | ENHO | Groin | Munich, Germany | |
EN-673 | ENHO | Stool | Munich, Germany | |
VIII | EN-30 | ENST | Trachea | Munich, Germany |
EN-285 | ENST | Wound | Regensburg, Germany | |
EN-288 | ENST | Wound | Regensburg, Germany | |
EN-305 | ENST | Urine | Växjö, Schweden | |
EN-311 | ENST | NS | Hannover, Germany | |
EN-315 | ENST | NS | Hannover, Germany | |
EN-320 | ENST | Wound | Stockholm, Sweden | |
EN-323 | ENST | Wound | Stockholm, Sweden | |
EN-325 | ENST | Urine | Stockholm, Sweden | |
EN-331 | ENST | BAL | Gelsenkirchen, Germany | |
EN-349 | ENST | Urine | Jena, Germany | |
EN-352 | ENST | Sputum | Jena, Germany | |
EN-359 | ENST | Urine | Jena, Germany | |
EN-365 | ENST | Puncture fluid | Lausanne, Switzerland | |
EN-369 | ENST | Blood | Mallorca, Spain | |
EN-370 | ENST | Blood | Mallorca, Spain | |
EN-371 | ENST | Blood | Mallorca, Spain | |
EN-380 | ENST | NS | Marseille, France | |
EN-384 | ENST | Urine | Berlin, Germany | |
EN-400 | ENST | Throat | Frankfurt, Germany | |
EN-403 | ENST | Skin | Frankfurt, Germany | |
EN-407 | ENST | Wound | Frankfurt, Germany | |
EN-466 | ENST | Trachea | Kiel, Germany | |
EN-468 | ENST | Central venous line | Kiel, Germany | |
EN-474 | ENST | Vagina | Kiel, Germany | |
EN-490 | ENST | Lung biopsy | Tübingen, Germany | |
EN-562T | ENST | Wound | Brüssel, Belgium | |
I | ATCC 35953T | ENAS | Lochia exudate | United States |
II | CDC 1347-71R | Blood | United States | |
ATCC BAA-260T | ENKO | Blood | Kobe City, Japan | |
XI | ATCC 13047T | ENCL | Cerebrospinal fluid | United States |
XII | ATCC 23373T | ENDI | Maize | United States |
ATCC 33241T | ENCA | Plant | Czech Republic | |
X | ATCC 9912T | ENNI | Elm tree | United States |
Genetic cluster denominations according to a recent population genetic study (7).
ENHO, Enterobacter hormaechei subsp. hormaechei; ENOH, Enterobacter hormaechei subsp. oharae; ENST, Enterobacter hormaechei subsp. steigerwaltii; ENAS, Enterobacter asburiae; ENKO, Enterobacter kobei; ENCL, Enterobacter cloacae; ENDI, Enterobacter dissolvens; ENCA, Enterobacter cancerogenus; ENNI, Enterobacter nimipressuralis.
NS, not specified; BAL, bronchoalveolar lavage fluid.
Bacterial strains were cultured aerobically on Columbia agar with 5% sheep blood and in Luria-Bertani broth at 37°C for 18 to 24 h. They underwent phenotypic testing with the API20E and Biotype 100 systems (bioMérieux, Marcy l'Etoile, France) and additional tests, i.e., motility testing, testing of acid production from mucate, and growth in medium containing potassium cyanide (KCN). The motility test was performed with SIM-agar (Becton Dickinson, Sparks, Md.) and the mucate fermentation test was performed with mucate broth (Fluka-Sigma-Aldrich, Steinheim, Switzerland), with both tests being performed according to the manufacturers' instructions. The ability to grow in the presence of KCN was tested in a peptone broth (1% peptone, 0.5% NaCl, 22.5‰ KH2PO4, 0.5% Na2HPO4 · H2O) at pH 7.6 containing 75‰ KCN. Arginine dihydrolase and ornithine decarboxylase activities were tested in Moeller's broth (pH 6.5), consisting of 0.5% peptone, 0.5% meat extract, 0.05% glucose, 0.5% pyridoxal, bromcresol purple, cresol red, and 1% respective amino acids (2). The esculin hydrolase test was performed with a broth containing 0.3% NaCl, 0.2% K2HP4, 0.3% Lab-Lemco medium, 1% peptone, and 0.1% esculin (2). Citrate activity was tested on Simmon's agar (Oxoid, Basingstoke, Hampshire, United Kingdom). The Voges-Proskauer test was performed according to the guidelines given by Chapin and Lauderdale (3). Urease activity was tested in a broth (pH 7.1) consisting of 0.5% NaCl, 0.2% KH2PO4, 0.1% glucose, 0.1% peptone, phenol red, and 2% urea (2). Additionally, urease activity was tested on Christensen's agar.
Antimicrobial susceptibilities to ampicillin, amoxicillin plus clavulanic acid, piperacillin, piperacillin plus tazobactam, cefoxitin, ceftazidime, cefotaxime, cefepime, meropenem, ciprofloxacin, gentamicin, and trimethoprim plus sulfamethoxazole were determined by disk diffusion tests on Mueller-Hinton agar on the basis of the quantitative interpretation criteria recommended by the Clinical and Laboratory Standards Institute (11). All phenotypic and susceptibility tests were performed at 37°C in ambient air. DNA preparations and DNA-DNA hybridizations in microplates were performed as described by Mehlen et al. (10).
Nucleotide sequence accession numbers.
Nucleotide sequence data are available at the EMBL/GenBank/DDBJ database under accession numbers AJ853889 and AJ853890 for 16S rRNA genes and under accession numbers AJ417108, AJ417124, AJ417129, AJ417141, AJ417142, AJ417143, AJ543761, AJ543765, AJ543766, AJ543771, AJ543777, AJ543779, AJ543782, AJ543783, AJ543788, AJ543790, AJ543791, AJ543795, AJ543796, AJ543798, AJ543810, AJ543811, AJ543815, AJ543821, AJ543822, AJ543825, AJ543826, AJ543827, AJ543835, AJ543836, AJ543846, AJ543849, AJ543851, AJ543854, AJ543863, AJ543908, AJ567895, AJ567899, AJ567900, AJ862841, AJ862866, and AJ862867 for hsp60 genes.
RESULTS AND DISCUSSION
In order to analyze the systematics of the E. hormaechei metacluster (7), we performed cross-hybridization experiments. The DNAs of strains EN-314 of genetic cluster VI, EN-562 of genetic cluster VIII, and E. hormaechei ATCC 49162T of genetic cluster VII were labeled and hybridized with two or three strains from each of the clusters as well as with the type strains of the other species of the E. cloacae complex (Table 2). In our recent phylogenetic analysis (7), the within-group sequence divergences of clusters VI and VII approximated zero, while that of cluster VIII was a bit higher (0.6 ± 0.4). The within-group heterogeneities were considered during the selection of strains for DNA-DNA hybridizations. The ΔTm values resulting from the hybridizations are presented in Table 2. The close DNA-DNA relatedness within clusters VI and VII was reflected by ΔTm values below 0.5. The relatively higher heterogeneity of cluster VIII was indicated by higher within-group ΔTm values of up to 2.7. By evaluating the DNA relatedness among the clusters, we found that clusters VI and VIII are closely related (mean ΔTm value = 2.2), while a relatively longer distance for E. hormaechei cluster VII from the members of clusters VI and VIII was indicated by the mean ΔTm value of 4.0. However, all three genetic clusters could still be assigned to the same species (14). They could be genetically distinguished from the other species of the E. cloacae complex, which had ΔTm values of 5.6 to 10.3 (Table 2). Phenotypic characterizations allowed a clear distinction of the E. hormaechei metacluster from the rest of the E. cloacae complex, e.g., by the lack of growth on 3-hydroxy-butyrate (Table 3). Similar to the original species description, the six E. hormaechei strains of cluster VII were negative in the esculin (0%), d-sorbitol (17%), and α-d-melibiose (17%) tests and positive in the dulcitol test (100%). However, some strains grew on raffinose (50%) and lactose (67%), which was inconsistent with the results of the study set used for the original species description, as none of those strains were raffinose positive and only 9% were lactose positive. Genetic clusters VI, VII, and VIII were differentiable from each other by their growth on dulcitol and adonitol and by other test results (Table 3).
TABLE 2.
Cluster or speciesa | Strainb | Δ Tm for hybridization with labeled DNAc
|
||
---|---|---|---|---|
ENOH EN-314 | ENHO ATCC 49162T | ENST EN-562 | ||
Cluster VI (ENOH) | EN314T | 0 | 3.2 | 1.8 |
EN18 | 0.3 | 3.2 | 1.0 | |
Cluster VII (ENHO) | ATCC 49162T | 4.0 | 0 | 4.3 |
EN280T | 4.8 | 0 | 4.3 | |
Cluster VIII (ENST) | EN562 | 2.2 | 3.7 | 0 |
EN331 | 4.0 | 4.8 | 2.7 | |
EN30 | 2.2 | 3.6 | 1.3 | |
E. cloacae | ATCC 13047T | 5.9 | 5.8* | 7.7 |
E. dissolvens | ATCC 23373T | 6.0 | 6.0* | 8.0 |
E. kobei | ATCC BAA260T | 6.6 | 6.7* | 6.6 |
CDC 1347-71R | 7.3 | 5.7* | 7.5 | |
E. asburiae | ATCC 35953T | 5.6 | 5.7* | 6.2 |
E. cancerogenus | ATCC 33241T | 7.0 | 6.6* | 6.8 |
E. nimipressuralis | ATCC 9912T | 9.2 | 10.3* | 8.5 |
ENOH, E. hormaechei subsp. oharae subsp. nov.; ENHO, E. hormaechei subsp. hormaechei comb. nov.; ENST, E. hormaechei subsp. steigerwaltii subsp. nov.
T, type strain; R, reference strain.
*, arithmetic mean from two hybridizations.
TABLE 3.
Test | % Positive isolates (cumulative)b
|
|||||
---|---|---|---|---|---|---|
E. hormaechei subsp. oharae (n = 15)
|
E. hormaechei subsp. hormaechei (n = 6)
|
E. hormaechei subsp. steigerwaltii (n = 27)
|
||||
48 h | 7 days | 48 h | 7 days | 48 h | 7 days | |
Voges-Proskauer (37°C)c | 100 | 100 | 100 | |||
Voges-Proskauer (37°C)d | 100 | 100 | 100 | |||
Motility in SIM-agar at 37°Cd | 90 | 83 | 88 | |||
Growth in KCNd | 30 | 40 | 67 | 19 | 38 | |
Mucate acid productiond | 100 | 100 | 89 | 96 | ||
β-Galactosidase (ONPG)c | 100 | 100 | 100 | |||
Arginine dihydrolasec | 100 | 100 | 100 | |||
Arginine dihydrolased | 100 | 100 | 100 | |||
Ornithine decarboxylasec | 100 | 100 | 100 | |||
Ornithine decarboxylased | 100 | 100 | 100 | |||
Lysine decarboxylasec | 0 | 0 | 0 | |||
H2S productionc | 0 | 0 | 0 | |||
Urease (API20E and broth)c | 0 | 0 | 0 | |||
Urease (Christensen's agar)d | 100 | 83 | 100 | 81 | ||
Indole productionc | 0 | 0 | 0 | |||
Citratec | 100 | 100 | 100 | |||
Citrate (Simmons agar)d | 100 | 100 | 100 | |||
Gelatinasec | 0 | 0 | 0 | |||
Esculine hydrolysationd | 0 | 27 | 0 | 0 | 7 | |
α-Methyl-d-gycoside, acid productiond | 80 | 90 | 100 | 77 | 100 | |
Growth on substrates | ||||||
1-0-Methyl-α-d-glucopyranoside | 53 | 100 | 100 | 52 | 100 | |
1-0-Methyl-α-galactopyranoside | 93 | 100 | 0 | 0 | 93 | 100 |
1-0-Methyl-β-d-glucopyranoside | 93 | 100 | 100 | 100 | ||
1-0-Methyl-β-galactopyranoside | 93 | 100 | 100 | 85 | 93 | |
3-0-Methyl-d-glucopyranose | 0 | 7 | 67 | 89 | 93 | |
3-Hydroxybutyrate | 0 | 0 | 0 | 0 | 0 | 0 |
5-Keto-d-gluconate | 0 | 0 | 17 | 0 | 0 | |
α-d-Fucose | 46 | 50 | 75 | 80 | ||
α-d-Fucose (acid from)d | 50 | 50 | 79 | |||
α-d-Melibiose | 100 | 17 | 100 | |||
α-d-Melibiose (acid from)d | 100 | 50 | 100 | |||
Adonitol | 13 | 17 | 93 | |||
Adonitol (acid from)d | 0 | 0 | 100 | |||
α-Ketoglutarate | 0 | 7 | 17 | 35 | 7 | 26 |
α-Lactose | 80 | 100 | 50 | 67 | 63 | 89 |
α-l-Fucose | 67 | 73 | 100 | 96 | 100 | |
α-l-Fucose (acid from)d | 30 | 50 | 98 | |||
α-l-Rhamnose | 93 | 100 | 100 | |||
α-l-Rhamnose (acid from)d | 100 | 100 | 100 | |||
β-d-Fructose | 100 | 100 | 100 | |||
β-Gentiobiose | 100 | 100 | 100 | |||
Caprate | 0 | 0 | 0 | 0 | 0 | 0 |
Caprylate | 0 | 0 | 0 | 0 | 0 | 0 |
cis-Aconitate | 100 | 67 | 83 | 100 | ||
d(−)-Ribose | 100 | 100 | 100 | |||
d(−)-Tartrate | 0 | 0 | 0 | 17 | 0 | 0 |
d(+)-Arabitol | 7 | 17 | 96 | |||
d(+)-Cellobiose | 100 | 100 | 100 | |||
d(+)-Galactose | 100 | 100 | 100 | |||
d(+)-Malate | 0 | 7 | 0 | 0 | 7 | 15 |
d(+)-Mannose | 100 | 100 | 100 | |||
d(+)-Trehalose | 100 | 100 | 100 | |||
d(+)-Turanose | 27 | 17 | 0 | 19 | ||
d(+)-Xylose | 93 | 100 | 100 | 100 | ||
d-Galacturonate | 100 | 100 | 96 | |||
d-Glucosamine | 100 | 100 | 96 | 100 | ||
d-Glucose | 100 | 100 | 100 | |||
d-Glucuronate | 100 | 100 | 96 | 100 | ||
dl-Glycerate | 0 | 0 | 0 | 33 | 0 | 0 |
d-Lyxose | 93 | 67 | 89 | 93 | ||
d-Mannitol | 100 | 100 | 100 | |||
d-Saccharate | 87 | 100 | 89 | 93 | ||
d-Sorbitol | 87 | 93 | 17 | 96 | ||
d-Tagatose | 0 | 7 | 0 | 0 | 0 | 0 |
Dulcitol | 0 | 0 | 100 | 0 | 4 | |
Fumarate | 100 | 100 | 100 | |||
Glycerol | 100 | 100 | 100 | |||
i-Erythritol | 7 | 17 | 0 | |||
l(+)-Arabinose | 100 | 100 | 100 | |||
l(+)-Tartrate | 0 | 0 | 0 | 0 | 0 | 0 |
Lactulose | 47 | 53 | 33 | 37 | 41 | |
l-Alanine | 100 | 100 | 100 | |||
Malonate | 0 | 13 | 0 | 17 | 0 | 19 |
Maltitol | 80 | 100 | 100 | 67 | 93 | |
Maltose | 100 | 100 | 100 | |||
meso-Tartrate | 0 | 0 | 17 | 0 | 0 | |
myo-Inositol | 7 | 20 | 0 | 7 | 15 | |
Palatinose | 100 | 100 | 96 | |||
Phenylacetate | 100 | 100 | 78 | 93 | ||
Protocatechuate | 0 | 0 | 0 | 0 | 0 | 4 |
Putrescine | 0 | 0 | 0 | 0 | 0 | 4 |
Raffinose | 93 | 50 | 96 | |||
Succinate | 100 | 100 | 96 | 100 | ||
Sucrose | 93 | 100 | 100 | |||
trans-Aconitate | 100 | 67 | 83 | 100 | ||
Xylitol | 0 | 7 | 0 | 0 | 22 | 63 |
The following tests were 100% positive for all strains analyzed at 24 h: growth on 4-aminobutyrate, 5-aminovalerate, l(−)-arabitol, benzoate, betain, m-coumarate, ethanolamine, gentisate, glutarate, histamine, l-histidine, m-hydroxybenzoate, p-hydroxybenzoate, hydroxyquinoline-b-glucuronide, itaconate, d(+)-melezitose, 3-phenylpropionate, propionate, (−)quinate, l(+)-sorbose, l-tryptophan, l-tyrosine, tricaballylate, trigonelline, tryptamine, and xylotol. The following tests were 100% negative for all strains analyzed at 24 h: growth on d-alanine, l-aspartate, d-gluconat, l-glutamate, 2-keto-d-gluconate, dl-lactate, l(−)-malate, maltotriose, N-acetyl-d-glucosamine, l-proline, and l-serine. If not otherwise stated, the Biotype 100 system was used.
A blank space indicates that the test was not read or that the results did not change at this time period.
API20E system.
Conventional test.
Our data correspond well to those reported by Grimont and Grimont (6), who found a group with DNA relatedness (DNA-relatedness group) around the E. hormaechei type strain, which they described as being “slightly heterogeneous with ΔTm values ranging from 0.0 to 4.0.” The authors observed seven biogroups within the DNA-relatedness group. Two of them were positive for growth on adonitol, d-arabitol, and d-sorbitol, corresponding to phylogenetic cluster VIII. Four were negative for growth on adonitol and d-arabitol but positive for growth on d-sorbitol, corresponding to phylogenetic cluster VI, and one was negative in all three tests, corresponding to E. hormaechei cluster VII. Davin-Regli et al. (4) reported an outbreak with an “E. cloacae strain with the E. hormaechei genotype” but an aberrant biotype. The strain exhibited all of the characteristics of E. hormaechei and was 80% related to the type strain in DNA-DNA reassociation experiments but was positive for growth on d-sorbitol and α-d-melibiose. Obviously, this outbreak was caused by a strain of genetic cluster VI. Hence, these studies are in agreement with our observation that genetic clusters VI and VIII belong to the species E. hormaechei (4, 6). Therefore, we propose that these clusters are new subspecies of E. hormaechei and we consequently reassign the species itself to E. hormaechei subsp. hormaechei comb. nov., which more or less keeps the original characteristics of the species.
Emended description of Enterobacter hormaechei O'Hara et al. 1989.
Enterobacter hormaechei (hor.maé′che.i. N.L. gen. m. Hormaeche, after Estenio Hormaeche, a Uruguayan microbiologist, who, together with P. D. Edwards, proposed and defined the genus Enterobacter [9]).
This emended description is based on phylogenetic sequence data and DNA-DNA hybridization data collected from 48 strains during the course of the present study and a previous population genetic study (7). Phenotypic characterization was performed by using the API20E and Biotype 100 systems and a series of conventional tests performed during the course of the present study. E. hormaechei strains are gram-negative rods which are 83% motile, catalase positive, oxidase and DNase negative, fermentative, and nonpigmented and exhibit the general characteristics of the family Enterobacteriaceae, the genus Enterobacter, and the E. cloacae complex. Growth occurs as nonpigmented colonies after 18 to 24 h at 15 to 42°C, with an optimum at 36°C, on all nonselective media, such as Colombia agar with 5% sheep blood, chocolate agar, tryptic soy agar, Luria-Bertani agar, and brain heart infusion agar, as well as on semiselective media such as MacConkey and ENDO agar. A detailed biochemical profiling of the species is given in Table 3. Growth on the following substances is subspecies specific: d-sorbitol, d-fucose, α-d-melibiose, 1-0-methyl-α-galactopyranoside, d-arabitol, dulcitol, d-(+)-raffinose, adonitol, and 3-methyl-d-glucopyranose. Table 4 shows the tests used for the differentiation of E. hormaechei and its subspecies from the other species of the genus. The G+C content of the DNA is 58.3 ± 0.3 mol% (Tm).
TABLE 4.
Species or subspecies | Biochemical test resulta
|
||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
d-Sorbitol | d-Fucose | l-Fucose | α-d-Melibiose | 1-0-Methyl-α-galactopyranoside | Esculin | α-Lactose | d-Arabitol | Dulcitol | d(+)-Raffinose | Adonitol | 3-Methyl-d-gluco-pyranose | 3-Hydroxy-butyrate | |
E. hormaechei subsp. oharae | + | V | V | + | + | V | +− | − | − | + | −+ | − | − |
E. hormaechei subsp. hormaechei | −+ | V | + | −+ | − | − | V | −+ | + | V | −+ | V | − |
E. hormaechei subsp. steigerwaltii | + | V | + | + | + | − | V | + | − | + | + | + | − |
E. asburiaeb | + | − | − | +− | +− | + | +− | − | − | + | − | − | + |
E. kobeib | + | − | − | + | + | − | +− | − | V | + | − | − | +− |
E. cloacaeb | + | − | − | + | + | − | V | − | −+ | + | − | − | + |
E. dissolvensb | + | V | V | + | + | + | + | − | − | + | V | − | + |
Incubation was done at 36°C. Symbols: −, 0 to 10%; −+, 10 to 20%; v, 20 to 80%; +−, 80 to 90%; +, 90 to 100%.
Results are according to reference 8.
The type strain is ATCC 49162 (equivalent to CIP 103441T and CCUG 27126T).
Description of Enterobacter hormaechei subsp. hormaechei comb. nov (12).
The description of Enterobacter hormaechei subsp. hormaechei comb. nov is based on the particular properties given in Table 3. E. hormaechei subsp. hormaechei corresponds to genetic cluster VII of the population structure of the E. cloacae complex which was presented recently (7). The biochemical tests used for the differentiation of this subspecies from the other E. hormaechei subspecies and from other Enterobacter species are subsumed in Table 4. All strains produced a Bush class 1 beta-lactamase at a low level, conferring resistance to ampicillin, amoxicillin plus clavulanic acid, and cefoxitin, but not to cefotaxime, ceftazidime, and cefepime, in agar diffusion tests. All strains were susceptible to meropenem, ciprofloxacin, trimethoprim plus sulfamethoxazole, and gentamicin.
The type strain, ATCC 49162 (equivalent to CIP 103441T and CCUG 27126T), was isolated from the sputum of a male patient from California (12).
Description of Enterobacter hormaechei subsp. oharae subsp. nov.
E. hormaechei subsp. oharae (o.há'rae. N.L. gen. f. Ohara, in honor of Caroline M. O'Hara, an American microbiologist who is affiliated at this time with the Centers for Disease Control, Atlanta, Ga., and who originally described the species E. hormaechei [12] and has contributed greatly to the taxonomy of various Enterobacteriaceae).
This description is based on the particular properties given in Table 3. E. hormaechei subsp. oharae corresponds to genetic cluster VI of the population structure of the E. cloacae complex which was presented recently (7). The biochemical tests used for the differentiation of this subspecies from the other E. hormaechei subspecies and from other Enterobacter species are subsumed in Table 4. All strains produced a Bush class 1 beta-lactamase (AmpC), with 69% producing the enzyme at a low level, conferring resistance to ampicillin, amoxicillin plus clavulanic acid, and cefoxitin, but not to cefotaxime, ceftazidime, and cefepime, in agar diffusion tests. Twenty-five percent of the strains hyperproduced the AmpC protein, conferring additional resistance to piperacillin, piperacillin plus tazobactam, cefotaxime, and ceftazidime, but not to cefepime, gentamicin, and trimethoprim plus sulfamethoxazole. One strain (EN-312) produced an extended-spectrum beta-lactamase and was resistant to all beta-lactam antibiotics, cephalosporins, and trimethoprim plus sulfamethoxazole. All strains were susceptible to meropenem and ciprofloxacin.
The type strain is EN-314, which is available at the German Collection of Microorganisms and Cell Cultures (DSMZ 16687T) and the Collection de l'Institut Pasteur (CIP 108490T). It was isolated from a mouth swab of a 2-year-old infant. The GenBank accession number for its 16S rRNA gene sequence is AJ853889.
Description of Enterobacter hormaechei subsp. steigerwaltii subsp. nov.
E. hormaechei subsp. steigerwaltii (stei.ger.wál'ti.i. N.L. gen. m. Steigerwalt, in honor of Arnold G. Steigerwalt, an American microbiologist who contributed to the species descriptions of E. asburiae and E. hormaechei).
This description is based on the particular properties given in Table 3. E. hormaechei subsp. steigerwaltii corresponds to genetic cluster VIII of the population structure of the E. cloacae complex which was presented recently (7). The biochemical tests used for the differentiation of this subspecies from the other E. hormaechei subspecies and from other species of the E. cloacae complex are subsumed in Table 4. All strains produced a Bush class 1 beta-lactamase (AmpC), with 54% producing the enzyme at a low level, conferring resistance to ampicillin, amoxicillin plus clavulanic acid, and cefoxitin, but not to the rest of the antibiotics tested. Forty-two percent of the strains hyperproduced the AmpC protein, conferring additional resistance to piperacillin, piperacillin plus tazobactam, cefotaxime, and ceftazidime, but not to cefepime, gentamicin, and trimethoprim plus sulfamethoxazole. One strain (EN-331) produced an extended-spectrum beta-lactamase and was resistant to all beta-lactam antibiotics, cephalosporins, and trimethoprim plus sulfamethoxazole. All strains were susceptible to meropenem and ciprofloxacin.
The type strain is EN-562, which is available at the German Type Cell Collection (DSMZ 16691T) and the Collection de l'Institut Pasteur (CIP 108489T). It was recovered from an infected surgical skin wound of a 49-year-old patient with tonsillar carcinoma. The GenBank accession number for its 16S rRNA gene sequence is AJ853890.
Acknowledgments
This study was funded by a grant from the German Research Association (DFG) in the frame of a special research field (SFB no. 576) and in part by the Friedrich Bauer Foundation.
We particularly thank Mrs. Kleinhuber and ESCMID for the publication of our “call for strains.” Many thanks go to all colleagues who collected and sent strains for this study, i.e., S. Alberti, Palma de Mallorca, Spain, J. Bille, Lausanne, Switzerland, D. Bitter-Suermann, Hannover, Germany, A. Dierkes-Kersting and P. Breuer, Gelsenkirchen, Germany, H. Erichsen, Kiel, Germany, S. Lukas, Regensburg, Germany, C. E. Nord, Stockholm, Sweden, W. Pfister, Jena, Germany, K. Poschinger, Munich, Germany, V. Schäfer, Frankfurt, Germany, R. Smyth, Växjö, Sweden, M. Stark and I. Authenrieth, Tübingen, Germany, J. Wagner, Berlin, Germany, and A. Wenger, Lausanne, Switzerland, and to all other colleagues who provided us with strains.
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