Enteric Gram-Negative Rods

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231 
15Enteric Gram-Negative Rods (Enterobacteriaceae)
C H A P T E R 
The Enterobacteriaceae are a large, heterogeneous group of 
gram-negative rods whose natural habitat is the intestinal 
tract of humans and animals. The family includes many gen-
era (Escherichia, Shigella, Salmonella, Enterobacter, Klebsi-
ella, Serratia, Proteus, and others). Some enteric organisms, 
such as Escherichia coli, are part of the normal microbiota 
and incidentally cause disease, but others, the salmonel-
lae and shigellae, are regularly pathogenic for humans. The 
Enterobacteriaceae are facultative anaerobes or aerobes, fer-
ment a wide range of carbohydrates, possess a complex anti-
genic structure, and produce a variety of toxins and other 
virulence factors. Enterobacteriaceae, enteric gram-negative 
rods, and enteric bacteria are the terms used in this chapter, 
but these bacteria may also be called coliforms.
CLASSIFICATION
The Enterobacteriaceae are the most common group of gram-
negative rods cultured in clinical laboratories and along with 
staphylococci and streptococci are among the most common 
bacteria that cause disease. The taxonomy of the Enterobac-
teriaceae is complex and rapidly changing since the introduc-
tion of techniques that measure evolutionary distance, such 
as nucleic acid hybridization and nucleic acid sequencing. 
According to the National Library of Medicine’s Internet 
Taxonomy database (available at http://www.ncbi.nlm.nih 
.gov//Taxonomy//Browser/wwwtax.cgl?id=543), 63 genera have 
been defined; however, the clinically significant Entero-
bacteriaceae comprise 20–25 species, and other species are 
encountered infrequently. In this chapter, taxonomic refine-
ments will be minimized, and the names commonly used in 
the medical literature are generally used. A comprehensive 
approach to identification of the Enterobacteriaceae is pre-
sented in Chapters 33, 37, and 38 of Jorgensen et al, 2015.
Members of the family Enterobacteriaceae have the fol-
lowing characteristics: They are gram-negative rods, either 
motile with peritrichous flagella or nonmotile; grow on pep-
tone or meat extract media without the addition of sodium 
chloride or other supplements; grow well on MacConkey 
agar; grow aerobically and anaerobically (are facultative 
anaerobes); ferment rather than oxidize glucose, often with 
gas production; are catalase positive, oxidase negative (except 
for Plesiomonas) and reduce nitrate to nitrite; and have a 
39–59% G + C DNA content. They can be differentiated to 
species level by a vast array of biochemical tests. In the United 
States, commercially prepared kits or automated systems are 
used to a large extent for this purpose. However, these are 
largely being replaced by other methods. The implementation 
of matrix-assisted laser desorption ionization time of flight 
mass spectroscopy (MALDI-TOF MS) for identification of 
culture isolates is replacing the more traditional panels of 
biochemicals currently in use in most clinical microbiology 
laboratories. This new technology seems to work quite well 
for identification of most of the common Enterobacteriaceae 
encountered in clinical material except for Shigella species. 
This technology is unable to differentiate Shigella from E coli.
The major groups of Enterobacteriaceae are described 
and discussed briefly in the following paragraphs. Specific 
characteristics of salmonellae, shigellae, and the other medi-
cally important enteric gram-negative rods and the diseases 
they cause are discussed separately later in this chapter.
Morphology and Identification
A. Typical Organisms
The Enterobacteriaceae are short gram-negative rods 
(Figure 15-1A). Typical morphology is seen in growth on 
solid media in vitro, but morphology is highly variable in 
clinical specimens. Capsules are large and regular in Klebsiella 
species, less so in Enterobacter species, and uncommon in the 
other species.
B. Culture
E coli and most of the other enteric bacteria form circular, 
convex, smooth colonies with distinct edges. Enterobacter 
colonies are similar but somewhat more mucoid. Klebsiella 
colonies are large and very mucoid and tend to coalesce with 
prolonged incubation. The salmonellae and shigellae produce 
colonies similar to E coli but do not ferment lactose. Some 
strains of E coli produce hemolysis on blood agar.
C. Growth Characteristics
Carbohydrate fermentation patterns and the activity of 
amino acid decarboxylases and other enzymes are used 
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232 SECTION III Bacteriology
in biochemical differentiation. Some tests, such as the pro-
duction of indole from tryptophan, are commonly used in 
rapid identification systems, but others, such as the Voges-
Proskauer reaction (production of acetylmethylcarbinol 
from dextrose), are used less often. Culture on “differen-
tial” media that contain special dyes and carbohydrates (eg, 
eosin-methylene blue [EMB], MacConkey, or deoxycholate 
medium) distinguishes lactose-fermenting (colored) from non–
lactose-fermenting colonies (nonpigmented) and may allow 
rapid presumptive identification of enteric bacteria (Table 15-1).
Many complex media have been devised to help in iden-
tification of the enteric bacteria. One such medium is triple 
sugar iron (TSI) agar, which is often used to help differentiate 
salmonellae and shigellae from other enteric gram-negative 
rods in stool cultures. The medium contains 0.1% glucose, 1% 
sucrose, 1% lactose, ferrous sulfate (for detection of H2S pro-
duction), tissue extracts (protein growth substrate), and a pH 
indicator (phenol red). It is poured into a test tube to produce 
a slant with a deep butt and is inoculated by stabbing bac-
terial growth into the butt. If only glucose is fermented, the 
slant and the butt initially turn yellow from the small amount 
of acid produced; as the fermentation products are subse-
quently oxidized to CO2 and H2O and released from the slant 
and as oxidative decarboxylation of proteins continues with 
formation of amines, the slant turns alkaline (red). If lactose 
or sucrose is fermented, so much acid is produced that the 
slant and butt remain yellow (acid). Salmonellae and shigellae 
typically yield an alkaline slant and an acid butt. Although 
Proteus, Providencia, and Morganella species produce an 
alkaline slant and acid butt, they can be identified by their 
rapid formation of red color in Christensen’s urea medium. 
Organisms producing acid on the slant and acid and gas 
(bubbles) in the butt are other enteric bacteria.
1. Escherichia—E coli typically produces positive test results 
for indole, lysine decarboxylase, and mannitol fermentation 
and produces gas from glucose. An isolate from urine can be 
quickly identified as E coli by its hemolysis on blood agar, typ-
ical colonial morphology with an iridescent “sheen” on dif-
ferential media such as EMB agar, and a positive spot indole 
test result. More than 90% of E coli isolates are positive for 
β-glucuronidase using the substrate 4-methylumbelliferyl-β-
glucuronide (MUG). Isolates from anatomic sites other than 
urine, with characteristic properties (above plus negative 
A B
Lipopolysaccharide
O side chains (O)
Capsule (K)
Flagella (H)
Cell envelope (cytoplasmic membrane, 
peptidoglycan, outer membrane)
FIGURE 15-1 A: Gram stain of Escherichia coli. Original magnification ×1000. (Courtesy of H Reyes.) B: Antigenic structure of 
Enterobacteriaceae.
TABLE 15-1 Rapid, Presumptive Identification of 
Gram-Negative Enteric Bacteria
Lactose fermented rapidly
Escherichia coli: metallic sheen on differential media; motile; flat, 
nonviscous colonies
Enterobacter aerogenes: raised colonies, no metallic sheen; often 
motile; more viscous growth
Enterobacter cloacae: similar to Enterobacter aerogenes
Klebsiella pneumoniae: very viscous, mucoid growth; nonmotile
Lactose fermented slowly
Edwardsiella, Serratia, Citrobacter, Arizona, Providencia, ErwiniaLactose not fermented
Shigella species: nonmotile; no gas from dextrose
Salmonella species: motile; acid and usually gas from dextrose
Proteus species: “swarming” on agar; urea rapidly hydrolyzed (smell 
of ammonia)
Pseudomonas species (see Chapter 16): soluble pigments, blue-green 
and fluorescing; sweetish smell
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CHAPTER 15 Enteric Gram-Negative Rods (Enterobacteriaceae) 233
oxidase test results) often can be confirmed as E coli with a 
positive MUG test result.
2. Klebsiella–Enterobacter–Serratia group—Klebsiella 
species exhibit mucoid growth, large polysaccharide cap-
sules, and lack of motility, and they usually give positive test 
results for lysine decarboxylase and citrate. Most Entero-
bacter species give positive test results for motility, citrate, 
and ornithine decarboxylase and produce gas from glucose. 
Enterobacter aerogenes has small capsules. Some species of 
Enterobacter have been moved into the genus Cronobacter. 
Serratia species produces DNase, lipase, and gelatinase. 
Klebsiella, Enterobacter, and Serratia species usually give 
positive Voges-Proskauer reactions.
3. Proteus–Morganella–Providencia group—The 
members of this group deaminate phenylalanine, are motile, 
grow on potassium cyanide medium (KCN), and ferment 
xylose. Proteus species move very actively by means of perit-
richous flagella, resulting in “swarming” on solid media 
unless the swarming is inhibited by chemicals, such as 
phenylethyl alcohol or CLED (cystine-lactose-electrolyte-
deficient) medium. Whereas Proteus species and Morganella 
morganii are urease positive, Providencia species usually are 
urease negative. The Proteus–Providencia group ferments lac-
tose very slowly or not at all.
4. Citrobacter—These bacteria typically are citrate posi-
tive and differ from the salmonellae in that they do not decar-
boxylate lysine. They ferment lactose very slowly if at all.
5. Shigella—Shigellae are nonmotile and usually do not 
ferment lactose but do ferment other carbohydrates, produc-
ing acid but not gas. They do not produce H2S. The four Shi-
gella species are closely related to E coli. Many share common 
antigens with one another and with other enteric bacteria (eg, 
Hafnia alvei and Plesiomonas shigelloides).
6. Salmonella—Salmonellae are motile rods that charac-
teristically ferment glucose and mannose without producing 
gas but do not ferment lactose or sucrose. Most salmonellae 
produce H2S. They are often pathogenic for humans or animals 
when ingested. Organisms originally described in the genus 
Arizona are included as subspecies in the Salmonella group.
7. Other Enterobacteriaceae—Yersinia species are dis-
cussed in Chapter 19. Other genera occasionally found in 
human infections include Cronobacter, Edwardsiella, Ewing-
ella, Hafnia, Cedecea, Plesiomonas, and Kluyvera.
Antigenic Structure
Enterobacteriaceae have a complex antigenic structure. They 
are classified by more than 150 different heat-stable somatic 
O (lipopolysaccharide) antigens, more than 100 heat-labile K 
(capsular) antigens, and more than 50 H (flagellar) antigens 
(Figure 15-1B). In Salmonella serotype Typhi, the capsular 
antigens are called Vi antigens. The antigenic classification of 
Enterobacteriaceae often indicates the presence of each spe-
cific antigen; for example, the antigenic formula of an E coli 
may be O55:K5:H21.
O antigens are the most external part of the cell wall 
lipopolysaccharide and consist of repeating units of polysac-
charide. Some O-specific polysaccharides contain unique 
sugars. O antigens are resistant to heat and alcohol and 
usually are detected by bacterial agglutination. Antibodies to 
O antigens are predominantly IgM.
Although each genus of Enterobacteriaceae is associated 
with specific O groups, a single organism may carry several 
O antigens. Thus, most shigellae share one or more O antigens 
with E coli. E coli may cross-react with some Providencia, Kleb-
siella, and Salmonella species. Occasionally, O antigens may be 
associated with specific human diseases (eg, specific O types 
of E coli are found in diarrhea and in urinary tract infections).
K antigens are external to O antigens on some but not all 
Enterobacteriaceae. Some are polysaccharides, including the 
K antigens of E coli; others are proteins. K antigens may inter-
fere with agglutination by O antisera, and they may be associ-
ated with virulence (eg, E coli strains producing K1 antigen 
are prominent in neonatal meningitis, and K antigens of 
E coli cause attachment of the bacteria to epithelial cells 
before gastrointestinal or urinary tract invasion).
Klebsiellae form large capsules consisting of polysac-
charides (K antigens) covering the somatic (O or H) antigens 
and can be identified by capsular swelling tests with specific 
antisera. Human infections of the respiratory tract are caused 
particularly by capsular types 1 and 2 and those of the uri-
nary tract by types 8, 9, 10, and 24.
H antigens are located on flagella and are denatured 
or removed by heat or alcohol. They are preserved by treat-
ing motile bacterial variants with formalin. Such H antigens 
agglutinate with anti-H antibodies, mainly IgG. The determi-
nants in H antigens are a function of the amino acid sequence 
in flagellar protein (flagellin). Within a single serotype, flagel-
lar antigens may be present in either or both of two forms, 
called phase 1 (conventionally designated by lowercase letters) 
and phase 2 (conventionally designated by Arabic numerals), 
as shown in 15-3. The organism tends to change from one 
phase to the other; this is called phase variation. H antigens 
on the bacterial surface may interfere with agglutination by 
anti-O antibody.
There are many examples of overlapping antigenic struc-
tures between Enterobacteriaceae and other bacteria. Most 
Enterobacteriaceae share the O14 antigen of E coli. The type 2 
capsular polysaccharide of Klebsiella is very similar to the 
polysaccharide of type 2 pneumococci. Some K antigens cross-
react with capsular polysaccharides of Haemophilus influen-
zae or Neisseria meningitidis. Thus, E coli O75:K100:H5 can 
induce antibodies that react with H influenzae type b.
Toxins and Enzymes
Most gram-negative bacteria possess complex lipopolysac-
charides in their cell walls. These substances, cell envelope 
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234 SECTION III Bacteriology
(cytoplasmic membrane, peptidoglycan, outer membrane) 
endotoxins, have a variety of pathophysiologic effects that 
are summarized in Chapter 9. Many gram-negative enteric 
bacteria also produce exotoxins of clinical importance. Some 
specific toxins are discussed in subsequent sections.
DISEASES CAUSED BY 
ENTEROBACTERIACEAE OTHER THAN 
SALMONELLA AND SHIGELLA
Causative Organisms
E coli are members of the normal intestinal microbiota (see 
Chapter 10). Other enteric bacteria (Proteus, Enterobacter, 
Klebsiella, Morganella, Providencia, Citrobacter, and Serratia 
species) are also found as members of the normal intestinal 
microbiota but are considerably less common than E coli. The 
enteric bacteria are sometimes found in small numbers as 
part of the normal microbiota of the upper respiratory and 
genital tracts. The enteric bacteria generally do not cause dis-
ease, and in the intestine, they may even contribute to normal 
function and nutrition. When clinically important infections 
occur, they are usually caused by E coli, but the other enteric 
bacteria are causes of hospital-acquired infections and occa-
sionally cause community-acquired infections. The bacteria 
become pathogenic only when they reach tissues outside of 
their normal intestinal or other less common normal micro-
biota sites. The most frequent sites of clinically important 
infection are the urinary tract, biliary tract, and other sites in 
the abdominal cavity, but any anatomic site (eg, bloodstream,prostate gland, lung, bone, meninges) can be the site of dis-
ease. Some of the enteric bacteria (eg, Serratia marcescens, 
E aerogenes) are opportunistic pathogens. When normal host 
defenses are inadequate—particularly in infancy or old age, 
in the terminal stages of other diseases, after immunosup-
pression, or with indwelling venous or urethral catheters—
localized clinically important infections can result, and the 
bacteria may reach the bloodstream and cause sepsis.
Pathogenesis and Clinical Findings
The clinical manifestations of infections with E coli and the 
other enteric bacteria depend on the site of the infection and 
cannot be differentiated by symptoms or signs from processes 
caused by other bacteria.
A. E coli
1. Urinary tract infection—E coli is the most common 
cause of urinary tract infection and accounts for approxi-
mately 90% of first urinary tract infections in young women 
(see Chapter 48). The symptoms and signs include urinary 
frequency, dysuria, hematuria, and pyuria. Flank pain is asso-
ciated with upper tract infection. None of these symptoms or 
signs is specific for E coli infection. Urinary tract infection 
can result in bacteremia with clinical signs of sepsis.
Most of the urinary tract infections that involve the blad-
der or kidney in an otherwise healthy host are caused by a 
small number of O antigen types that have specifically elabo-
rated virulence factors that facilitate colonization and sub-
sequent clinical infections. These organisms are designated 
as uropathogenic E coli. Typically, these organisms produce 
hemolysin, which is cytotoxic and facilitates tissue invasion. 
Strains that cause pyelonephritis express K antigen and elab-
orate a specific type of pilus, P fimbriae, which binds to 
the P blood group antigen.
Over the last decade, a pandemic clone, E coli O25b/
ST131, has emerged as a significant pathogen. This organ-
ism has been successful largely as a result of its acquisition 
of plasmid-mediated resistance factors that encode resistance 
to β-lactam antibiotics (elaboration of extended spectrum 
β-lactamases), fluoroquinolones, and aminoglycosides (see 
the review by Johnson et al, 2010).
2. E coli–associated diarrheal diseases—E coli that 
cause diarrhea are extremely common worldwide. These E coli 
are classified by the characteristics of their virulence proper-
ties (see later discussion), and each group causes disease by a 
different mechanism—at least six of which have been char-
acterized. The small or large bowel epithelial cell adherence 
properties are encoded by genes on plasmids. Similarly, the 
toxins often are plasmid or phage mediated. Some clinical 
aspects of diarrheal diseases are discussed in Chapter 48.
Enteropathogenic E coli (EPEC) are an important 
cause of diarrhea in infants, especially in developing coun-
tries. EPEC adhere to the mucosal cells of the small bowel. 
Pathogenicity requires two important factors, the bundle 
forming pilus encoded by a plasmid EPEC adherence factor 
(EAF) and the chromosomal locus of enterocyte effacement 
(LEE) pathogenicity island that promote the tight adherence 
characteristic of EPEC (attachment and effacement). After 
attachment, there is loss of microvilli (effacement); forma-
tion of filamentous actin pedestals or cuplike structures; 
and, occasionally, entry of the EPEC into the mucosal cells. 
Characteristic lesions can be seen on electron micrographs 
of small bowel biopsy lesions. The result of EPEC infection in 
infants is characterized by severe, watery diarrhea, vomiting, 
and fever, which are usually self-limited but can be prolonged 
or chronic. EPEC diarrhea has been associated with multiple 
specific serotypes of E coli; strains are identified by O anti-
gen and occasionally by H antigen typing. A two-stage infec-
tion model using HEp-2 or HeLa cells also can be performed. 
Tests to identify EPEC are performed in reference laborato-
ries. The duration of the EPEC diarrhea can be shortened and 
the chronic diarrhea cured by antibiotic treatment.
Enterotoxigenic E coli (ETEC) are a common cause of 
“traveler’s diarrhea” and a very important cause of diarrhea 
in children less than 5 years of age in developing countries. 
ETEC colonization factors (pili known as colonization factor 
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CHAPTER 15 Enteric Gram-Negative Rods (Enterobacteriaceae) 235
antigens [CFAs]) specific for humans promote adherence 
of ETEC to epithelial cells of the small bowel. Some strains 
of ETEC produce a heat-labile enterotoxin (LT) (molecu-
lar weight [MW], 80,000) that is under the genetic control 
of a plasmid and is closely related to cholera toxin. Its sub-
unit B attaches to the GM1 ganglioside in the apical mem-
brane of enterocytes and facilitates the entry of subunit A 
(MW, 26,000) into the cell, where the latter activates adenylyl 
cyclase. This markedly increases the local concentration of 
cyclic adenosine monophosphate (cAMP) after which ensues 
a complex cascade that involves the cystic fibrosis transmem-
brane conductance regulator. The end result is an intense and 
prolonged hypersecretion of water and chlorides and inhibi-
tion of the reabsorption of sodium. The gut lumen is distended 
with fluid, and hypermotility and diarrhea ensue, lasting for 
several days. LT is antigenic and cross-reacts with the entero-
toxin of Vibrio cholerae, which has an identical mechanism of 
action. LT stimulates the production of neutralizing antibod-
ies in the serum (and perhaps on the gut surface) of persons 
previously infected with enterotoxigenic E coli. Persons resid-
ing in areas where such organisms are highly prevalent (eg, 
in some developing countries) are likely to possess antibod-
ies and are less prone to develop diarrhea on reexposure to 
the LT-producing E coli. Assays for LT include (1) fluid accu-
mulation in the intestines of laboratory animals, (2) typical 
cytologic changes in cultured Chinese hamster ovary cells 
or other cell lines, (3) stimulation of steroid production in 
cultured adrenal tumor cells, (4) binding and immunologic 
assays with standardized antisera to LT, and (5) detection of 
the genes that encode the toxins. These assays are done only 
in reference laboratories.
Some strains of ETEC produce the heat-stable entero-
toxin STa (MW, 1500–4000), which is under the genetic 
control of a heterogeneous group of plasmids. STa activates 
guanylyl cyclase in enteric epithelial cells and stimulates fluid 
secretion. Many STa-positive strains also produce LT. The 
strains with both toxins produce a more severe diarrhea.
The plasmids carrying the genes for enterotoxins (LT, 
ST) also may carry genes for the CFAs that facilitate the 
attachment of E coli strains to intestinal epithelium. Recog-
nized colonization factors occur with particular frequency in 
some serotypes. Certain serotypes of ETEC occur worldwide; 
others have a limited recognized distribution. It is possible 
that virtually any E coli may acquire a plasmid encoding for 
enterotoxins. There is no definite association of ETEC with 
the EPEC strains causing diarrhea in children. Likewise, 
there is no association between enterotoxigenic strains and 
those able to invade intestinal epithelial cells.
Care in the selection and consumption of foods poten-
tially contaminated with ETEC is highly recommended to 
help prevent traveler’s diarrhea. Antimicrobial prophylaxis 
can be effective but may result in increased antibiotic resis-
tance in the bacteria and probably should not be uniformly 
recommended. When diarrhea develops, antibiotic treatment 
effectively shortens the duration of disease.
Shiga toxin-producing E coli (STEC) are named for the 
cytotoxic toxins they produce. There are at least two antigenic 
forms of the toxin referred to as Shiga-like toxin 1 and Shiga-
like toxin 2. STEC has been associated with mild non-bloody 
diarrhea, hemorrhagic colitis, a severe form of diarrhea, 
and with hemolyticuremic syndrome, a disease resulting in 
acute renal failure, microangiopathic hemolytic anemia, and 
thrombocytopenia. Shiga-like toxin 1 is identical to the Shiga 
toxin of Shigella dysenteriae type 1, and Shiga-like toxin 2 also 
has many properties that are similar to the Shiga toxin; how-
ever, the two toxins are antigenically and genetically distinct. 
A low infectious dose (< 200 CFU) is associated with infec-
tion. Of the more than 150 E coli serotypes that produce Shiga 
toxin, O157:H7 is the most common and is the one that can be 
identified most readily in clinical specimens. STEC O157:H7 
does not use sorbitol, unlike most other E coli, and is negative 
(clear colonies) on sorbitol MacConkey agar (sorbitol is used 
instead of lactose); O157:H7 strains also are negative on MUG 
tests (see earlier discussion). Many of the non-O157 serotypes 
may be sorbitol positive when grown in culture. Specific anti-
sera are used to identify the O157:H7 strains. Tests for the 
detection of both Shiga toxins using commercially available 
enzyme immunoassays (EIAs) are done in many laboratories. 
Other sensitive test methods include cell culture cytotoxin 
testing using Vero cells and polymerase chain reaction for the 
direct detection of toxin genes directly from stool samples. 
Many cases of hemorrhagic colitis and its associated compli-
cations can be prevented by thoroughly cooking ground beef 
and by avoiding unpasteurized products such as apple cider. 
In 2011, the largest outbreak of hemorrhagic colitis attrib-
uted to a non-O157 serotype—namely, E coli O104:H4—was 
related to consumption of contaminated sprouts in Germany. 
This organism had increased virulence characterized by 
enhanced adherence as well as the production of shiga-like 
toxins (see reference by Buchholz et al, 2011).
Enteroinvasive E coli (EIEC) produce a disease very 
similar to shigellosis. The disease occurs most commonly 
in children in developing countries and in travelers to these 
countries. Similar to Shigella, EIEC strains are nonlactose or 
late lactose fermenters and are nonmotile. EIEC produce dis-
ease by invading intestinal mucosal epithelial cells.
Enteroaggregative E coli (EAEC) causes acute and 
chronic diarrhea (>14 days in duration) in persons in develop-
ing countries. These organisms also are the cause of foodborne 
illnesses in industrialized countries and have been associated 
with traveler’s diarrhea and persistent diarrhea in patients with 
HIV. They are characterized by their specific patterns of adher-
ence to human cells. This group of diarrheagenic E coli is quite 
heterogeneous, and the exact pathogenic mechanisms are still 
not completely elucidated. Some strains of EAEC produce ST-
like toxin (see earlier discussion on E coli O104:H11); others 
a plasmid-encoded enterotoxin that produces cellular dam-
age; and still others, a hemolysin. Diagnosis can be suspected 
clinically but requires confirmation by tissue culture adhesion 
assays not readily available in most clinical laboratories.
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236 SECTION III Bacteriology
3. Sepsis—When normal host defenses are inadequate, 
E coli may reach the bloodstream and cause sepsis. Newborns 
may be highly susceptible to E coli sepsis because they lack 
IgM antibodies. Sepsis may occur secondary to urinary tract 
infection and often the major clone associated with invasion 
is E coli O25b/ST131.
4. Meningitis—E coli and group B streptococci are the 
leading causes of meningitis in infants. Approximately 80% 
of E coli from meningitis cases have the K1 antigen. This anti-
gen cross-reacts with the group B capsular polysaccharide 
of N meningitidis. The mechanisms of virulence associated 
with the K1 antigen are reviewed in the reference by Kim et 
al (2005).
B. Klebsiella–Enterobacter–Serratia; Proteus–
Morganella–Providencia; and Citrobacter
The pathogenesis of disease caused by these groups of enteric 
gram-negative rods is similar to that of the nonspecific fac-
tors in disease caused by E coli.
1. Klebsiella—Klebsiella pneumoniae is present in the 
respiratory tract and feces of about 5% of normal individuals. 
It causes a small proportion (~1%) of bacterial pneumonias. 
K pneumoniae can produce extensive hemorrhagic necrotiz-
ing consolidation of the lung. It produces urinary tract infec-
tion and bacteremia with focal lesions in debilitated patients. 
Other enterics also may produce pneumonia. Recently a par-
ticular clone of K pneumoniae has emerged as a cause of com-
munity acquired pyogenic liver abscess that is seen mostly 
among Asian males worldwide. This particular K1 encapsu-
lated strain phenotypically appears hypermucoviscous when 
grown in culture. Klebsiella species rank among the top 10 
bacterial pathogens responsible for hospital-acquired infec-
tions. Multilocus sequencing typing has identified global 
emergence of two particularly important clones. Sequence 
type 16 has elaborated extended spectrum β-lactamases 
resulting in resistance to a broad range of penicillins and 
cephalosporins (but not carbapenem antibiotics). ST 258 is a 
multidrug resistant strain called a “carbapenamase producer” 
because it is resistant to all β-lactam antibiotics including 
the broad spectrum carbapenem agents. Typically it is resis-
tant to other antimicrobial agents as a result of acquisition 
of plasmids that carry multiple resistance genes. Two other 
Klebsielleae are associated with inflammatory conditions of 
the upper respiratory tract: K pneumoniae subspecies ozae-
nae has been isolated from the nasal mucosa in ozena, a fetid, 
progressive atrophy of mucous membranes; and K pneu-
moniae subspecies rhinoscleromatis form rhinoscleroma, a 
destructive granuloma of the nose and pharynx. Klebsiella 
granulomatis (formerly Calymmatobacterium granuloma-
tis) causes a chronic genital ulcerative disease, granuloma 
inguinale, an uncommon sexually transmitted disease. The 
organism grows with difficulty on media containing egg yolk. 
Ampicillin or tetracycline is effective treatment.
2. Enterobacter—Three species/complexes of Enterobac-
ter—Enterobacter cloacae complex, E aerogenes complex, and 
Enterobacter sakazakii (now in the genus Cronobacter)—cause 
the majority of Enterobacter infections. These bacteria ferment 
lactose, may contain capsules that produce mucoid colonies, 
and are motile. These organisms cause a broad range of hos-
pital-acquired infections such as pneumonia, urinary tract 
infections, and wound and device infections. Most strains pos-
sess a chromosomal β-lactamase called ampC, which renders 
them intrinsically resistant to ampicillin and first- and sec-
ond-generation cephalosporins. Mutants may hyperproduce 
β-lactamase, conferring resistance to third-generation cepha-
losporins. Like K pneumoniae, some hospital-acquired strains 
have plasmids that make them multidrug resistant including 
the carbapenem class of antimicrobial agents.
3. Serratia—Serratia marcescens is a common opportu-
nistic pathogen in hospitalized patients. Serratia (usually 
nonpigmented) causes pneumonia, bacteremia, and endo-
carditis (especially in narcotics addicts) and in hospitalized 
patients. Only about 10% of isolates form the red pigment 
(prodigiosin) that has long characterized S marcescens. 
S marcescens is often multiply resistant to aminoglycosides and 
penicillins; infections can be treated with third-generation 
cephalosporins.
4. Proteus—Proteus species produce infections in humans 
only when the bacteria leave the intestinal tract. They are 
found in urinary tract infections and produce bacteremia, 
pneumonia, and focal lesions in debilitated patients or those 
receiving contaminated intravenous infusions. P mirabilis 
causes urinary tract infections and occasionally other infec-
tions. Proteus vulgaris and M morganii are also important 
nosocomial pathogens.
Proteus species produce urease, resulting in rapid hydro-
lysis of urea with liberation ofammonia. Thus, in urinary 
tract infections with Proteus species, the urine becomes alka-
line, promoting stone formation and making acidification 
virtually impossible. The rapid motility of Proteus may con-
tribute to its invasion of the urinary tract.
Strains of Proteus vary greatly in antibiotic susceptibility. 
P mirabilis is often inhibited by penicillins; the most active 
antibiotics for other members of the group are aminoglyco-
sides and cephalosporins.
5. Providencia—Providencia species (Providencia rett-
geri, Providencia alcalifaciens, and Providencia stuartii) are 
members of the normal intestinal microbiota. All cause uri-
nary tract infections and occasionally other infections and 
are often resistant to antimicrobial therapy.
6. Citrobacter—Citrobacter species can cause urinary tract 
infections and sepsis principally among debilitated hospital-
ized patients. In addition, Citrobacter koseri has been associ-
ated with meningitis in infants less than 2 months of age.
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CHAPTER 15 Enteric Gram-Negative Rods (Enterobacteriaceae) 237
Diagnostic Laboratory Tests
A. Specimens
Specimens include urine, blood, pus, spinal fluid, sputum, or 
other material, as indicated by the localization of the disease 
process.
B. Smears
The Enterobacteriaceae resemble each other morphologi-
cally. The presence of large capsules is suggestive of Klebsiella 
species.
C. Culture
Specimens are plated on both blood agar and differential 
media. With differential media, rapid preliminary identifica-
tion of gram-negative enteric bacteria is often possible (see 
Chapter 47).
D. Nucleic Acid Amplification Tests (NAATs)
A variety of multiplex NAATs designed to detect the most 
common pathogens responsible for particular syndromes, 
are currently available and many more are entering clinical 
trials. These panel tests detect members of the Enterobacte-
riaceae in specimens such as positive blood cultures, cere-
brospinal fluid, respiratory specimens, and stool. In some of 
these assays resistance markers are also detected. The reader 
should consult the literature for the most up to date informa-
tion on these assays.
Immunity
Specific antibodies develop in systemic infections, but it is 
uncertain whether significant immunity to the organisms 
follows.
Treatment
No single specific therapy is available. The sulfonamides, 
ampicillin, cephalosporins, fluoroquinolones, and aminogly-
cosides have marked antibacterial effects against the enterics, 
but variation in susceptibility is great, and laboratory tests 
for antibiotic susceptibility are essential. Multiple drug resis-
tance is common and is under the control of transmissible 
plasmids.
Certain conditions predisposing to infection by these 
organisms require surgical correction, such as relief of uri-
nary tract obstruction, closure of a perforation in an abdomi-
nal organ, or resection of a bronchiectatic portion of lung.
Treatment of gram-negative bacteremia and impending 
septic shock requires rapid institution of antimicrobial ther-
apy, restoration of fluid and electrolyte balance, and treat-
ment of disseminated intravascular coagulation.
Various means have been proposed for the prevention of 
traveler’s diarrhea, including daily ingestion of bismuth sub-
salicylate suspension (bismuth subsalicylate can inactivate E 
coli enterotoxin in vitro) and regular doses of tetracyclines or 
other antimicrobial drugs for limited periods. Because none 
of these methods are entirely successful or lacking in adverse 
effects, it is widely recommended that caution be observed 
in regard to food and drink in areas where environmental 
sanitation is poor and that early and brief treatment (eg, with 
ciprofloxacin or trimethoprim–sulfamethoxazole) be substi-
tuted for prophylaxis.
Epidemiology, Prevention, and Control
The enteric bacteria establish themselves in the normal intes-
tinal tract within a few days after birth and from then on 
constitute a main portion of the normal aerobic (facultative 
anaerobic) microbial flora. E coli is the prototype. Enterics 
found in water or milk are accepted as proof of fecal contami-
nation from sewage or other sources.
Control measures are not feasible as far as the normal 
endogenous microbiota is concerned. Enteropathogenic E coli 
serotypes should be controlled like salmonellae (see below). 
Some of the enterics constitute a major problem in hospi-
tal infection. It is particularly important to recognize that 
many enteric bacteria are “opportunists” that cause illness 
when they are introduced into debilitated patients. Within 
hospitals or other institutions, these bacteria commonly are 
transmitted by personnel, instruments, or parenteral medi-
cations. Their control depends on handwashing, rigorous 
asepsis, sterilization of equipment, disinfection, restraint in 
intravenous therapy, and strict precautions in keeping the 
urinary tract sterile (ie, closed drainage). For control of the 
multidrug-resistant pathogens, especially carbapenamase 
producers, surveillance of hospitalized patients with prompt 
implementation of contact precautions for colonized patients 
is often employed.
THE SHIGELLAE
The natural habitat of shigellae is limited to the intestinal 
tracts of humans and other primates, where they produce 
bacillary dysentery.
Morphology and Identification
A. Typical Organisms
Shigellae are slender gram-negative rods; coccobacillary 
forms occur in young cultures.
B. Culture
Shigellae are facultative anaerobes but grow best aerobically. 
Convex, circular, transparent colonies with intact edges reach 
a diameter of about 2 mm in 24 hours.
C. Growth Characteristics
All shigellae ferment glucose. With the exception of Shigella 
sonnei, they do not ferment lactose. The inability to ferment 
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238 SECTION III Bacteriology
lactose distinguishes shigellae on differential media. Shigel-
lae form acid from carbohydrates but rarely produce gas. 
They may also be divided into those organisms that ferment 
mannitol and those that do not (Table 15-2).
Antigenic Structure
Shigellae have a complex antigenic pattern. There is great 
overlapping in the serologic behavior of different species, and 
most of them share O antigens with other enteric bacilli.
The somatic O antigens of shigellae are lipopolysaccha-
rides. Their serologic specificity depends on the polysaccha-
ride. There are more than 40 serotypes. The classification of 
shigellae relies on biochemical and antigenic characteristics. 
The pathogenic species are S sonnei, Shigella flexneri, S dysen-
teriae, and Shigella boydii (Table 15-2).
Pathogenesis and Pathology
Shigella infections are almost always limited to the gastro-
intestinal tract; bloodstream invasion is quite rare. Shigel-
lae are highly communicable; the infective dose is on the 
order of 103 organisms (it usually is 105–108 for salmonellae 
and vibrios). The essential pathologic process is invasion of 
the mucosal epithelial cells (eg, M cells) by induced phago-
cytosis, escape from the phagocytic vacuole, multiplication 
and spread within the epithelial cell cytoplasm, and pas-
sage to adjacent cells. Microabscesses in the wall of the large 
intestine and terminal ileum lead to necrosis of the mucous 
membrane, superficial ulceration, bleeding, and formation of 
a “pseudomembrane” on the ulcerated area. This consists of 
fibrin, leukocytes, cell debris, a necrotic mucous membrane, 
and bacteria. As the process subsides, granulation tissue fills 
the ulcers, and scar tissue forms.
Toxins
A. Endotoxin
Upon autolysis, all shigellae release their toxic lipopolysac-
charide. This endotoxin probably contributes to the irritation 
of the bowel wall.
B. Shigella Dysenteriae Exotoxin
S dysenteriae type 1 (Shiga bacillus) produces a heat-labile 
exotoxin that affects both the gut and the central nervous 
system. The exotoxin isa protein that is antigenic (stimulat-
ing production of antitoxin) and lethal for experimental ani-
mals. Acting as an enterotoxin, it produces diarrhea as does 
the E coli Shiga-like toxin, perhaps by the same mechanism. 
In humans, the exotoxin also inhibits sugar and amino acid 
absorption in the small intestine. Acting as a “neurotoxin,” 
this material may contribute to the extreme severity and fatal 
nature of S dysenteriae infections and to the central nervous 
system reactions observed in them (ie, meningismus, coma). 
Patients with S flexneri or S sonnei infections develop anti-
toxin that neutralizes S dysenteriae exotoxin in vitro. The 
toxic activity is distinct from the invasive property of shi-
gellae in dysentery. The two may act in sequence, the toxin 
producing an early nonbloody, voluminous diarrhea and the 
invasion of the large intestine, resulting in later dysentery 
with blood and pus in stools.
Clinical Findings
After a short incubation period (1–2 days), there is a sud-
den onset of abdominal pain, fever, and watery diarrhea. 
A day or so later, as the infection involves the ileum and 
colon, the number of stools increases; they are less liquid 
but often contain mucus and blood. Each bowel movement 
is accompanied by straining and tenesmus (rectal spasms), 
with resulting lower abdominal pain. In more than half of 
adult cases, fever and diarrhea subside spontaneously in 
2–5 days. However, in children and elderly adults, loss of 
water and electrolytes may lead to dehydration, acidosis, 
and even death. The illness caused by S dysenteriae may be 
particularly severe.
On recovery, most persons shed dysentery bacilli for only 
a short period. Upon recovery from the infection, most per-
sons develop circulating antibodies to shigellae, but these do 
not protect against reinfection.
Diagnostic Laboratory Tests
A. Specimens
Specimens include fresh stool, mucus flecks, and rectal swabs 
for culture. Large numbers of fecal leukocytes and some red 
blood cells often are seen microscopically.
B. Culture
The materials are streaked on differential media (eg, MacCo-
nkey or EMB agar) and on selective media (Hektoen enteric 
agar or xylose-lysine-deoxycholate agar), which suppress 
other Enterobacteriaceae and gram-positive organisms. Col-
orless (lactose-negative) colonies are inoculated into TSI agar. 
Organisms that fail to produce H2S, that produce acid but not 
gas in the butt and an alkaline slant in TSI agar medium, and 
that are nonmotile, should be subjected to slide agglutination 
by specific Shigella antisera. Shigella and E coli cannot be dif-
ferentiated by MALDI-ToF MS.
TABLE 15-2 Pathogenic Shigella Species
Present Designation
Group and 
Type Mannitol
Ornithine 
Decarboxylase
Shigella dysenteriae A − −
Shigella flexneri B + −
Shigella boydii C + −
Shigella sonnei D + +
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CHAPTER 15 Enteric Gram-Negative Rods (Enterobacteriaceae) 239
C. Serology
Healthy persons often have agglutinins against several Shi-
gella species. However, serial determinations of antibody 
titers may show a rise in specific antibody. Serology is not 
used to diagnose Shigella infections.
D. Nucleic Acid Amplification Tests
There are several commercial NAATs that directly detect shi-
gellae in fecal samples along with some of the other major 
enteric pathogens.
Immunity
Infection is followed by a type-specific antibody response. 
Injection of killed shigellae stimulates production of antibod-
ies in serum but fails to protect humans against infection. IgA 
antibodies in the gut may be important in limiting reinfec-
tion; these may be stimulated by live attenuated strains given 
orally as experimental vaccines. Serum antibodies to somatic 
Shigella antigens are IgM.
Treatment
Ciprofloxacin, ampicillin, doxycycline, and trimethoprim–
sulfamethoxazole are most commonly inhibitory for Shigella 
isolates and can suppress acute clinical attacks of dysentery 
and shorten the duration of symptoms. Azithromycin is often 
used to treat children with shigellosis. Some antibiotics may 
fail to eradicate the organisms from the intestinal tract. Mul-
tiple drug resistance can be transmitted by plasmids, and 
resistant infections are widespread. Many cases are self-
limited. Opioids should be avoided in Shigella dysentery.
Epidemiology, Prevention, and Control
Shigellae are transmitted by “food, fingers, feces, and flies” 
from person to person. A low infectious dose (1–100 organ-
isms) is capable of causing disease. Most cases of Shigella 
infection occur in children younger than 10 years of age. 
Shigellosis, caused primarily by S sonnei, has become an 
important problem in daycare centers in the United States. 
S dysenteriae can spread widely. Because humans are the 
main recognized host of pathogenic shigellae, control efforts 
must be directed at eliminating the organisms from this res-
ervoir by (1) sanitary control of water, food, and milk; sewage 
disposal and fly control; (2) isolation of patients and disinfec-
tion of excreta; (3) detection of subclinical cases and carri-
ers, particularly food handlers; and (4) antibiotic treatment of 
infected individuals.
THE SALMONELLAE
Salmonellae are often pathogenic for humans or animals 
when acquired by the oral route. They are transmitted from 
animals and animal products to humans, where they cause 
enteritis, systemic infection, and enteric fever.
Morphology and Identification
Salmonellae vary in length. Most isolates are motile with 
peritrichous flagella. Salmonellae grow readily on simple 
media, but they almost never ferment lactose or sucrose. They 
form acid and sometimes gas from glucose and mannose. 
They usually produce H2S. They survive freezing in water for 
long periods. Salmonellae are resistant to certain chemicals 
(eg, brilliant green, sodium tetrathionate, sodium deoxycho-
late) that inhibit other enteric bacteria; such compounds are 
therefore useful for inclusion in media to isolate salmonellae 
from feces.
Classification
The classification of salmonellae is complex because the 
organisms are a continuum rather than a defined species. The 
members of the genus Salmonella were originally classified on 
the basis of epidemiology; host range; biochemical reactions; 
and structures of the O, H, and Vi (when present) antigens. 
The names (eg, S typhi, Salmonella typhimurium) were writ-
ten as if they were genus and species; this form of the nomen-
clature remains in widespread but incorrect use. DNA–DNA 
hybridization studies have demonstrated that there are seven 
evolutionary groups. Currently, the genus Salmonella is 
divided into two species each with multiple subspecies and 
serotypes. The two species are Salmonella enterica and Sal-
monella bongori (formerly subspecies V). S enterica contains 
five subspecies, which are subspecies enterica (subspecies I), 
subspecies salamae (subspecies II), subspecies arizonae 
(subspecies IIIa), subspecies diarizonae (subspecies IIIb), 
subspecies houtenae (subspecies IV), and subspecies indica 
(subspecies VI). Most human illness is caused by the subspe-
cies I strains, written as S enterica subspecies enterica. Rarely 
human infections may be caused by subspecies IIIa and IIIb 
or the other subspecies frequently found in cold-blooded 
animals. Frequently, these infections are associated with 
exotic pets such as reptiles. It seems probable that the widely 
accepted nomenclature for classification will be as follows: 
S enterica subspecies enterica serotype Typhimurium, which 
can be shortened to S Typhimurium with the genus name in 
italics and the serotype name in roman type. National and 
international reference laboratories may use the antigenic 
formulas following the subspecies name because they impart 
more precise information about the isolates (see Table 15-3).
There are more than 2500 serotypes of salmonellae, 
including more than 1400 in DNA hybridization group I 
that can infect humans.Four serotypes of salmonellae that 
cause enteric fever can be identified in the clinical laboratory 
by biochemical and serologic tests. These serotypes should 
be routinely identified because of their clinical significance. 
They are as follows: Salmonella paratyphi A (serogroup A), 
S paratyphi B (serogroup B), Salmonella choleraesuis (sero-
group C1), and S typhi (serogroup D). Salmonella serotypes 
Enteritidis and Typhimurium are the two most common 
serotypes reported in the United States. The more than 1400 
other salmonellae that are isolated in clinical laboratories are 
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240 SECTION III Bacteriology
serogrouped by their O antigens as A, B, C1, C2, D, and E; 
some are nontypeable with this set of antisera. The isolates 
are then sent to reference laboratories for definitive serologic 
identification. This allows public health officials to monitor 
and assess the epidemiology of Salmonella infections on a 
statewide and nationwide basis.
Variation
Organisms may lose H antigens and become nonmotile. Loss 
of O antigen is associated with a change from smooth to 
rough colony form. Vi antigen may be lost partially or com-
pletely. Antigens may be acquired (or lost) in the process of 
transduction.
Pathogenesis and Clinical Findings
S serotype Typhi, S serotype, and perhaps S serotype Para-
typhi and S serotype Paratyphi B are primarily infective for 
humans, and infection with these organisms implies acquisi-
tion from a human source. The vast majority of salmonellae, 
however, are chiefly pathogenic in animals that constitute 
the reservoir for human infection; these include poultry, 
pigs, rodents, cattle, pets (from turtles to parrots), and many 
others.
The organisms almost always enter via the oral route, 
usually with contaminated food or drink. The mean infective 
dose to produce clinical or subclinical infection in humans 
is 105–108 salmonellae (but perhaps as few as 103 S serotype 
Typhi organisms). Among the host factors that contribute to 
resistance to salmonella infection are gastric acidity, normal 
intestinal microbiota, and local intestinal immunity (see later).
Salmonellae produce three main types of disease in 
humans, but mixed forms are frequent (Table 15-4).
A. The “Enteric Fevers” (Typhoid Fever)
This syndrome is produced by only a few of the salmonellae, 
of which S serotype Typhi (typhoid fever) is the most impor-
tant. The ingested salmonellae reach the small intestine, from 
which they enter the lymphatics and then the bloodstream. 
They are carried by the blood to many organs, including the 
intestine. The organisms multiply in intestinal lymphoid tis-
sue and are excreted in stools.
After an incubation period of 10–14 days, fever, malaise, 
headache, constipation, bradycardia, and myalgia occur. The 
fever rises to a high plateau, and the spleen and liver become 
enlarged. Rose spots, usually on the skin of the abdomen or 
chest, are seen briefly in rare cases. The white blood cell count 
is normal or low. In the preantibiotic era, the chief complica-
tions of enteric fever were intestinal hemorrhage and perfo-
ration, and the mortality rate was 10–15%. Treatment with 
antibiotics has reduced the mortality rate to less than 1%.
The principal lesions are hyperplasia and necrosis of 
lymphoid tissue (eg, Peyer’s patches); hepatitis; focal necrosis 
of the liver; and inflammation of the gallbladder, periosteum, 
lungs, and other organs.
B. Bacteremia With Focal Lesions
This is associated commonly with S serotype choleraesuis but 
may be caused by any salmonella serotype. After oral infection, 
there is early invasion of the bloodstream (with possible focal 
TABLE 15-3 Representative Antigenic Formulas of 
Salmonellae
O Group Serotype Antigenic Formulaa
D Salmonella Typhi 9, 12 (Vi):d:—
A Salmonella Paratyphi A 1, 2, 12:a—
C
1
Salmonella Choleraesuis 6, 7:c:1,5
B Salmonella Typhimurium 1, 4, 5, 12:i:1, 2
D Salmonella Enteritidis 1, 9, 12:g, m:—
aO antigens: boldface numerals.
(Vi): Vi antigen if present.
Phase 1 H antigen: lowercase letter.
Phase 2 H antigen: numeral.
TABLE 15-4 Clinical Diseases Induced by Salmonellae
 Enteric Fevers Septicemias Enterocolitis
Incubation period 7–20 days Variable 8–48 hours
Onset Insidious Abrupt Abrupt
Fever Gradual; then high plateau with “typhoidal” 
state
Rapid rise; then spiking “septic” 
temperature
Usually low
Duration of disease Several weeks Variable 2–5 days
Gastrointestinal symptoms Often early constipation; later, bloody 
diarrhea
Often none Nausea, vomiting, diarrhea 
at onset
Blood culture results Positive in first to second weeks of disease Positive during high fever Negative
Stool culture results Positive from second week on; negative 
earlier in disease
Infrequently positive Positive soon after onset
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CHAPTER 15 Enteric Gram-Negative Rods (Enterobacteriaceae) 241
lesions in lungs, bones, meninges, etc), but intestinal mani-
festations are often absent. Blood culture results are positive.
C. Enterocolitis
This is the most common manifestation of salmonella infec-
tion. In the United States, S Typhimurium and Salmonella 
Enteritidis are prominent, but enterocolitis can be caused by 
any of the more than 1400 group I serotypes of salmonel-
lae. Eight to 48 hours after ingestion of salmonellae, there is 
nausea, headache, vomiting, and profuse diarrhea, with few 
leukocytes in the stools. Low-grade fever is common, but the 
episode usually resolves in 2–3 days.
Inflammatory lesions of the small and large intestine are 
present. Bacteremia is rare (2–4%) except in immunodeficient 
persons. Blood culture results are usually negative, but stool 
culture results are positive for salmonellae and may remain 
positive for several weeks after clinical recovery.
Diagnostic Laboratory Tests
A. Specimens
Blood for culture must be taken repeatedly. In enteric fevers 
and septicemias, blood culture results are often positive in 
the first week of the disease. Bone marrow cultures may be 
useful. Urine culture results may be positive after the second 
week.
Stool specimens also must be taken repeatedly. In enteric 
fevers, the stools yield positive results from the second or 
third week on; in enterocolitis, the stools yield positive results 
during the first week.
A positive culture of duodenal drainage establishes the 
presence of salmonellae in the biliary tract in carriers.
B. Bacteriologic Methods for Isolation of 
Salmonellae
1. Differential medium cultures—EMB, MacConkey, 
or desoxycholate medium permits rapid detection of lactose 
nonfermenters (not only salmonellae and shigellae but also 
Proteus, Serratia, Pseudomonas, etc). Gram-positive organ-
isms are somewhat inhibited. Bismuth sulfite medium permits 
rapid detection of salmonellae, which form black colonies 
because of H2S production. Many salmonellae produce H2S.
2. Selective medium cultures—The specimen is plated 
on salmonella-shigella (SS) agar, Hektoen enteric agar, xylose-
lysine desoxycholate (XLD) agar, or desoxycholate-citrate 
agar, which favor growth of salmonellae and shigellae over 
other Enterobacteriaceae. Chromogenic agars specifically for 
salmonella recovery are also available.
3. Enrichment cultures—The specimen (usually stool) 
also is put into selenite F or tetrathionate broth, both of which 
inhibit replication of normal intestinal bacteria and permit 
multiplication of salmonellae. After incubation for 1–2 days, 
this is plated on differential and selective media.
4. Final identification—Suspect colonies from solid 
media are identified by biochemical reaction patterns and 
slide agglutination tests with specific sera.
C. Serologic Methods
Serologic techniques are used to identify unknown cultures 
with known sera (see later discussion) and may also be used 
to determine antibody titers in patients with unknown ill-
ness, although the latteris not very useful in the diagnosis of 
Salmonella infections.
1. Agglutination test—In this test, known sera and 
unknown culture are mixed on a slide. Clumping, when it 
occurs, can be observed within a few minutes. This test is par-
ticularly useful for rapid preliminary identification of cultures. 
There are commercial kits available to agglutinate and sero-
group salmonellae by their O antigens: A, B, C1, C2, D, and E.
2. Tube dilution agglutination test (Widal test)—
Serum agglutinins rise sharply during the second and third 
weeks of S serotype Typhi infection. The Widal test to detect 
these antibodies against the O and H antigens has been in use 
for decades. At least two serum specimens, obtained at inter-
vals of 7–10 days, are needed to prove a rise in antibody titer. 
Serial dilutions of unknown sera are tested against antigens 
from representative salmonellae. False-positive and false-
negative results occur. The interpretive criteria when single 
serum specimens are tested vary, but a titer against the O anti-
gen of greater than 1:320 and against the H antigen of greater 
than 1:640 is considered positive. High titer of antibody to the 
Vi antigen occurs in some carriers. Alternatives to the Widal 
test include rapid colorimetric and EIA methods. There are 
conflicting reports in the literature regarding superiority of 
these methods to the Widal test. Results of serologic tests 
for Salmonella infection cannot be relied upon to establish a 
definitive diagnosis of typhoid fever and are most often used 
in resource poor areas of the world where blood cultures are 
not readily available.
D. Nucleic Acid Amplification Tests
As mentioned above for the shigellae, several commercial 
NAATs are available for direct detection of salmonellae in 
fecal samples of patients with acute diarrhea. Since these 
assays are new, performance characteristics of the assays 
and their impact on public health surveillance are still under 
investigation.
Immunity
Infections with S serotype Typhi or S Paratyphi usually con-
fer a certain degree of immunity. Reinfection may occur but 
is often milder than the first infection. Circulating antibodies 
to O and Vi are related to resistance to infection and disease. 
However, relapses may occur in 2–3 weeks after recovery 
despite antibodies. Secretory IgA antibodies may prevent 
attachment of salmonellae to intestinal epithelium.
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242 SECTION III Bacteriology
Persons with S/S hemoglobin (sickle cell disease) are 
exceedingly susceptible to Salmonella infections, particularly 
osteomyelitis. Persons with A/S hemoglobin (sickle cell trait) 
may be more susceptible than normal individuals (those with 
A/A hemoglobin).
Treatment
Although enteric fevers and bacteremias with focal lesions 
require antimicrobial treatment, the vast majority of cases of 
enterocolitis do not. Antimicrobial treatment of Salmonella 
enteritis in neonates is important. In enterocolitis, clinical 
symptoms and excretion of the salmonellae may be prolonged 
by antimicrobial therapy. In severe diarrhea, replacement of 
fluids and electrolytes is essential.
Antimicrobial therapy of invasive Salmonella infec-
tions is with ampicillin, trimethoprim–sulfamethoxazole, or 
a third-generation cephalosporin. Multiple drug resistance 
transmitted genetically by plasmids among enteric bacteria 
is a problem in Salmonella infections. Susceptibility testing is 
an important adjunct to selecting a proper antibiotic.
In most carriers, the organisms persist in the gallbladder 
(particularly if gallstones are present) and in the biliary tract. 
Some chronic carriers have been cured by ampicillin alone, 
but in most cases cholecystectomy must be combined with 
drug treatment.
Epidemiology
The feces of persons who have unsuspected subclinical dis-
ease or are carriers are a more important source of contami-
nation than frank clinical cases that are promptly isolated, 
such as when carriers working as food handlers are “shed-
ding” organisms. Many animals, including cattle, rodents, 
and fowl, are naturally infected with a variety of salmonel-
lae and have the bacteria in their tissues (meat), excreta, or 
eggs. The high incidence of salmonellae in commercially pre-
pared chickens has been widely publicized. The incidence of 
typhoid fever has decreased, but the incidence of other Salmo-
nella infections has increased markedly in the United States. 
The problem probably is aggravated by the widespread use of 
animal feeds containing antimicrobial drugs that favor the 
proliferation of drug-resistant salmonellae and their poten-
tial transmission to humans.
A. Carriers
After manifest or subclinical infection, some individuals 
continue to harbor salmonellae in their tissues for variable 
lengths of time (ie, convalescent carriers or healthy perma-
nent carriers). Three percent of survivors of typhoid become 
permanent carriers, harboring the organisms in the gallblad-
der, biliary tract, or, rarely, the intestine or urinary tract.
B. Sources of Infection
The sources of infection are food and drink that have been 
contaminated with salmonellae. The following sources are 
important:
1. Water—Contamination with feces often results in 
explosive epidemics
2. Milk and other dairy products (ice cream, 
cheese, custard)—Contamination with feces and inad-
equate pasteurization or improper handling; some outbreaks 
are traceable to the source of supply
3. Shellfish—From contaminated water
4. Dried or frozen eggs—From infected fowl or con-
taminated during processing
5. Meats and meat products—From infected animals 
(poultry) or contamination with feces by rodents or humans
6. “Recreational” drugs—Marijuana and other drugs
7. Animal dyes—Dyes (eg, carmine) used in drugs, foods, 
and cosmetics
8. Household pets—Turtles, dogs, cats, exotic pets such 
as reptiles, and so on
Prevention and Control
Sanitary measures must be taken to prevent contamination 
of food and water by rodents or other animals that excrete 
salmonellae. Infected poultry, meats, and eggs must be thor-
oughly cooked. Carriers must not be allowed to work as food 
handlers and should observe strict hygienic precautions.
Two typhoid vaccines are currently available in the 
United States: an oral live, attenuated vaccine (Ty 21a) and a 
Vi capsular polysaccharide vaccine (Vi CPS) for intramuscu-
lar use. Vaccination is recommended for travelers to endemic 
regions, especially if the traveler visits rural areas or small 
villages where food choices are limited. Both vaccines have 
an efficacy of 50–80%. The time required for primary vacci-
nation and age limits for each vaccine varies, and individuals 
should consult the Centers for Disease Control and Preven-
tion’s Web site or obtain advice from a travel clinic regarding 
the latest vaccine information.
CHAPTER SUMMARY
•	 Members of the Enterobacteriaceae are short gram-
negative rods that grow rapidly on standard laboratory 
media.
•	 Members of this group are catalase positive; nitrate posi-
tive and, with the exception of Plesiomonas, are cyto-
chrome oxidase negative. Organisms can be identified 
readily by the ability to ferment lactose on MacConkey 
and by other biochemical reactions or newer technolo-
gies such as MALDI-TOF MS.
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CHAPTER 15 Enteric Gram-Negative Rods (Enterobacteriaceae) 243
•	 Enterobacteriaceae express a variety of antigens that 
include somatic or O antigens (cell wall lipopolysaccha-
ride), capsular or K antigens, and H or flagellar antigens. 
Salmonella express the Vi antigens. These antigens are 
virulence factors and can be used to serotype those organ-
isms that possess them.
•	 Enterobacteriaceae cause a variety of human infections 
that can be broadly classified as either enteric diseases 
or extraintestinal infections such as urinary tract infec-
tions, bacteremia, and meningitis.•	 Genera associated with enteric illnesses include Salmo-
nella, Shigella, and diarrheagenic E coli, of which there 
are six types based on the mechanism of disease (eg, toxi-
genic or invasive or both).
•	 The most common extraintestinal infections caused by 
these organisms are urinary tract infections. E coli pre-
dominates, but the urea-positive organisms such as Pro-
teus species can cause bladder and kidney stones.
•	 Enterobacteriaceae acquired in the hospital environment 
are often resistant to many antimicrobial agents usually 
mediated by plasmid-encoded resistance determinants.
REVIEW QUESTIONS
1. A 20-year-old college student goes to the student health center 
because of dysuria, frequency, and urgency on urination for 
24 hours. She has recently become sexually active. On urinalysis, 
many polymorphonuclear cells are seen. The most likely organ-
ism responsible for these symptoms and signs is
(A) Staphylococcus aureus
(B) Streptococcus agalactiae
(C) Gardnerella vaginalis
(D) Lactobacillus species
(E) Escherichia coli
2. A 27-year-old woman is admitted to the hospital because of 
fever, with increasing anorexia, headache, weakness, and altered 
mental status of 2 days’ duration. She works for an airline as a 
cabin attendant, flying between the Indian subcontinent and 
other places in Southeast Asia and the West Coast of the United 
States. Ten days before admission, she had a diarrheal illness 
that lasted for about 36 hours. She has been constipated for the 
past 3 days. Her temperature is 39°C, heart rate is 68 beats/
min, blood pressure is 120/80 mm Hg, and respirations are 
18 breaths/min. She knows who she is and where she is but does 
not know the date. She is picking at the bedclothes. Rose spots 
are seen on her trunk. The remainder of the physical examina-
tion is normal. Blood cultures are done, and an intravenous line 
is placed. The most likely cause of her illness is
(A) Enterotoxigenic Escherichia coli (ETEC)
(B) Shigella sonnei
(C) Salmonella enterica subspecies enterica serotype Typhimurium 
(Salmonella Typhimurium)
(D) Salmonella enterica subspecies enterica serotype Typhi 
(Salmonella Typhi)
(E) Enteroinvasive Escherichia coli (EIEC)
3. Blood cultures from the patient in question 2 grow a non–
lactose-fermenting gram-negative bacillus. Which of the 
following is likely to be a constituent of this organism?
(A) O antigen 157, H antigen 7 (O157:H7)
(B) Vi antigen (capsule; virulence antigen)
(C) O antigen 139 (O139)
(D) Urease
(E) K1 (capsular type 1)
4. A 37-year-old woman with a history of urinary tract infections 
comes to the emergency department with burning on urination 
along with frequency and urgency. She says her urine smells like 
ammonia. The cause of her urinary tract infection is likely to be
(A) Enterobacter aerogenes
(B) Proteus mirabilis
(C) Citrobacter freundii
(D) Escherichia coli
(E) Serratia marcescens
5. An 18-year-old student has abdominal cramps and diarrhea. A 
selective agar plate is inoculated and grows suspicious gram-
negative rods. Triple sugar iron agar is used to identify the 
isolates as salmonellae or shigellae. A result suggesting one of 
these two pathogens would be
(A) Production of urease
(B) Motility in the medium
(C) Inability to ferment lactose and sucrose
(D) Fermentation of glucose
(E) Production of gas in the medium
6. An uncommon serotype of Salmonella enterica subspecies 
enterica was found by laboratories in the health departments 
of adjacent states. The isolates were all from a small geographic 
area on either side of the border between the states, suggest-
ing a common source for the isolates. (All of the isolates were 
from otherwise healthy young adults who smoked marijuana; 
the same Salmonella was isolated from a specimen of the mari-
juana.) By what method did the public health laboratories 
determine that these isolates were the same?
(A) Capsular (K antigen) typing
(B) O antigen and H antigen typing
(C) DNA sequencing
(D) Sugar fermentation pattern determination
(E) Decarboxylase reaction pattern determination
7. A 43-year-old man with diabetes has a 4-cm nonhealing foot 
ulcer. Culture of the ulcer yields Staphylococcus aureus, Bacte-
roides fragilis, and a gram-negative bacillus that swarms across 
the blood agar plate covering the entire surface of the agar after 
36 hours. The gram-negative bacillus is a member of the genus
(A) Escherichia
(B) Enterobacter
(C) Serratia
(D) Salmonella
(E) Proteus
8. A 4-year-old boy from Kansas City who recently started attend-
ing preschool and after-school daycare is brought to his pedia-
trician for a diarrheal illness characterized by fever to 38.2°C, 
severe lower abdominal pain, and initially watery diarrhea. His 
mother became concerned because the stools are now blood 
tinged 24 hours into the illness, and the child appears quite 
ill. The mother reports that two other children who attend the 
same after-school daycare have recently had diarrheal disease, 
one of whom likewise had bloody stools. Which of the fol-
lowing is the most likely pathogen causing the illness in these 
children?
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244 SECTION III Bacteriology
(A) An enterotoxigenic strain of Escherichia coli
(B) Salmonella enterica subspecies enterica serotype Typhi 
(Salmonella Typhi)
(C) Shigella sonnei
(D) Edwardsiella tarda
(E) Klebsiella oxytoca
9. A 5-year-old girl attended a birthday party at a local fast food 
restaurant. About 48 hours later, she developed cramping 
abdominal pain and a low-grade fever and had five episodes of 
loose, bloody stools. She is taken to a local emergency depart-
ment the next evening because the diarrhea has continued, and 
she now appears pale and lethargic. On presentation, she has a 
temperature of 38°C, and she is hypotensive and tachycardic. 
The abdominal examination reveals tenderness in the lower 
quadrants. Laboratory work is remarkable for a serum creati-
nine of 2.0 mg/dL, a serum hemoglobin of 8.0 mg/dL, thrombo-
cytopenia, and evidence of hemolysis. What is the most likely 
pathogen causing this child’s illness?
(A) Escherichia coli O157:H7
(B) Salmonella enterica subspecies enterica serotype Typhimurium
(C) Enteropathogenic Escherichia coli
(D) Edwardsiella tarda
(E) Plesiomonas shigelloides
10. A 55-year-old homeless man with alcoholism presents with 
severe multilobar pneumonia. He requires intubation and 
mechanical ventilation. A Gram stain of his sputum reveals 
numerous polymorphonuclear leukocytes and gram-negative 
rods that appear to have a capsule. The organism is a lactose 
fermenter on MacConkey agar and is very mucoid. It is nonmo-
tile and lysine decarboxylase positive. What is the most likely 
organism causing this man’s illness?
(A) Serratia marcescens
(B) Enterobacter aerogenes
(C) Proteus mirabilis
(D) Klebsiella pneumoniae
(E) Morganella morganii
11. Which of the following statements regarding O antigens is 
correct?
(A) All Enterobacteriaceae possess identical O antigens.
(B) They are found in the polysaccharide capsules of enteric 
bacteria.
(C) They are covalently linked to a polysaccharide core.
(D) They do not stimulate an immune response in the host.
(E) They are not important in the pathogenesis of infection 
caused by enteric bacteria.
12. Which of the following test methods is the least sensitive pro-
cedure for diagnosis of colitis caused by Shiga toxin–producing 
Escherichia coli?
(A) Culture on sorbitol MacConkey agar
(B) Toxin testing using an enzyme immunoassay
(C) Cell culture cytotoxin assay using Vero cells
(D) Polymerase chain reaction for detection of the genes that 
encode Shiga toxin
13. An HIV-positive man recently traveled to the Caribbean for 
a 2-week vacation. He developed acute watery diarrhea and 
abdominal pain without fever during the second week of his 
vacation. Three weeks later, he is seen in clinic for persistent 
symptoms, and he is concerned because he is beginning to lose 
weight. Given this history,you suspect:
(A) Enteroinvasive Escherichia coli
(B) Salmonella typhi
(C) Enteropathogenic Escherichia coli
(D) Shigella flexneri
(E) Enteroaggregative Escherichia coli
14. Heat-labile toxin of ETEC acts by which of the following 
mechanisms?
(A) Attachment and effacement
(B) Activation of adenylyl cyclase
(C) Aggregative adherence
(D) Ribosomal dysfunction
(E) None of the above
15. A young woman presents with recurrent urinary tract infec-
tions caused by the same Proteus mirabilis strain. What is the 
major concern?
(A) She does not take her medication.
(B) She is pregnant because pregnant patients are more suscep-
tible to UTIs.
(C) She has a bladder or kidney stone.
(D) Her partner is infected.
(E) She has occult diabetes and should have a glucose tolerance 
test.
Answers
 1. E
 2. D
 3. B
 4. B
 5. C
 6. B
 7. E
 8. C
 9. A
10. D
11. C
12. A
13. E
14. B
15. C
REFERENCES
Buchholz U, et al: German outbreak of Escherichia coli O104:H4 
associated with sprouts. N Engl J Med 2011;365:1763–1770.
Donnenberg MS: Enterobacteriaceae.  In Bennett JE, Dolin R, 
Blaser MJ (editors). Mandell, Douglas and Bennett’s Principles 
and Practice of Infectious Diseases, 8th ed. Elsevier, 2015.
Eigner U, et al: Performance of a matrix-assisted laser desorption 
ionization-time-of-flight mass spectrometry system for the 
identification of bacterial isolates in the clinical routine labora-
tory. Clin Lab 2009;55:289–296.
Forsythe SS, Pitout J, Abbott S: Klebsiella, Enterobacter, Citro-
bacter, Cronobacter, Serratia, Plesiomonas, and other Entero-
bacteriaceae.  In Jorgensen JH, Pfaller MA, Carroll KC, et al 
(editors). Manual of Clinical Microbiology, 11th ed. ASM Press, 
2015.
Johnson JR, et al: E coli sequence type ST131 as the major cause 
of serious multidrug-resistant E. coli infections in the United 
States. Clin Infect Dis 2010;51:286–294.
Kim BY, Kang J, Kim KS: Invasion processes of pathogenic 
Escherichia coli. Int J Med Microbiol 2005;295:463–470.
Strockbine NA, et al: Escherichia, Shigella, and Salmonella. In 
 Jorgensen JH, Pfaller, MA, Carroll KC, et al (editors). Manual 
of Clinical Microbiology, 11th ed. ASM Press, 2015.
Pegues DA, Miller SI: Salmonella species.  In Bennett JE, Dolin R, 
Blaser MJ (editors). Mandell, Douglas and Bennett’s Principles 
and Practice of Infectious Diseases, 7th ed. Elsevier, 2010.
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	Section III: Bacteriology
	15. Enteric Gram-Negative Rods (Enterobacteriaceae)
	Classification
	Diseases Caused By Enterobacteriaceae Other Than Salmonella and Shigella
	The Shigellae
	The Salmonellae
	Chapter Summary
	Review Questions