Description of Enterobacter ludwigii sp. nov., a novel Enterobacter species of clinical relevance

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Systematic and Applied Microbiology 28 (2005) 206–212 www.elsevier.de/syapm

Description of Enterobacter ludwigii sp. nov., a novel Enterobacter species of clinical relevance Harald Hoffmanna,,1, Sibylle Stindlb,1, Anita Stumpfc, Andre Mehlenb, Daniel Mongetd, Ju¨rgen Heesemannc, Karl H. Schleiferb, Andreas Roggenkampc a

Institute of Microbiology and Laboratory Medicine, Pneumatological Hospital, Robert-Koch-Allee 2, D-82131 Gauting, Germany Lehrstuhl fu¨r Mikrobiologie, Technische Universita¨t Mu¨nchen, D-85353 Freising, Germany c Max von Pettenkofer Institute for Hygiene and Medical Microbiology, Ludwig Maximilian University Munich, Klinikum Großhadern, Marchioninistrasse 17, D-81377 Munich, Germany d BioMe´rieux, R&D Microbiology, Marcy I’Etoile, France b

Received 20 August 2004

Abstract A new species, Enterobacter ludwigii, is presented on the basis of the characteristics of 16 strains, which were isolated from clinical specimens. These bacteria form a distinct genetic cluster in phylogenetic analyses of the population structure of the Enterobacter cloacae complex. As determined by DNA–DNA cross-hybridization experiments in microplates, this genetic cluster can be delineated from the other species of the E. cloacae complex with DT m values equal to or above 5 1C with Enterobacter hormaechei being the closest relative. The bacteria are gram-negative, fermentative, motile rods with the general characteristics of the genus Enterobacter and the E. cloacae complex in particular. E. ludwigii can be differentiated from the other Enterobacter species by its growth on myo-inositol and 3-0-methyl-D-glucopyranose. The type strain is EN-119 ( ¼ DSM 16688T ¼ CIP 108491T). r 2005 Elsevier GmbH. All rights reserved. Keywords: Enterobacter ludwigii; Taxonomy; Genetic cluster; Population genetic structure

Introduction The genus Enterobacter was first described by Hormaeche and Edwards in 1960 [13]. Since Enterobacter aerogenes is considered a homotypic synonym to Klebsiella mobilis [26], and Enterobacter agglomerans is transferred to the genus Pantoea [9], 13 species are left in the genus Enterobacter [1], i.e. Enterobacter amnigenus [17], Enterobacter cowanii [15], Enterobacter gergoviae Corresponding author. Tel.: +49 89 85791 8230; fax: +49 89 85791 8350. E-mail address: [email protected] (H. Hoffmann). 1 Contributed equally to the study.

0723-2020/$ - see front matter r 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.syapm.2004.12.009

[18], Enterobacter intermedius [16,18,27], Enterobacter pyrinus [5], Enterobacter sakazakii [7], and seven species which are combined in the so-called Enterobacter cloacae complex [10]. These are Enterobacter asburiae [2], E. cloacae [13], Enterobacter dissolvens [2], Enterobacter hormaechei [25], Enterobacter kobei [19], Enterobacter nimipressuralis [3], and Enterobacter cancerogenus [6,11], the senior synonym of Enterobacter taylorae [8]. In three independent DNA–DNA-hybridization studies [2,11,20], E. dissolvens [2] fell into the same DNA–DNA reassociation group as E. cloacae. Therefore, it has been recently transferred to E. cloacae as a subspecies (Hoffmann et al. Reassignment of E. dissolvens to E. cloacae as E. cloacae subspecies

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dissolvens comb. nov. and emended description of E. asburiae and E. kobei. Syst. Appl. Microbiol. In press). In a recent population genetic study of the E. cloacae complex [12], six genovars could be delineated beside the named species, which we called genetic clusters III, IV, V, VI, VIII and IX. The classification of cluster V was subject of the present study. DNA–DNA hybridizations and extensive phenotypic characterizations were performed in order to elucidate the taxonomic position of this genovar. It turned out to be genetically and phenotypically distinct from the established Enterobacter species. We propose it as new taxon named Enterobacter ludwigii sp. nov.

Material and methods Sixteen study strains and the type and reference strains of the 13 established Enterobacter species were included in this study (Table 1). Strains were assigned to their respective genetic cluster of the E. cloacae complex by partial sequence comparison of the hsp60 gene as Table 1.

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previously described [12]. Type strains were purchased from the American Type Culture Collection (ATCC) or the Collection de I’lnstitut Pasteur (CIP). Reference strain CDC 134771R was purchased from the Center of Disease Controle (CDC), Atlanta, Georgia. Bacterial strains were cultured aerobically on Columbia agar with 5% sheep blood and in Luria–Bertani (LB) broth at 37 1C for 18–24 h. They underwent phenotypic testing with the API 20E system, the Biotype 100 system (BioMe´rieux, Marcy I’Etoile, France) and conventional tests, i.e. motility, acid production from mucate and growth in medium containing potassium cyanide (KCN). The motility test was performed in SIM agar (BD, Sparks, MD) and the mucate fermentation test in Mucate Broth (Fluka–Sigma–Aldrich, Steinheim, Switzerland), both following the manufacturer’s 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% Na2 HPO4 dH2 O) at pH 7.6 containing 75% KCN. Arginine dehydrolase and ornithine decarboxylase activities were tested in Moeller’s broth (pH6.5) consisting of 0.5% peptone, 0.5% meat extract, 0.05% glucose, 0.5% pyridoxal, bromkresolpurpur, kresol-red and 1% of the respective amino

Studied strains of the E. cloacae complex

Strain numbera

Cluster/speciesb

Materialc

Origin

EN-119 EN-187 EN-227 EN-243 EN-250 EN-259 EN-279 EN-289 EN-303 EN-319 EN-338 EN-340 EN-342 EN-493 EN-495 EN-517 ATCC 49162T ATCC 35953T ATCC 29941R ATCC BAA-260T ATCC 13047T ATCC 23373T ATCC 33241T ATCC 9912T

V V V V V V V V V V V V V V V V VII/ENHO I/ENAS II/ENKO II/ENKO XI/ENCL XII/ENDI ENCA ENNI

Urine Trachea Fat tissue of left thigh Venous line Sputum Blood Stool BAL Urine Biopsy Urine Throat Skin NS Throat Swab Sputum Lochia exsudate Blood Blood Cerebrospinal fluid Maize Plant Elm tree

Munich, Germany Munich, Germany Munich, Germany Munich, Germany Munich, Germany Munich, Germany Munich, Germany Regensburg, Germany Va¨xjo¨, Sweden Stockholm, Sweden Gelsenkirchen, Germany Aachen, Germany Aachen, Germany Tu¨bingen, Germany Tu¨bingen, Germany Freiburg, Germany California, USA USA Kobe City, Japan USA USA USA Czechoslovakia USA

a

T, type strain; R, reference strain. Genetic cluster denominations according to population genetic study [12]. ENHO, Enterobacter hormaechei, ENAS, Enterobacter asburiae, ENKO, Enterobacter kobei, ENCL, Enterobacter cloacae, ENDI, Enterobacter dissolvens, ENCA, Enterobacter cancerogenus, ENNI, Enterobacter nimipressuralis. c NS ¼ not specified, BAL ¼ bronchoalveolar lavage. b

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acids. The esculin hydrolase test was performed in a broth containing 0.3% NaCl, 0.2% K2HP4, 0.3% lablemco, 1% peptone and 0.1% esculin. Citrate activity was tested on Simmon’s agar (OXOID, Basingstoke, Hampshire, UK). The Voges-Proskauer test was performed following the guidelines given by Chapin and Lauderdale [4]. Antimicrobial susceptibilities to ampicillin, amoxicillin plus clavulanic acid, piperacillin, piperacillin plus tazobactam, cefoxitin, ceftazidime, cefotaxime, cefepime, meropenem, ciprofloxacin, gentamicin and trimethoprim plus sulphamethoxazole were determined by disk diffusion tests on Mueller–Hinton agar on the basis of the quantitative interpretation criteria recommended by the NCCLS [24]. All phenotypic and susceptibility tests were performed at 37 1C in room air. DNA preparations and DNA–DNA hybridizations in microplates were performed as described by Mehlen et al. [23]. The complete 16S rDNA of strain EN-119 was determined as described previously [14] using an ABI PRISM 3100 Genetic Analyser. It is available at GenBank with the accession number AJ85389.

Results and discussion Partial hsp60 sequences of the strains were analysed as exemplified in our recent population genetic study [12] and yielded a robust genetic cluster (sequence identities 499%), which was well delineable from the other clusters of the E. cloacae complex. It was denominated genetic cluster V. The high phylogenetic homogeneity of this cluster was reflected by DT m values below 3.0 1C in DNA–DNA hybridizations performed in microplates with the three representatives EN-119, EN-319, EN-495. Hybridization of labelled DNA of EN-119 with the type

strains of the six species of the E. cloacae complex resulted in DT m values between 5.0 and 7.3 1C (Table 1). The species E. hormaechei was the closest relative. Cross-hybridization with labeled DNA of its type strain with EN-119 yielded a DT m value of 5.9 1C. Hence, cluster V met the genetic species criteria by yielding DT m values above 5 1C in cross-hybridization experiments with the other taxa of the E. cloacae complex (Table 2). The species and genovars of the E. cloacae complex are genetically as well as phenotypically closely related to each other. They have been combined as complex and delineated from the other Enterobacter species in DNA–DNA hybridization studies [10,20] as well as phylogenetic analyses [12]. For these reasons, only members of the E. cloacae complex were considered in the DNA–DNA hybridization experiments of the present study. As cluster V could be genetically delineated within the E. cloacae complex, it is also different from the residual species of the genus Enterobacter. The biochemical properties of the 16 the representatives of genetic cluster V tested displayed in Table 3. Phenotypic tests proposed for the identification of genetic cluster V are listed in Table 4. The most differentiating characteristics were the ability of cluster V strains to grow on 3-0-methy-D-glucopyranose and myo-inositol. Taken together, cluster V fulfilled the genotypic and phenotypic criteria postulated for species descriptions and is proposed as novel species E. ludwigii sp. nov.

Description of E. ludwigii sp. nov. E. ludwigii [lud.wi’gi.i N.L. gen. n. of Ludwig, named in honour to Wolfgang Ludwig, a microbiologist who contributed to the general understanding of bacterial

Table 2. DTm values for DNA – DNA-hybridization experiments with representatives of E. ludwigii sp. nov. and type and reference strains of the other species of the E. cloacae complex Cluster, speciesa

E. ludwigii sp. nov. (cluster V)

E. E. E. E. E.

asburiae cancerogenus cloacae hormaechei kobei

E. nimipressuralis a

Cluster denomination according to [12]. T, type strain; R, reference strain.

b

Strainb

EN-119T EN-319 EN-495 ATCC 35953T ATCC 33241T ATCC 13047T ATCC 49162T ATCC BAA-260T CDC 134771R ATCC 9912T

Source of labelled DNA E. ludwigii sp. nov. EN-119

E. hormaechei hormaechei ATCC 49162T

0 2.6 0.5 6.0 6.7 5.3 5.0 5.3 5.1 7.3

5.9 — — 4.8 5.6 5.1 0 4.5 4.6 11.7

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Table 3. Biochemical reactions of 16 Enterobacter ludwigii sp. nov. strains in the API20E system, Biotype 100 system and in conventional tests Test

Table 3. (continued ) Test

Voges–Proskauerb Voges–Proskauerc Motility in SIM-agar at 37 1Cc Mucate, acid productionc Growth in KCNc b-galactosidaseb Arginine dihydrolaseb Arginine dihydrolase, Moeller’sc Ornithine decarboxilaseb Ornithine decarboxilase, Moeller’sc DNAsec Lysine decarboxilaseb H2S productionb Ureaseb Indole productionb Gelatinaseb Citrateb Citrate, Simmon’sc Esculin hydrolyzationb a-methyl-D-glucosidec

100 100 100 90 30 100 100 100 100 100 0 14 0 0 0 0 100 100 20 90

Growth on D-glucose b-D-fructose D(+)-galactose D(+)-trehalose D(+)-mannose L(+)-sorbose a-D-melibiose a-D-melibiose, acid fromc Sucrose D(+)-raffinose Maltotriose Maltose a-lactose Lactulose 1-0-methyl-b-galactopyranoside 1-0-methyl-a-galactopyranoside D(+)-cellobiose b-gentiobiose 1-0-methyl-b-glucopyranoside D()-ribose L(+)-arabinose c L(+)-arabinose, acid from D(+)-xylose Palatinose a-L-rhamnosec a-L-fucosec a-L-fucose, acid fromc a-D-fucose, acid fromc D(+)-melezitose D(+)-arabitol c D(+)-arabitol, acid from L()-arabitol Xylotol Dulcitol D-tagatose Gycerol Myo-Inositol Myo-Inositol, acid fromc D-mannitol Maltitol D(+)-turanose D-sorbitol c D-sorbitol, acid from Adonitol

100 100 100 100 100 0 100 100 100 100 100 100 60 7 87 93 100 100 100 100 100 100 100 93 100 40 33 8 0 0 0 0 0 0 0 100 100 100 100 93 7 100 100 0

7 days

0 0 0 0 0

27

0

100 100

58 0 0 0 0 0 0 0

Hydroxyquinoline-b-glucuronide D-lyxose i-erythritol 1-0-methyl-a-D-glucopyranoside 3-0-methyl-D-glucopyranose D-sacharate Mucate L(+)-tartrate D()-tartrate meso-tartrate D(+)-malate L()-malate cis-aconitate trans-aconitate Tricaballylate Citrate D-glucuronate D-galacturonate 2-keto-D-gluconate 5-keto-D-gluconate L-tryptophan N-acetyl-D-glucosamine D-gluconat Phenylacetate Protocatechuate p-hydroxybenzoate ()quinate Gentisate m-hydroxybenzoate Benzoate 3-phenylpropionate m-coumarate Trigonelline Betain Putrescine 4-aminobutyrate Histamine DL-lactate Caprate Caprylate L-histidine Succinate Fumarate Glutarate DL-glycerate 5-aminovalerate Ethanolamine Tryptamine D-glucosamine Itaconate 3-hydroxybutyrate L-aspartate L-glutamate L-proline D-alanine L-alanine L-serine Malonate Propionate L-tyrosine a-ketoglutarate a

33

0

Cumulative % positive ata 48 h

Cumulative % positive ata 48 h

209

0 87 0 93 87 93 93 7 0 0 0 100 100 100 0 100 100 100 100 0 0 100 100 100 0 0 0 0 0 0 0 0 0 0 0 0 0 100 0 0 0 100 100 0 0 0 0 0 100 0 67 100 100 100 100 100 100 0 0 0 0

7 days 0 93 0 93

0 0 7

0

0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 13 0 0 0 0 93

40 0 0 13

A blank space indicates that the test was not read or that results did not change at this time period. b API20E system used. c Conventional tests [4] used. If not otherwise stated, the Biotype 100 system was used.

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Table 4.

Key tests for phenotypic differentiation Enterobacter ludwigii sp. nov. from the other Enterobacter species

Species

Biochemical testa

a



V

+

+

V

+





+



+



+

+

   V  V +f f f c f f f f

   +  V +e e e c Ve Ve e e

+ + + + + + b +b +b +c +b +b +d +b

+ + +  + + f f +f +f +f +f f +f

+ +    + +b +b +b +c +b +b +d +b

+ + + + + + +e +e +e +c +e +e +d +e

   +   e e e c +e e +e e

  V +   b b b +c b +b d b

+ + +  + + e e e c Ve e +d Ve

   +  V e e e c e e d e

+   V   +e f f f f f f f

 + V  + + +e e e f Ve e +f +e

+ + +  + + e e e f Ve e f e

+ V V V V + +e +e +e +c e +e e e

Incubation at 36 1C. Symbols: , 0–10%; +, 10–20%; V, 20–80%; +, 80–90%; +, 90–100%. Sources of data: Acc. to [25]. c Acc. to [15]. d Acc. to [5]. e Acc. to [10]. f Type strain considered only, no information in literature. b

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E. ludwigii sp. nov. (cluster V) Type strain EN-119T E. asburiae E. kobei E. hormaechei E. cloacae E. dissolvens E. cancerogenus E. nimipressuralis E. amnigenus E. cowanii E. gergoviae E. intermedius E. pyrinus E. sakazakii

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DLa-D1-0-methyl- Esculin aDDulcitol MyoAdonitol 3-0-methyl- Putrescine 3-hydroxy- Mucate fucose fucose melibiose a-galactoinositol L-rhamnose arabitol D-glucobutyrate pyranoside pyranose

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systematics [21], especially by developing the ARB data bases and opening them to the public [22]]. This description is based on phylogenetic analyses of partial hsp60 sequence data collected in a recent population genetic study [12] as well as on DNA–DNA-hybridization assays and phenotypic characterizations performed in the frame of the present study. Phenotypic characterization was performed using API20E, Biotype 100, and a series of conventional tests. E. ludwigii strains are gram-negative rods, which are motile, catalase positive, oxidase and DNAase negative, fermentative, and non-pigmented. They exhibit the general characteristics of the family Enterobacteriaceae, the genus Enterobacter, and the E. cloacae complex. Growth occurs after 18–24 h at 15–42 1C with an optimum at 36 1C on all non-selective media like Columbia agar with 5% sheep-blood, chocolate-, TSA, Luria–Bertani-, or Brain–Heart agar as well as on semi-selective media like MacConkey- or ENDO-agar in non-pigmented colonies. A detailed biochemical profiling of the species is given in Table 3. Table 4 shows tests in use for differentiating E. ludwigii from the other species of the genus. Its identification is mainly possible by the ability to grow on 3-0-methyl-D-gluco-pyranose and on myo-inositol. All strains analysed produced a Bush class 1 beta-lactamase rendering resistance to ampicillin, amoxicillin plus clavulanic acid and cefoxitin in the disk diffusion tests performed. 20% of strain displayed a resistance pattern typical for AmpC hyperproduction (resistance to piperacillin, piperacillin plus tazobactam, cefoxitin, cefotaxime, ceftazidime, and susceptibility to cefepime). All strains were susceptible to trimethoprim plus sulphamethoxazole, gentamicin, meropenem and ciprofloxacin. The type strain EN-119T ( ¼ DSM 16688T ¼ CIP 108491T) was isolated from mid-stream urine of an 18year old male patient with a nosocomial urinary tract infection, while he was hospitalized at the GrosshadernUniversity-Hospital Munich, Germany. The GenBank accession number of the 16S rDNA of strain EN-119T is AJ853891.

Acknowledgements This study was funded by a grant from the Friedrich Bauer Foundation of the LMU, Munich, Germany. We are very grateful to Mrs. Kleinhuber and ESCMID for the publication of our ‘‘Call for strains’’. Special thanks go to all colleagues who have collected and sent strains for this study, i.e. F. Allerberger, Innsbruck, J. Bille, Lausanne, D. Bitter-Suermann, Hannover, M. BreuerWera, Aachen, A. Dierkes-Kersting and P. Breuer, Gelsenkirchen, H. Erichsen, Kiel, S. Lukas, Regensburg, K. Poschinger, Munich, V. Scha¨fer, Frankfurt, R. Smyth, Va¨xjo¨, M. Stark and I. Authenrieth,

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Tu¨bingen, J. Wagner, Berlin, A. Wenger, Lausanne, Mrs. Zwilling and M. Kist, Freiburg and all other colleagues who have not specified their names but provided strains for the study.

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