A short selection from:

The Enteric Bacteria
© 2004 Kenneth Todar University of Wisconsin-Madison Department of Bacteriology

ntroduction to the Family Enterobacteriaceae

Enterobacteriaceae are Gram-negative, oxidase-negative, rod-shaped bacteria, 0.3-1.0 x 1.0-6.0 um. Typically, they are
motile by peritrichous flagella. They are facultative anaerobes, being  chemoorganotrophs that exhibit both respiratory
and fermentative metabolism. Most grow well between 22 and 35°C on media containing peptone  or beef extract. They
also grow on MacConkey's agar which may be used for their selective isolation. Most grow on glucose as a sole carbon
source, although some require vitamins and/or amino acids for growth. They produce mixed acids and often gas from
fermentation of sugars. With very few exceptions they are catalase-positive, and most strains reduce nitrate to nitrite.

Escherichia coli is the type species. E. coli is considered the most thoroughly studied of all species of bacteria, and the
family Enterobacteriaceae, as a whole, is the best studied group of microorganisms. Among the reasons for their
popularity are their medical and economic importance, ease of isolation and cultivation, rapid generation time, and their
ability to be genetically manipulated.

Enterobacteriaceae are distributed worldwide. They are found in water and soil and as normal intestinal flora in humans
and many animals. They live as .saprophytically, as symbionts, epiphytes, and parasites. Their host range includes
animals ranging from insects to humans, as well as fruits, vegetables, grains, flowering plants, and trees.

Economic and Medical Importance

As stated above, one of the reasons that the enterobacteriaceae have been so widely studied is due to their obvious
impact on human and animal health and on agricultural practice. The enterobacteriaceae include agents of food
poisoning and gastroenteritis, hospital-acquired infections, enteric fevers (e.g. typhoid fever) and plague. They also
cause infections in domestic, farm and zoo animals and include an important group of plant pathogens. Some of these
bacteria are discussed below.

Plant Pathogens
Many species of Enterobacteriaceae are responsible for significant economic losses in agriculture.  Erwinia species
cause blight, wilt, or soft-rot in numerous trees, flowers, and crops, often destroying substantial amounts of crops.  
Among the plants affected are walnut and oak trees, rose, orchid and chrysanthemum flowers, and crops such as  corn,
wheat, potato, carrot, sugar beet, sugar cane and pineapple.

Animal Pathogens
Enterobacteriaceae cause disease in all sorts of animals, ranging from nematodes and insects through primates.
Salmonella alone has been associated with disease in more than 125 species. Infections frequently cause problems in
zoos, often in snakes and lizards. In regional primate centers in the United States, the most frequently diagnosed
diarrheal diseases were caused by Enterobacteriaceae, most often by Shigella, E. coli and Salmonella. Klebsiella
pneumoniae is a frequent cause of respiratory disease in primates, and Yersinia pseudotuberculosis is associated with
enterocolitis and peritonitis.

Pets and farm animals are affected by a variety of enterobacterial diseases. Cats and dogs are susceptible to cystitis
and other urogenital infections caused by E. coli. Proteus species cause other diseases in cats and dogs, and these
animals can be carriers of Salmonella. Salmonellae, especially S. typhimurium, S. newport, and S. anatum, cause
enteritis with high fatality and septic abortion in horses, and K. pneumoniae causes metritis in mares and pneumonia in

Septicemia caused by E. coli is an important cause of death in chickens. Serotypes of Salmonella enterica are
pathogenic and highly fatal for turkeys and other poultry, causing a characteristic diarrheal syndrome. Pullorum disease,
caused by Salmonella  pullorum, is highly fatal to eggs and chicks. Fowl typhoid, a septicemic disease of poultry,
especially chickens, is caused by Salmonella gallinarum. Both pullorum disease and fowl typhoid can be largely
eradicated if infected adult birds are slaughtered.  Nearly 200 Salmonella serotypes had been isolated from fowl in the
United States. The distribution of salmonellosis in poultry is worldwide.  As in human disease, certain serotypes are
prevalent in some regions and absent in others. A mortality rate of 10-20% is normal in young birds, mostly in the first
two weeks after hatching.

Sheep suffer from a variety of illnesses caused by Enterobacteriaceae. Infant diarrhea in lambs, is usually caused by
strains of E. coli producing a heat-stable enterotoxin. Most of these strains also contain the K-99 fimbrial adhesin.
Salmonella abortion is usually caused by Salmonella abortusovis, S. typhimurium, or S. dublin, which also cause
stillbirths and wool damage.

Calves are susceptible to both systemic colibacillosis and neonatal diarrhea (calf scours), which are usually fatal if not
promptly treated. Specific heat-stable enterotoxigenic E. coli serotypes containing K99 fimbrial adhesin are the
causative agents. Bovine mastitis has become a very prevalent disease since the advent of antibiotics. The most
prevalent causative agents are E. coli and Serratia species, and less often, Klebsiella species and Citrobacter freundii.
Salmonellosis is frequent in cattle. Most cases are due to Salmonella dublin and S. typhimurium, although more than
100 serotypes have been isolated. As with other animal infections, Salmonella is frequently introduced through
contaminated feed.

Swine are subject to infection with several species of Enterobacteriaceae. E. coli infection may present as diarrhea in
piglets, or as edema preceded by mild diarrhea. Both forms are acute and highly fatal. As in sheep and cows, the
causative strains produce a heat-stable enterotoxin, but they may also produce a heat-labile enterotoxin. Swine strains  
usually possess a K88 fimbrial adhesin, which is antigenically distict from K99. Sows are susceptible to mastitis and
metritis caused by K. pneumoniae, and to enteritis and lymphadenitis caused by Yersinia enterocolitica. More than 100
Salmonella serotypes have been isolated from pigs.  However, only two serotypes, S. choleraesuis and S. typhisuis,
have pigs as their primary host. S. choleraesuis has a wide host range, including humans, but S. typhisuis is rarely
pathogenic to animals other than pigs. Salmonella typhimurium and S. derby are also frequently isolated from porcine

Substantial losses in fishing industries are caused by enterobacterial diseases. Yersinia ruckeri is the cause of
outbreaks of redmouth disease in salmon and trout hatcheries. Edwardsiella tarda is pathogenic for eels, catfish, and
goldfish, and Edwardsiella ictaluri is pathogenic for catfish.

The host range for species of Enterobacteriaceae varies greatly. For example Proteus myxofaciens has been isolated
only from larvae of gypsy moths and Escherichia blattae has been isolated only from the hindgut of cockroaches.  
Shigellae are seen only in primates. Others, including E. coli, many salmonellae, and yersiniae, infect or are carried by
hosts ranging from insects to humans.

Human Pathogens
Enterobacteriaceae as a group were originally divided into pathogens and nonpathogens based on their ability to cause
diarrheal disease of humans. The pathogenic genera were Salmonella and Shigella. However, it is now known that E.
coli causes at least five types of gastrointestinal disease in humans. Pathogenicity in E. coli strains is due to the
presence of one or more virulence factors, including invasiveness factors (invasins), heat-labile and heat-stable
enterotoxins, verotoxins, and colonization factors or adhesins. Pathogenic strains are usually identified by detection of a
specific virulence factor or of a serotype associated with a virulence factor.  The most recently identified E. coli disease
is hemorrhagic colitis caused by strains of serotype 0157:H7. The disease, characterized by painful abdominal cramping
and bloody diarrhea, is caused by strains that produce verotoxin, and the same strains are associated with hemolytic
uremic syndrome (HUS).

Yersinia enterocolitica causes diarrhea, probably by a combination of invasiveness and the presence of a heat-stable
enterotoxin. Strains of Klebsiella pneumoniae and Enterobacter cloacae isolated from patients with tropical sprue
contained a heat-stable enterotoxin. Edwardsiella tarda and Citrobacter strains are occasionally associated with
diarrhea and have been shown to produce heat-stable or heat-labile enterotoxin.

Foodborne and waterborne disease outbreaks in the U.S. are frequently associated with Enterobacteriaceae.  
According to the Centers for Disease Control (CDC), 40-45% of such outbreaks are caused by Enterobacteriaceae, the
overwhelming majority by Salmonella. Meats, milk and milk products, and eggs are the most common vehicles of
transmission.  Such figures represent only a small fraction of total foodborne disease, since the etiologic agent is
identified in only about one-third of the outbreaks, and many outbreaks are undetected or are not reported to the
Centers for Disease Control. For Salmonella, it is estimated that each reported case represents about 100 total cases.
The largest outbreak of salmonellosis in the United States occurred in 1985 in Illinois and Wisconsin, where an
estimated 170,000 to almost 200,000 persons were infected with Salmonella typhimurium transmitted in pasteurized milk
from a single dairy plant.

The incidence and recognition of rheumatoid disease occurring secondary to foodborne and waterborne diarrheal
disease have also increased. These diseases include reactive arthritis, Reiter's syndrome, ankylosing spondylitis, septic
and aseptic arthritis, ulcerative colitis, Crohn's disease, and Whipple's disease. Y. enterocolitica, Y. pseudotuberculosis,
Shigella flexneri, Shigella dysenteriae, various salmonellae, E. coli, and K. pneumoniae have been associated with these
chronic conditions.

Waterborne disease outbreaks due to Enterobacteriaceae are usually due to contaminated wells. Cases of shigellosis
due to a contaminated wells have been reported; even typhoid fever has occurred fairly recently in community water
systems contaminated with human sewage.

Enterobacteriaceae not normally associated with the GI tract or diarrheal disease may still be pathogens of humans.
Most notably, Yersinia pestis, which does not have an intestinal habitat, is the etiologic agent of plague a highly fatal
disease that has dessimated whole populations of individuals at several times in the history of civilization. Furthermore,
most, if not all, Enterobacteriaceae are opportunistic pathogens. Once established, they can cause a variety of
infections, including urinary tract disease, pneumonia, septicemia, meningitis, and wound infection.

According to the CDC, Enterobacteriaceae are responsible for 40-50% of nosocomial infections occurring in the United
States. E. coli is the worst offender, followed by Klebsiella, Proteus-Providencia-Morganella, Serratia, and Citrobacter.
The compromised host is particularly susceptible to nosocomial infections. Catheterized patients, patients on
immunosuppressants, burn patients, cancer patients, and elderly patients are all especially vulnerable to opportunistic
pathogens. To make matters worse, many of these organisms acquired in the hospital setting are multiply drug resistant.

Taxonmy and Classification of Enteric Bacteria

In artificial classification schemes (e.g.  Bergey's Manual of Systematic Bacteriology, 1st edition, 1986)
Enterobacteriaceae is a family of bacteria in Section  8 - Gram-negative facultatively anaerobic rods. Because of the
large number and broad range of phenotypic properties that solidifiy the group, these traits being a reflection of their
genetic relatedness, these bacteria have remained unified in modern phylogenetic schemes based on 16S ribosomal
RNA comparison. Thus, Citrobacter, Edwardsiella, Enterobacter, Erwinia, Escherichia, Klebsiella, Proteus, Providencia,
Salmonella, Serratia, Shigella, and Yersinia (along with several other genera, including Hafnia, Morganella,
Photorhabdus,and Xenorhabdus) are  presently classified in the subclass Gammaproteobacteria, order
Enterobacteriales, family Enterobacteriaceae in Bergey's Manual of Systematic Bacteriology, 2nd Edition, 2001.

The classic definition of an enteric bacterium is one that is found in the intestinal tract of warm-blooded animals in health
and disease, but bacteriologists reserve the term for reference to E. coli and its relatives, even though some of the
relatives of E. coli rarely or never are found growing in the GI tract. But in the end, this is one of the most close-related
and cohesive groups of bacteria that can be brought together for discussion.

The Genus Escherichia

Theodor Escherich first described E. coli in 1885, as Bacterium coli commune, which he isolated from the feces of
neonates. It was later renamed Escherichia coli, and for many years the bacterium was simply considered to be a
commensal organism of the large intestine. It was not until 1935 that a strain of E. coli was shown to be the cause of an
outbreak of diarrhea among neonates.

Most investigations of enteric organisms at the turn of the 20th century were concerned with the problems of being able
to distinguish the "typhoid bacillus" and other types of Salmonella from non-Salmonella organisms. Early workers also
demonstrated that there were a number of types and subtypes of these organisms, which could easily be distinguished
from the typhoid bacillus and E. coli. Thus, the biochemical techniques that have become the basis for most taxonomic
studies came into being during the early 1900s. These studies led to the modern taxonomy of the group, which in
principle is still valid today.

Initially, the family Enterobacteriaceae was created by Rahn in 1937, for the genus Enterobacter, and despite some
debate about nomenclature among bacteriologists, the family name was maintained with the type genus becoming
Escherichia.  The family currently comprises Gram-negative, nonsporeforming, rod-shaped bacteria that are often motile
by means of peritrichous flagella. The majority of strains grow well on the usual laboratory media in both the presence
and absence of oxygen, and metabolism can be either respiratory or fermentative. The fermentation products of glucose
and other carbohydrate substrates include mixed acids and (usually) detectable gas. Most strains are oxidase-negative
and are able to reduce nitrate to nitrite.

The taxonomic distinctiveness of Escherichia has been confirmed by rRNA-DNA heteroduplex studies. On the basis of
DNA-DNA relatedness studies, the genera of enteric bacteria are placed into a series of groupings, with Escherichia and
Shigella forming a close group distinct from their nearest group, which includes the genera  Citrobacter, Enterobacter,
Klebsiella, and Salmonella.

Although most investigations of the genus Escherichia have centered on various aspects of the E. coli species, it should
not be forgotten that a number of other species have been described, including E. blattae, E. fergusonii, E. hermanii,
and E. vulneris. These species can be differentiated on the basis of a large battery of biochemical tests.

For many years it has been realized that there exists a close relationship between two genera of enterics,  Escherichia
and Shigella. This is true for their biochemical characteristics as well as various other phenotypic traits. Also, studies of
certain E. coli antigens have shown a close relationship ("cross reactivity") with Shigella antigens. The "O" antigens of
virtually all serotypes of Shigella are either identical with or closely related to those of E. coli.  The discovery that the
characteristic “invasiveness” of Shigella strains is also possessed by certain types of E. coli, which have become known
as enteroinvasive E. coli (EIEC), also suggests a close relationship.  EIEC can cause dysentery-like symptoms clinically
indistinguishable from those caused by strains of Shigella. Furthermore, antigens of E. coli strain O124 are shown to
have a very close relationship to Shigella dysenteriae type 3 antigens, and a serological relationship between E. coli
strain O129 and S. flexneri type 5 is also known. Finally, enterohemorrhagic strains of E. coli  (EHEC), specifically E. coli
O157:H7 produce the shiga (vero) toxin which is identical to the toxin produced by Shigella dysenteriae.The point being
that it has become apparent that the line dividing these two genera of enteric bacteria is exceedingly thin, and it should
be remembered that on the basis of  DNA relatedness alone, Shigella and E. coli have been considered one genus.

Detection and Isolation of Escherichia coli

E. coli as an Indicator of Fecal Pollution

For most of the 20th century, E. coli has been used as the principal indicator of fecal pollution in both tropical and
temperate countries. E. coli comprises about 1% of the total fecal bacterial flora of humans and most warm-blooded
animals. Sewage is always likely to contain E. coli in relatively large numbers. In addition, E. coli, being a typical member
of the Enterobacteriaceae, is presumed to have survival characteristics very similar to those of the well-known
pathogenic members of the family, Salmonella and Shigella. Thus, E. coli has been used world-wide as an indicator of
fecal microbiological contamination. As such an indicator organism, its value is significantly enhanced by the ease with
which it can be detected. and cultured.

Tests to identify isolates as E. coli have, of necessity, been simple tests designed predominantly to differentiate them
from organisms normally associated with uncontaminated water. Since full biochemical analyses are not generally
performed, the term  "coliform" has been coined to describe E. coli-like organisms that satisfy these limited tests. As a
result, regulations are promulgated throughout the world defining standards for water based on the so-called "coliform
count." For example, in the U.S., according to a regulation published in the Federal Register (1986), there is a
requirement that there be 0 coliforms/100 ml drinking water, as determined by any method for any sampling frequency.
Since not all organisms which meet the criteria of a coliform are associated with the intestinal tract (some may be
saprophytic), a further distinction must be made between "fecal coliforms" (E coli) and "nonfecal coliforms" (e.g.
Klebsiella and Enterobacter).

Early attempts to distinguish strains of E. coli from other related Enterobacteriaceae centered on being able to
distinguish them from the various pathogenic groups, since E. coli was initially not considered to be a pathogen. When
E. coli was recognized to be a useful marker for fecal pollution, it similarly became important to distinguish it from related
species likely to be found naturally in the environment. The realization that strains of E. coli generally ferment lactose,
while those of Salmonella and Shigella do not, led to an early method of preliminary differentiation. The IMViC tests were
developed in order to distinguish strains of E. coli from related species that also produced acid and gas from the
fermentation of lactose. IMViC is an acronym in which the capital letters stand for Indole, Methyl red, Voges-Proskauer,
and Citrate.) The IMViC set of tests examines: the ability of an organism to (1) produce Indole; (2) produce sufficient
acid to change the color of Methyl red indicator; (3) produce acetoin, an intermediate in ther butanediol fermentation
pathway (a positive result of the Voges-Proskauer test); and (4) the ability to grow on Citrate as the sole source of
carbon. Lactose fermenters are considered E. coli if they are positive in the first two tests and negative in the second