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1 Institute for Food Safety and Hygiene, Vetsuisse Faculty University of Zurich, Winterthurerstrasse 272, 8057 Zurich, Switzerland
2 BCCM/LMG Bacteria Collection, Laboratorium voor Microbiologie, Universiteit Gent, K. L. Ledeganckstraat 35, 9000 Gent, Belgium
3 Nestlé Research Centre, Nestec Ltd, Vers-chez-les-Blanc, Lausanne, Switzerland
Correspondence
Roger Stephan
stephanr{at}fsafety.uzh.ch
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-galactopyranoside and their negative reactions for D-sorbitol utilization and the Voges–Proskauer test. On the basis of the phylogenetic analyses, DNA–DNA hybridization data, and unique physiological and biochemical characteristics, it is proposed that the isolates represent a novel species of the genus Enterobacter, Enterobacter pulveris sp. nov. The type strain is 601/05T (=LMG 24057T=DSM 19144T).
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains 1160/04, 516/05, 601/05T, E441, E443 and E444 are DQ273686, DQ273682, DQ273684, EF472537, EF614996 and EF059835, respectively. The accession numbers for the rpoB gene sequences of strains 1160/04, 516/05, 601/05T, E441, E443 and E444 are EF486514, EF486513, EF486512, EF632062, EF632063 and EF632064, respectively.
A neighbour-joining tree of 16S rRNA gene sequences showing the estimated phylogenetic relationships between Enterobacter pulveris sp. nov. and related genera within the family Enterobacteriaceae is available with the online version of this paper.
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), but which could be mistaken phenotypically for E. sakazakii (Iversen et al., 2004b). Lehner and others also described isolates from fruit powder that gave colony morphologies on E. sakazakii isolation media that were typical for the target species, but the API 32E confirmation of these strains gave ambiguous results (Lehner et al., 2004). Subsequently, comparative analysis of 16S rRNA gene sequences revealed that these isolates were clearly distinct from E. sakazakii with sequence similarities of <97 % (Lehner et al., 2006).
There is no evidence that the presence of these novel species in food products represents a health concern, but they do pose a challenge for the design of isolation methods for E. sakazakii, a micro-organism that is occasionally reported as an opportunistic pathogen and has been associated with infant formula (Lehner & Stephan, 2004). These novel species share several typical characteristics with E. sakazakii, such as resistance to desiccation, production of a yellow pigment and constitutive metabolism of 5-bromo-4-chloro-3-indolyl--D-glucopyranoside, which is a significant trait used in the differentiation of presumptive E. sakazakii colonies (Iversen et al., 2004b).
Recently, two groups from the fruit powder isolates have been described as novel species, Enterobacter helveticus and Enterobacter turicensis (Stephan et al., 2007). The characterization of a third group of strains, with representative isolates from fruit powder, infant formula and an infant formula production environment, is described in this study and it is concluded that these isolates represent another novel Enterobacter species.
Apple fruit powder (150 g) samples (spray-dried powder of 100 % fruit pulp or vacuum-dried granules of fruit juice) were suspended in 1.35 l buffered peptone water (BPW) and incubated for 24 h at 37 °C. Samples (0.1 ml) of the BPW enrichment were subcultured in 9 ml Enterobacteriaceae enrichment broth (BBL EE broth; Becton Dickinson) for 24 h at 37 °C and subsequently plated on violet red bile glucose agar (Difco VRBG; Becton Dickinson) and chromogenic E. sakazakii agar (Oxoid CM1055). After incubation for 24 h at 37 °C, isolates 1160/04 (=LMG 24058=DSM 19146), 516/05 and 601/05T (=LMG 24057T=DSM 19144T) were recovered as typical turquoise colonies from E. sakazakii agar. All strains produced yellow-pigmented colonies on tryptone soy agar (TSA) plates.
In addition to the isolates obtained from fruit powder, three further isolates have been obtained from infant formula [E441 (=LMG 24059=DSM 19145) and E443] and an infant formula production environment (E444) using the ISO/TS for E. sakazakii (Anonymous, 2006).
For morphological and physiological studies, strains were grown on BHI medium (Oxoid CM0225). Physiological studies were performed using API 32E and Biotype 100 tests with Biotype Medium 1, according to the instructions of the manufacturer (bioMérieux). Motility testing and indole and H2S production were performed in SIM agar (BD) at 37 °C.
The six isolates consisted of facultatively anaerobic, motile, Gram-negative coccoid rods (0.9–1.0x1.5–3.0 µm). After 24 h aerobic incubation at 37 °C on sheep blood agar, colonies were non-haemolytic and yellow pigmented. Yellow pigmentation increased when colonies were exposed to light. All strains were negative or weakly positive for the oxidase reaction and were catalase-positive. The strains could grow at 10–44 °C. The minimal pH for growth at 37 °C was pH 5.0 for all strains. Generally, the six strains had the same biochemical profile, but they gave variable results for the utilization of dulcitol, aesculin, putrescine, turanose, DL--amino-n-butyrate, DL-glycerate, lactulose, maltitol, 1-O-methyl--D-glucopyranoside and L-tartrate as sole sources of carbon (see Table 1 and species description).
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. The almost complete 16S rRNA gene sequences comprising 1220 (1160/04), 1317 (516/05), 1325 (601/05T), 1420 (E441), 1412 (E443) and 1479 (E444) nucleotides were determined and aligned to 28 000 almost full-length 16S rRNA gene sequences using the alignment tool of the ARB package (Ludwig et al., 2004). Alignments were refined by visual inspection. Analysis of the 16S rRNA gene sequences was performed using the distance-matrix tool and a phylogenetic tree was estimated using the neighbour-joining method combined with a Felsenstein correction, all included in the ARB package. The significance of branchings was evaluated by bootstrap analysis of 1000 replicates. Phylogenetic analyses were performed using distance matrix and the neighbour-joining dendrogram tool included in the ARB software package employing special data structures (PT-servers) derived from the ssu-rRNA database (ssu_jan04.arb).
The 16S rRNA gene sequences of isolates 1160/04, 516/05, 601/05T, E441, E443 and E444 showed 99.3–99.5 % similarity to each other and formed a separate branch in the phylogenetic tree (see Supplementary Fig. S1 available in IJSEM Online). Based on the data of Stackebrandt & Ebers (2006), this suggests that these strains may belong to the same species. The strains grouped most closely with a cluster containing E. helveticus LMG 23732T (GenBank accession no. DQ273688; 98.6–99.2 % sequence similarity), E. sakazakii DSM 4485T (AB004746; 96.8–97.3 % sequence similarity) and E. sakazakii ATCC 51329 (AY752937; 96.7–97.3 % sequence similarity).
16S rRNA gene sequence analysis indicates that the strains belong to the family Enterobacteriaceae but, based on these results alone, they cannot unequivocally be allocated to a finer taxonomic level. Since rpoB sequence analysis has been used successfully for species discrimination within the family Enterobacteriaceae (Mollet et al., 1997; Drancourt et al., 2001; Li et al., 2004; Kämpfer et al., 2005; Stephan et al., 2007), this approach was also used to determine the taxonomic position of the six strains. Total DNA was prepared according to the protocol of Niemann et al. (1997). The rpoB gene was amplified and sequenced following the protocol of Mollet et al. (1997). Sequence assembly was performed by using the program AUTOASSEMBLER (Applied Biosystems). Phylogenetic analysis was performed using TREECON software (Van de Peer & De Wachter, 1994) after including the consensus sequence in a CLUSTAL W alignment (Thompson et al., 1994) of rpoB sequences collected from the EMBL nucleotide sequence library. Evolutionary distances were calculated using the Jukes–Cantor evolutionary model and a tree was constructed using the neighbour-joining method. Bootstrap values (500 replicates) were also calculated.
The rpoB sequences of the six isolates showed 99.6–100.0 % sequence similarity to each other, indicating that they probably belong to the same species. The highest rpoB sequence similarities were obtained with E. helveticus LMG 23732T (95.6–95.8 %), Enterobacter radicincitans CIP 108468T (91.5–91.7 %), E. turicensis LMG 23730T (91.3–91.6 %) and E. sakazakii LMG 5740T (91.2–91.5 %). The phylogenetic branch formed by E. helveticus, E. turicensis, E. sakazakii, E. radicincitans, Enterobacter cowanii and the six novel strains was supported by a high bootstrap value (95 %) (Fig. 1), demonstrating that they belong to the genus Enterobacter, taking into account that when using only one protein coding gene, a possible genetic transfer cannot be excluded. The similarity values found with their nearest neighbours were rather low (89.4–95.8 %) compared with the intraspecies similarity range of 98–100 % found in the family Enterobacteriaceae (Mollet et al., 1997), thus confirming that these strains represent a novel species within this family.
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and hybridizations were carried out at 45 °C according to the microtitre plate technique described by Ezaki et al. (1989). The DNA relatedness percentages are the means of a minimum of four hybridizations. Reciprocal reactions (i.e. AxB and BxA) were performed and the variation between them was within the acceptable limits of this method (Goris et al., 1998).
The DNA–DNA hybridization results revealed that the strains 1160/04, 516/05 and 601/05T show a very high DNA–DNA relatedness to each other (99–100 %), demonstrating that they belong to the same genomospecies. The DNA–DNA relatedness between strain 601/05T and E. helveticus LMG 23732T was 54 %, which is clearly below 70 %, the generally accepted limit for species delineation (Wayne et al., 1987). On the basis of these genotypic results, it is concluded that the six strains represent a novel genomic species.
The overall G+C content was also determined for three strains according to the HPLC method (Mesbah et al., 1989) using the DNA prepared for DNA–DNA hybridizations. The values (means of three independent analyses of the same DNA sample) for 1160/04, 516/05 and 601/05T were 57.0, 56.4 and 57.0 mol%, respectively. These values are consistent with the DNA G+C contents of other members of the genus Enterobacter (Richard, 1984; Inoue et al., 2000).
The results of this polyphasic analysis support the recognition of a novel Enterobacter species, for which the name Enterobacter pulveris sp. nov. is proposed. Details on the physiological and biochemical characteristics of the novel species are given below. It can be clearly differentiated from its nearest neighbours by several properties, including the utilization of melibiose, sucrose, D-sorbitol, D-arabitol, mucate and 1-O-methyl--galactopyranoside and the Voges–Proskauer reaction (see Table 1
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Description of Enterobacter pulveris sp. nov.
Enterobacter pulveris (pul've.ris. L. n. pulvis -eris dust, powder; L. gen. n. pulveris of powder).
Cells are Gram-negative coccoid rods that are facultatively anaerobic and motile. Cells are 0.9–1.0 µm wide by 1.5–3.0 µm long and occur singly or in pairs. After 24 h aerobic incubation at 37 °C on TSA medium, colonies are yellow pigmented and convex. Catalase-positive and negative or weakly positive for oxidase. Colonies grow poorly at 10 °C (within 3 days), but well at 44 °C. Negative for ornithine decarboxylase, malonate decarboxylase, urease, arginine dihydrolase and lysine decarboxylase. Voges–Proskauer test, indole production and H2S production are also negative. Acid is produced from galacturonate, D-mannitol, maltose, D-glucose, sucrose, D-arabitol, L-arabinose, trehalose, L-rhamnose and D-cellobiose. Acid is not produced from L-arabitol, 5-ketogluconate, adonitol, palatinose, inositol or D-sorbitol. The chromogenic substrates ONPG, 4-nitrophenyl-β-D-glucopyranoside, 4-nitrophenyl-β-D-galactopyranoside, 4-nitrophenyl--D-glucopyranoside, 5-bromo-3-indoxyl-nonanoate (weak reaction), 4-nitrophenyl--D-galactopyranoside and 4-nitrophenyl--D-maltopyranoside are hydrolysed. 4-Nitrophenyl-β-D-glucuronide and L-aspartic acid 4-nitroanilide are not hydrolysed. Positive for utilization of -D-glucose, β-D-fructose, D-galactose, trehalose, D-mannose, melibiose, sucrose, raffinose, maltotriose, maltose, -lactose, 1-O-methyl-β-galactopyranoside, 1-O-methyl--galactopyranoside, D-cellobiose, β-gentiobiose, 1-O-methyl-β-D-glucopyranoside, D-ribose, L-arabinose, D-xylose, -L-rhamnose, D-arabitol, glycerol, D-mannitol, D-saccharate, mucate, L-malate, cis-aconitate, trans-aconitate, D-glucuronate, D-galacturonate, 2-keto-D-gluconate, 5-keto-D-gluconate, N-acetyl-D-glucosamine, D-gluconate, protocatechuate, p-hydroxybenzoate, quinate, DL-lactate, fumarate, D-glucosamine, L-aspartate, L-glutamate, L-proline, L-alanine and L-serine. The following compounds are not utilized as sole sources of carbon: L-sorbose, -L-fucose, L-arabitol, xylitol, D-tagatose, myo-inositol, D-sorbitol, adonitol, hydroxyquinoline-β-glucuronide, i-erythritol, 3-O-methyl-D-glucopyranose, D-tartrate, meso-tartrate, tricarballylate, L-tryptophan, phenylacetate, gentisate, m-hydroxybenzoate, benzoate, 3-phenylpropionate, trigonelline, betaine, histamine, caprate, caprylate, L-histidine, glutarate, DL--amino-n-valerate, ethanolamine, tryptamine, itaconate, DL-β-hydroxybutyrate, malonate, propionate, L-tyrosine and -ketoglutarate. Variable for utilization of dulcitol, aesculin, putrescine, turanose, DL--amino-n-butyrate, DL-glycerate, lactulose, maltitol, 1-O-methyl--D-glucopyranoside and L-tartrate as sole sources of carbon. The DNA G+C contents of strain 601/05T and representative strains 1160/04 and 516/05 are 57.0, 56.4 and 57.0 mol%, respectively.
The type strain, 601/05T (=LMG 24057T=DSM 19144T), was isolated from fruit powder.
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