FAMILY I. ENTEROBACTERIACEAE 607 kazakii in most biochemical reactions, including production of a by Kosako et al. (1996) indicated that Enterobacter kobei is the yellow pigment. However, they were only 43% related to E. sa- closest relative of Enteric Group 69. kazakii by DNA–DNA hybridization. Recent DNA relatedness data Genus I. Escherichia Castellani and Chalmers 1919, 941TAL FLEMMING SCHEUTZ AND NANCY A. STROCKBINE Esch.er.iЈchi.a. M.L. fem. n. Escherichia named after Theodor Escherich, who isolated the type species of the genus. -lm, occurring singly cannot be used by E. coli and E. vulneris, whereas a smaller pro 6.0–2.0 ן Straight cylindrical rods, 1.1–1.5 or in pairs. Conform to the general definition of the family En- portion of E. fergusonii and E. hermannii exhibit immediate or terobacteriaceae. Gram negative. Motile by peritrichous flagella or delayed use of this substrate. Growth on Simmons’ citrate agar nonmotile. Aerobic and facultatively anaerobic having both a by E. blattae is variable and probably strain specific. respiratory and a fermentative type of metabolism, but anaero- Lysine is decarboxylated by the majority of strains. Exceptions genic biotypes occur. Oxidase negative. Chemoorganotrophic. include “metabolically inactive” E. coli strains, the majority of Both acid and gas are formed from most fermentable carbo- enteroinvasive E. coli strains (EIEC), and E. hermannii. Ornithine hydrates, but i-inositol is not utilized and d-adonitol is utilized is decarboxylated by all species except by E. vulneris and a little only by Escherichia fergusonii. Lactose is fermented by most strains less than half of E. coli strains. Indole is produced by all species of Escherichia coli, but fermentation may be delayed or absent except E. blattae and E. vulneris. in Escherichia blattae, Escherichia hermannii, Escherichia fergusonii, Other tests that help in differentiating between the species and Escherichia vulneris. Do not grow in KCN (with the exception of Escherichia include growth in potassium cyanide, malonate util- of E. hermannii and a small proportion of E. vulneris). Usually ization, and acid production from d-adonitol, d-arabitol, cello- do not produce H2S. E. coli occur naturally in the lower part of biose, dulcitol, lactose, d-mannitol, melibiose, d-sorbitol, and mu- the intestine of warm-blooded animals, E. blattae in the hind-gut cate. See Table BXII.c.197. of cockroaches, and E. fergusonii, E. hermannii, and E. vulneris are Molecular data Findings from the comparison of 16S rDNA found in the intestine, as well as extraintestinal sites of warm- sequences performed with strains of E. coli, Salmonella spp., and blooded animals. Seven copies of the rrn operon with genes C. freundii have shown a close phylogenetic relatedness between coding for 16S, 23S, and 5S rRNA are present on the chromo- these bacteria (Ahmad et al., 1990; Cilia et al., 1996; Chang et some of E. coli. Comparative sequence analysis between the genes al., 1997). Analysis of 16S rDNA sequences separates the two for 16S rRNA of E. coli, E. vulneris, and E. hermannii and ho- Salmonella species (S. enterica and S. bongori) from the complex mologous genes from all eubacteria places E. coli and E. vulneris of E. coli and Shigella, and shows that E. hermannii is more closely together in a tightly related cluster with shigellae, and E. her- related to Salmonella enterica and C. freundii (Christensen et al., mannii between Salmonella spp. and Citrobacter freundii (Cilia et 1998). This analysis is unable to separate inter-operon variation al., 1996). Based on 16S rRNA sequencing, escherichiae belong of E. coli from strains of E. coli and from Shigella. Patterns of in the Gammaproteobacteria. sequence heterogeneity in strains from the E. coli Collection of C of the DNA is: 48–59. Reference (ECOR) (Ochman and Selander, 1984) have been ם The mol% G Type species: Escherichia coli (Migula 1895) Castellani and located at regions V1 and V6 of cloned 16S rRNA genes (Mar- Chalmers 1919, 941 (Bacillus coli Migula 1895, 27.) tinez-Murcia et al., 1999). Average DNA relatedness assessed by DNA–DNA hybridization FURTHER DESCRIPTIVE INFORMATION among Escherichia species ranges from 29% to 94% (Table Cell morphology Escherichiae are straight, cylindrical, BXII.c.198). DNAs from different strains of E. coli are closely Gram-negative rods with rounded ends that are 1.1–1.5 lm in related (average, 84%; Table BXII.c.198). With the exception of diameter and 2.0–6.0 lm in length. They occur singly or in pairs S. boydii serotype 13, the DNAs of E. coli and the four Shigella and can be motile by peritrichous flagella or nonmotile. Fig. species show such a high degree of relatedness (average 80–87%, BXII.c.191 shows negatively stained preparations of each of the except S. boydii type 13, which is about 65%) that these species Escherichia species. See Nanninga (1985) for a comprehensive should be considered as a single species (Brenner et al., 1973a). treatment of the ultrastructure of E. coli. The distinction between these bacteria prevails, however, for rea- sons of historical/medical precedent and to avoid confusion in Phylogenetic and systematic treatment The genus consists of the literature and with existing surveillance systems. five species: E. coli, E. hermannii, E. fergusonii, E. vulneris, and E. Based on complete sequencing of the K-12 strain MG1655 blattae. Biochemical reactions that will help in differentiating be- (Blattner et al., 1997), it is estimated that the E. coli lineage tween the species of Escherichia are listed in Table BXII.c.197. diverged from the Salmonella lineage some 100 million years ago Biochemical reactions Escherichiae produce strong acids and (Lawrence and Ochman, 1998). This is a little less than the 120– usually gas from the fermentation of d-glucose (positive in the 160 million years estimated by calibration of the rate of 16S rRNA Methyl Red test) and do not produce acetyl-methyl carbinol (ace- evolution in bacteria (Ochman and Wilson, 1987). The chro- toin) (negative in the Voges–Proskauer test). Sodium acetate is mosome of E. coli strain MG1655 is 4,639,221 bp and contains frequently used as a sole carbon source, except by E. blattae and 4,288 open reading frames (ORFs). Approximately 18% of these a majority of E. vulneris strains. Citrate (Simmons’ citrate agar) ORFs represent genes that have been acquired and have persisted 608 FAMILY I. ENTEROBACTERIACEAE A B C D E FIGURE BXII.c.191. Electron micrographs of E. blattae strain 2928-78 (A), E. coli O157:H7 strain EDL933 (B), E. fergusonii strain 2460-89 (C), E. .nm 1000 ס hermannii strain 2456-88 (D), and E. vulneris strain 2485-88 (E) prepared by negatively staining in 0.5% (w/v) uranyl acetate. Bar (Micrograph courtesy of Charles D. Humphrey, CDC.) since divergence. This is similar to estimates based on analysis serotype O157:H7, the genome of which is 5.5 Mb in size, 859 of codon usage, indicating that 16% of sequenced genes arose Kb larger than that of the laboratory strain K-12 (Hayashi et al., through horizontal transfer (Me´digue et al., 1991). The ability 2001; Perna et al., 2001). The genome sizes of 14 E. coli strains of individual strains and lineages to acquire foreign DNA results from the five major subgroups in the ECOR (Ochman and Se- in great heterogeneity in both individual genes and in the size lander, 1984) range from 4.66–5.30 Mb (Bergthorsson and Och- of the E. coli chromosome. This is exemplified by the unusual man, 1995), and the uropathogenic E. coli strain J96 genome is GENUS I. ESCHERICHIA 609 TABLE BXII.c.197. Differentiation of the five species of Escherichiaa,b Test E. coli E. coli (metabolically inactive strains) E. blattae E. fergusonii E. hermannii E. vulneris מ ם ם מ [ם] ם Indole מ cמ [מ] d מ מ Citrate, Simmons [ם] dמ ם ם d ם Lysine decarboxylase מ ם ם ם [מ] Ornithine decarboxylase d ם ם ם eמ מ ם Motility [מ] ם מ מ מ מ KCN, growth [ם] מ d ם מ מ Malonate utilization ם ם ם ם מ ם D-Glucose, gas Acid production from: מ מ ם מ מ מ d-Adonitol מ מ ם מ מ מ d-Arabitol ם ם ם מ מ מ Cellobiose מ [מ] d מ Dulcitol d d f[מ] f dמ מ [מ] ם Lactose ם ם ם מ ם ם d-Mannitol ם מ מ מ d [ם] Melibiose מ מ מ מ d ם d-Sorbitol [ם] ם מ d d ם Mucate d [ם] ם מ d ם Acetate utilization d ם מ מ מ מ Yellow pigmentation aData compiled from references Farmer (1999), Cowan et al. (1995), Holt et al. (1994), and Richard (1989). Reactions for indole for E. fergusonii and melibiose for E. coli differ slightly in these references. The reactions listed in this table are supported by our own unpublished data. .1ЊC ע positive. Results are for 48 h incubation at 36Њ 100%–90 ,ם ;positive 89%–76 ,[ם] ;positive; d, 26–75% positive 25%–11 ,[מ] ;positive 10%–0 ,מ :bSymbols cDelayed positive in approximately a fifth of E. hermannii strains. dDelayed positive in a third of E. hermannii strains. e75% of E. blattae strains will become motile after incubation of more than 2 d. fDelayed positive in approximately two thirds of E. fergusonii and E. vulneris strains. TABLE BXII.c.198. DNA relatedness among escherichiaea Labeled Average percent relatedness DNA from E. coli E. blattae E. fergusonii E. hermannii E. vulneris E. coli 84 42 64 38 43 E. blattae 90 39 29 E. fergusonii 57 94 59 E.