Taxonomy
Members of the genus Escherichia are classified in the
family Enterobacteriaceae, which is
addressed in chapter
37 of this Manual (32,
66, 139, 175). There are six species in this
genus: Escherichia albertii, Escherichia blattae, Escherichia
coli, Escherichia fergusonii,
Escherichia hermannii, and Escherichia vulneris. The G+C content
is 48 to 59 mol%, and the
type species is E. coli (Migula 1895) (39).
Average DNA relatedness between the type
species and other Escherichia species, as assessed by
DNA-DNA hybridization, ranges from
38% to 64%. As more genomes become sequenced, refinements to the
classification of
members of the familyEnterobacteriaceae will be possible. A
recent publication proposes to
reclassify E. blattae in a newly formed genus as Shimwellia
blattae, based on sequence
analysis of the 16S rRNA gene and four protein coding genes (162).
E. blattae exhibits about
43% DNA relatedness to E. coli by DNA-DNA hybridization.
Members of the genus Shigella are phenotypically similar to
Escherichia coli and, with the
exception of Shigella boydii serotype 13, would be
considered the same species by DNA-DNA
hybridization analysis (29) and whole-genome
sequence analysis (82). Findings from recent
phylogenetic studies with nucleotide sequences of internal
fragments from 14 housekeeping
genes show that S. boydii 13 strains cluster in a
neighbor-joining tree with E. albertii.
Comparative genomic analysis has provided important insights into
the structure and
organization of bacterial genomes and the mechanisms by which they
evolved. The genomes
of more than 20 E. coli strains representative of commensal
and different pathogenic groups
(pathotypes) have been sequenced and compared (2,
26, 46, 60, 146, 167). They range in
size from 4,639,221 base pairs for E. coli K-12 (26)
to 5,945,000 base pairs for a strain of
Shiga toxin-producing E. coli (STEC) serotype O26:H11 (146).
In a comparison of 17
genomes, the number of genes per genome ranged from 4,238 to
5,589, with an average of
2,344 (46.7%) of these being conserved (167).
Strains representative of a pathotype
contained shared genes as well as unique genes. Enterohemorrhagic E.
coli (EHEC) strains
shared the largest number of genes, with 122 group-specific genes,
while
uropathogenic E. coli (UPEC) strains shared 45 to 56 genes;
enteropathogenicE. coli (EPEC),
enteroaggregative E. coli (EAEC), and enterotoxigenic E.
coli (ETEC) strains each shared 3 to
5 genes; and commensal strains shared 11 genes. The number of strain-specific
genes
varied widely and was not consistent within a pathotype (167).
Pathogenomic analysis of the numerous plasmids present within
representative strains of
each of the E. colipathotypes and commensal E. coli has
revealed considerable diversity and
plasticity within these genetic elements (105).
In contrast to the overall plasmid diversity,
there is limited diversity in the virulence plasmids, which are
restricted to a few plasmid
backbones, with conservation and linkage of their core components
(105). These plasmids
contain distinct regions for genetic exchange that appear to
evolve via insertion sequencemediated
site-specific recombination. Many such plasmids have also acquired
multidrug
resistance-encoding islands. In addition to plasmids, bacteriophages
play a major role in
generating genome diversity by promoting homologous recombination
and horizontal gene
transfer between bacteria (11, 95).
For example, the Sakai strain of E. coli O157:H7 contains
18 prophages and 6 prophage-like elements (16% of the total
genome), which carry a wide
range of virulence genes, including the genes for Shiga toxins 1
and 2 (92, 147). Although
many of these prophages carry genetic defects, some are able to be
induced and recombine
with each other to generate new phages capable of transferring
virulence genes to other
bacteria (11). The dynamic nature of the Shiga
toxin-converting phages has implications for
diagnostic testing for STEC. Since STEC strains can lose critical
virulence genes, some
researchers have proposed that multiple virulence-associated
genes, as well as conserved
genes, be used to diagnose infections by these bacteria (22,
23). This concept would also
apply to other pathotypes of E. coli, as most of them carry
critical virulence genes on mobile
genetic elements.
Description of the Genus
The genus Escherichia is composed of motile or nonmotile
bacteria that conform to the
definitions of the familyEnterobacteriaceae (66,
175). Species in this genus are gramnegative,
oxidase-negative rods that grow well on MacConkey agar (MAC). When
these
organisms are motile, it is by peritrichous flagella. They can
grow aerobically or
anaerobically. All ferment D-glucose, and most produce gas from
the fermentation of this
substrate and other fermentable carbohydrates. Lactose is
fermented by most strains of E.
coli, but its fermentation may be delayed or absent in all or most
strains of E. albertii, E.
blattae, E. fergusonii, and E. vulneris.
Epidemiology and Transmission
E. coli occurs naturally in the lower part of the intestines of humans and
warm-blooded
animals. In humans, it typically colonizes an infant’s
gastrointestinal tract within hours of
birth and subsequently becomes a prominent facultative anaerobe in
the human colonic
microbiota, where it exists in a mutually beneficial relationship
with its host. E. coli generally
remains confined to the intestinal lumen; however, in a
debilitated or immunosuppressed
host or when bacteria are introduced to other tissues following
trauma or surgical
procedures, even commensal, “nonpathogenic” strains of E. coli can
cause infection. E. coli is
typically transmitted through ingestion of contaminated food and
water, person-to-person
contact, contact with animals, or contact with environments or
fomites contaminated with
fecal material. Convincing evidence of respiratory transmission
has not been reported. Most
strains of E. coli do not cause disease in healthy persons;
however, there are specific
pathogenic groups, addressed below, whose members are capable of
causing disease in
humans and animals. The infectious dose for diarrheagenic E.
coli varies by strain and
pathotype and is estimated to range from 10 to 100 bacteria for E.
coli O157:H7 to at least
108 bacteria for ETEC (72).
Because E. coli is ubiquitous in human and animal feces,
the presence of this species in
water is considered an indicator of fecal contamination. It can be
isolated from fecescontaminated
foods or water but probably does not occur as a free-living
organism in the
environment.
E. fergusonii, E. hermannii, E. vulneris, and E. albertii have been isolated from a
wide variety
of human clinical specimens (stool, urine, sputum, blood, spinal
fluid, peritoneal dialysis
fluid, and wounds) (13, 33,
70, 100,119, 157, 179). E. fergusonii and E. albertii have
also
been isolated from wild and domestic birds (15,
145). Recently, E. vulneris strains with
traits
common to mammalian pathogens were found actively multiplying
within legumes, raising
the possibility that plants may represent a niche for transmission
of clinically important
strains (137). In addition, E. vulneris was recovered
in combination with other members of
theEnterobacteriaceae from neonatal enteral feeding tubes,
highlighting the potential
importance of these devices as risk factors for neonatal
infections (99). E. blattae occurs
naturally in the hindgut of cockroaches and is not known to cause
disease in humans.
Clinical Significance
Of the six Escherichia species, E. coli is the
species usually isolated from human specimens
and is the one that we know the most about. We know little about
the pathogenesis of the
other escherichiae; however, it is interesting that strains of E.
albertii, which have been
associated with diarrheal disease in children, contain the locus
for enterocyte effacement
(LEE) pathogenicity island that is also present in EPEC and EHEC (100,101,
187).
Extraintestinal E. coli
Pathogenic E. coli strains are broadly grouped into two
categories, namely, extraintestinal
pathogenic E. coli(ExPEC) and intestinal or diarrheagenic E.
coli, depending on whether they
cause disease outside or within the intestinal tract. Commensal E.
coli strains, which
comprise the majority of the facultative anaerobic intestinal
biota in most humans and other
mammals, typically do not cause disease but can be opportunistic
pathogens when certain
conditions exist, such as the presence of a foreign body (e.g.,
urinary catheter), host
compromise (e.g., local anatomical or functional abnormalities,
such as urinary or bile tract
obstruction or immunocompromise), or a breach in normally sterile
sites causing the
introduction of feces or high concentrations of mixed bacteria.
ExPEC strains carry a distinct
set of virulence genes that enable them to cause disease outside
the intestine. This category
contains at least two well-recognized pathogenic groups or
pathotypes—UPEC and
meningitis/sepsis-associated E. coli (MNEC)—and a variety
of disease-associated strains not
yet classified into specific pathotypes.
UPEC
UPEC strains are a major cause of community-acquired urinary tract
infections and possess a
variety of chromosomally and plasmid-encoded virulence factors
that are present in various
combinations. Members of this group have a limited number of O
antigens (six O groups
cause 75% of urinary tract infections) and show combinations of
traits, including expression
of adhesins (P [Pap], type 1, and other fimbriae), toxins
(hemolysin, cytotoxic necrotizing
factor, and an autotransported protease [Sat]), or aerobactin,
serum resistance, and
encapsulation, that are epidemiologically associated with cystitis
and acute pyelonephritis in
individuals with normal urinary tracts. No single phenotypic
profile defining UPEC has
emerged. UPEC strains possess large and small pathogenicity
islands containing blocks of
genes not found in the chromosomes of fecal strains. For a review
of the virulence genes and
a proposed model of the pathogenesis of UPEC, see the work of
Kaper et al. (108) and
Johnson and Russo (104).
MNEC
MNEC strains are the most common gram-negative organisms causing
neonatal meningitis,
which is associated with high morbidity and mortality. The MNEC
pathotype is comprised of
strains with a limited number of O antigens, and about 80% are
positive for the K1
antigen. E. coli strains that cause meningitis are spread
hematogenously. Levels of
bacteremia correlate with the development of meningitis: levels of
>103CFU/ml of blood are
significantly associated with the development of meningitis (59).
After entering the blood,
these bacteria invade brain microvascular endothelial cells
through membrane-bound
vacuoles. Within these vacuoles, the organisms control
intracellular trafficking to avoid
lysosomal fusion and to gain access to the central nervous system
without causing apparent
damage to the blood-brain barrier. Recent studies have identified
several bacterial
determinants (IbeA, IbeB, IbeC, AslA, CNF1, FimH, and OmpA) which
contribute to the
pathogenesis of MNEC in vitro. For reviews, see the work of Kim et
al. (113) and Kaper et al.
(108).
Diarrheagenic E. coli
There are at least five categories of recognized diarrheagenic E.
coli: STEC, which includes a
subset of strains referred to as enterohemorrhagic E. coli (EHEC)
for their ability to cause
bloody diarrhea and hemorrhagic colitis; ETEC; EPEC; EAEC; and
enteroinvasive E.
coli (EIEC) (108, 139). The clinical significance of several other
groups of putative
diarrheagenic E. coli, particularly diffusely adherent E.
coli (DAEC), is unclear.
STEC
We refer to the STEC category of diarrheagenic E. coli according
to the toxins that the
organisms produce, i.e., as STEC rather than EHEC, because the
essential genetic features
that define organisms capable of causing hemorrhagic colitis and
hemolytic-uremic syndrome
(HUS) are not clear. E. coli serotypes O157:H7 and
O157:nonmotile (O157:NM) (collectively
called O157 STEC) produce one or more Shiga toxins, also called
verocytotoxins, and are the
most frequently identified diarrheagenic E. coli serotypes
in North America and Europe. Each
year, an estimated 73,000 cases of illness and 60 deaths are
caused by O157 STEC in the
United States (133).
E. coli O157:H7 and other STEC serotypes cause illness that can present as
mild nonbloody
diarrhea, severe bloody diarrhea (hemorrhagic colitis), or HUS
(reviewed in reference 84).
Additional symptoms of E. coliO157:H7 infection include
abdominal cramps and lack of a high
fever. Among patients with O157 STEC diarrhea, 4% or more develop
HUS (165), a condition
characterized by microangiopathic hemolytic anemia,
thrombocytopenia, and acute renal
failure. The fatality rate of HUS has declined in recent years due
to improvements in case
management.
O157 STEC is thought to cause at least 80% of cases of HUS in
North America and is
recognized as a common cause of bloody diarrhea in developed
countries (165). In the
United States, the rate of isolation of O157 STEC from fecal
specimens is highest in the
Northern Tier states, where it may approach the rates for common
diarrheal pathogens.
Many U.S. clinical laboratories do not routinely culture or
otherwise test stools for O157
STEC; as a result, many illnesses are not detected (44,
204). O157 STEC colonizes dairy and
beef cattle, and therefore ground beef has caused more O157 STEC
outbreaks than any
other vehicle of transmission (165,184).
Other known vehicles of transmission include raw
milk, sausage, roast beef, unchlorinated municipal water, apple
cider, raw vegetables, and
sprouts; these vehicles are typically exposed to water
contaminated by bovine manure. O157
STEC spreads easily from person to person because the infectious
dose is low (<200 CFU);
outbreaks associated with person-to-person spread have occurred in
schools, long-term care
institutions, families, and day care facilities.
More than 150 non-O157 STEC serotypes have been isolated from
persons with diarrhea or
HUS (http://www.microbionet.com.au/frames/feature/vtec/brief01html). In some countries,
non-O157 STEC strains, particularly E. coli serotypes
O111:NM and O26:H11, are more
commonly isolated than O157 STEC strains, although most outbreaks
and cases of HUS are
attributed to the latter (serotypes characteristic of
diarrheagenic E. coli pathotypes are
presented in Table
2). In the United States, E.
coli O157:H7 is the most frequently isolated
STEC serotype, but increasingly, non-O157 STEC strains are
identified as causes of
outbreaks and sporadic illness (36). At the CDC E. coli Reference
Laboratory, 71% of all non-
O157 STEC isolates received between 2003 and 2008 belonged to six
serogroups (O26,
O103, O111, O121, O45, and O145) (N. A. Strockbine, unpublished
data). Because most
laboratory methods for the detection of O157 STEC do not detect
non-O157 STEC, the
numbers of infections with serotypes other than O157:H7 or O157:NM
are probably
underestimated.
EPEC
In the past, EPEC strains were defined as certain E. coli serotypes
that were
epidemiologically associated with infantile diarrhea but did not
produce enterotoxins or Shiga
toxins and were not invasive. The traditional EPEC serotypes are
listed in Table 2; typically,
these serotypes show a distinct pattern of localized adherence to
HeLa and HEp-2 cells
(203). These serotypes usually also demonstrate actin
aggregation in the fluorescent actin
stain test, which correlates with the attaching-and-effacing (A/E)
lesion in vivo (139).
Because of the lack of simple diagnostic methods for detection of
EPEC strains, few
laboratories attempt to identify these organisms. Full EPEC pathogenicity
requires two
genetic elements: the EPEC adherence factor (EAF) plasmid, which
encodes, most
importantly, the bundle-forming pilus, and the chromosomal LEE,
which mediates the A/E
phenotype. The term “typical EPEC” has been suggested for those
organisms harboring both
the EAF plasmid and the LEE pathogenicity island (see below).
Typical EPEC strains
correspond to EPEC strains of the classical serotypes and are
important causes of diarrhea in
developing countries (62, 139);
these organisms were implicated in highly lethal nursery
outbreaks in the United States and the United Kingdom before 1970.
The infection is
currently rare in the industrialized world. More recently,
atypical EPEC strains have been
implicated as enteric pathogens in the United States, including
implication in an outbreak of
diarrheal disease (94, 200).
These strains possess a functional LEE apparatus but do not
carry the EAF plasmid. The full role of these pathogens has yet to
be elucidated, but they
may be considered potential causes of diarrheal outbreaks when no
other pathogens are
identified.
Symptoms of severe, prolonged, and nonbloody diarrhea, vomiting,
and fever in infants or
young toddlers are characteristic of EPEC illness (139).
Infection with EPEC has been
associated with chronic diarrhea; sequelae may include
malabsorption, malnutrition, weight
loss, and growth retardation.
EIEC
EIEC strains invade cells of the colon and produce a generally
watery, but occasionally
bloody, diarrhea by a pathogenic mechanism similar to that of Shigella.
EIEC is rare in the
United States and is less common than ETEC or EPEC in the
developing world (139). EIEC
strains, like ETEC and EPEC, are associated with a few
characteristic serotypes (Table
2).
Three large outbreaks of diarrhea caused by EIEC have been
reported in the United States
(139).
EAEC
EAEC, originally defined by its specific pattern of aggregative
adherence to HEp-2 cells in
culture, has been associated with diarrhea in a variety of
clinical settings, including endemic
diarrhea in children of both impoverished and industrialized
countries, epidemic diarrhea,
diarrhea of travelers to developing countries, and persistent
diarrhea among patients with
human immunodeficiency virus infection (98).
The pathogenicity of EAEC has been confirmed
in volunteer studies (138) and by implication of
EAEC in diarrhea outbreaks (52). Early
studies frequently failed to find an association of EAEC with
pediatric diarrhea, but this
association has been strengthened by the use of molecular techniques
which discriminate the
true pathogens exhibiting the aggregative pattern (53,
174). The term “typical EAEC”
describes organisms harboring virulence genes under the control of
the global EAEC
regulator AggR (174). Typical EAEC may be a common cause of pediatric
diarrhea in U.S.
infants (53) and should be considered a potential cause of
food-borne outbreaks and
diarrhea in human immunodeficiency virus-infected patients (98).
EAEC diarrhea is
accompanied by signs and symptoms of mild inflammation (abdominal
pain and fever), but
stools usually do not contain blood or fecal leukocytes (98).
Putative Diarrheagenic E. coli
Several putative pathotypes have been described. Virulence has not
been demonstrated
clearly for any of these types by either volunteer studies or
outbreak investigations. DAEC
strains, which exhibit a characteristic diffuse pattern of
adherence to HEp-2 cells, have been
implicated as causes of diarrhea in some epidemiologic studies but
not others (139), and a
prototypical DAEC strain did not elicit diarrhea in adult
volunteers (193). In several studies,
DAEC infections were significantly associated with watery diarrhea
among children of 1 to 5
years of age but were not associated with illness among infants (118).
DAEC may occur in
industrialized countries (139). A complex signal
transduction cascade has been suggested as
the mechanism of DAEC pathogenesis (180).
Cytotoxic necrotizing factor (CNF)-producing E. coli strains
produce a toxin that induces
morphological alterations (multinucleation) and death in tissue
cultures (38). Two forms
have been described: CNF1 and CNF2. CNF1-producing strains were
originally detected in
infants with enteritis and later in humans with extraintestinal
infections (25, 38). Most CNF1-
producing strains are also hemolytic, although the toxin is
distinct from hemolysin. CNF2-
producing strains have been isolated from animals with diarrhea (57,
150, 185). The role of
these strains in human diarrheal disease has not been determined
definitively (139).
Cytolethal distending toxin-producing E. coli strains
produce a heat-labile factor that induces
cytotonic and cytotoxic changes in Chinese hamster ovary cells
similar to those caused by LT
(106). This factor does not affect Y-1 cells. The
results of one study in Bangladesh suggested
that cytolethal distending toxin-producing E. coli strains
are associated with diarrhea
(5, 106), but other studies are needed to establish their
status as etiologic agents.
Several diarrheal outbreaks have been linked to E. coli strains
that do not belong to any of
the established pathotypes. Some of these strains carry the gene
encoding the
enteroaggregative ST-like toxin (EAST1), which is related to the
ETEC ST enterotoxin.
Further studies are needed to prove the pathogenicity of these
strains, but the EAST1 gene
can be identified by use of molecular techniques (132).
Collection, Transport, and Storage of Specimens
Information on the collection, transport, and storage of specimens
from extraintestinal sites
is provided inchapter
16 of this Manual.
Fecal specimens should be collected in the early
stages of any enteric illness (preferably within 4 days of onset),
when pathogens are usually
present in the stool in the largest numbers and before
antimicrobial therapy has been
started. Whole stools are usually the specimen of choice, but
carefully collected rectal swabs
with visible fecal staining may be preferable for diagnosis of Shigella
(4, 91,190). Collection
of multiple specimens may enhance the recovery rate of E. coli,
Shigella, and Salmonella (65).
Transport of fecal specimens to the laboratory in a timely fashion
is critical, particularly for
more delicate organisms such as Shigella (210).
Ideally, fecal specimens should be examined
as soon as they are received in the laboratory, but if not
processed immediately, they should
be either refrigerated or frozen at–70°C. Fecal specimens that
will not be examined within 1
to 2 h of collection and all rectal swabs should be placed in cold
transport medium and kept
at 4°C (80). Transport and storage of fecal specimens at 4°C
are very important
for Shigella as well as Campylobacter spp.
Manufacturers of commercial transport media,
including the acceptable media listed below, commonly state that
fecal specimens may be
transported and stored at ambient temperature. For Shigella and
Campylobacter spp., this is
not advised because there are data showing that transport at
ambient temperature may
deleteriously affect recovery of these organisms (204,
207).
Many of the commercially available transport media (e.g.,
Cary-Blair, Stuart’s, and Amies
transport media) and buffered glycerol saline are satisfactory for
E. coli,
Salmonella, and Shigella. Although acceptable for the transport of the
organisms addressed
in this chapter, buffered glycerol saline should not be used for
specimens that must also be
tested for Campylobacter and Vibrio (58,
207).
Direct Examination
Microscopy
Gram stains of patient specimens from normally sterile body sites
can provide a preliminary
indication of which category of bacteria to cultivate from the
specimen and if inflammatory
cells or blood are present; however, E. coli and other Escherichia
species cannot readily be
distinguished from other gram-negative rods by staining or
microscopy methods.
Antigen Detection
Several commercial immunoassays are available to diagnose STEC
infections by detecting
Shiga toxin or the O157 antigen (lipopolysaccharide [LPS]) in
fecal specimens (Table 3).
These assays are usually more sensitive when performed on enriched
specimens than on
stool directly. Isolation of STEC from fecal specimens that are
positive by one of these rapid
diagnostic methods is important for public health purposes.
Determination of the subtype of
O157 STEC or the serotype of a non-O157 STEC isolate is valuable
for outbreak
investigations and
surveillance purposes (see “Typing Systems” and “Identification” below).
Nucleic Acid Detection
There are no U.S. Food and Drug Administration (FDA)-approved
nucleic acid detection
methods for the clinical diagnosis of pathogenic E. coli or
other Escherichia infections. Due to
the number of inhibitors and amount of competing DNA present in
stool specimens, most
researchers have found it necessary to perform an enrichment step
before testing these
specimens by PCR for pathogenic E. coli. One group recently
reported success for use of an
automated DNA extraction system to prepare target DNA (Shiga toxin
genes) from
unenriched stool specimens for amplification in a home-brew
real-time PCR assay (86).
Isolation Procedures
Isolation Procedures for Extraintestinal E.
coli
Isolation procedures for E. coli and other Escherichia species
from sites outside the intestines
are covered inchapter
16 in this Manual. Any
E. coli strain isolated in large numbers
(particularly >105 CFU/ml of urine) or from normally sterile
body sites should be considered
a potential pathogen. Although E. coli strains with
particular virulence in the urinary tract
cannot easily be distinguished on differential and selective
plating media from organisms of
lower virulence, they are commonly hemolytic on sheep blood agar
and express one or more
of several urinary tract adhesins. Several chromogenic media have
been proposed for use in
detecting UPEC; these have been compared in published studies (12,
47).
Isolation Procedures for Diarrheagenic E. coli
Isolation Procedures for STEC
Guidelines published in 2009 (83) recommend that all
stools submitted for testing for routine
enteric pathogens (Salmonella, Shigella, and Campylobacter)
at clinical diagnostic
laboratories and all patients with suspected HUS should be
cultured for O157 STEC on
selective and differential agar and assayed for non-O157 STEC with
a test that detects the
Shiga toxins or genes encoding these toxins. The recommendations
to isolate O157 STEC
and to detect non-O157 STEC were extended to all stools from
patients with acute,
community-acquired diarrhea because selective testing strategies
such as testing only bloody
stools or specimens from children, limiting testing to summer
months, or basing testing on
the presence of indicators such as white blood cells miss many
STEC infections. The absence
of blood in the stool does not negate the possibility of an
STEC-associated diarrheal illness
(111, 129). In several studies, most STEC isolates,
including both O157 and non-O157
strains, were from patients with apparently nonbloody diarrhea (78,
83, 152, 196,201). Also,
STEC strains are isolated more frequently from children, but
almost half of all isolates are
obtained from persons of >12 years of age (36,
41, 184, 201), so limiting STEC testing to
children would miss many infections. Although infections are more
common in summer
months, seasonality is not a reliable predictor of STEC infections
because infections and
outbreaks occur year-round (36, 184,
196). As a sign of STEC, white blood cells are often
but not always observed in the stools of patients with STEC
infection; thus, determination of
white blood cells in stool should not be used as a criterion for
STEC specimen selection
(87,184).
Because there is no selective isolation medium for non-O157 STEC
strains, testing for the
presence of Shiga toxin in fecal specimens is the best approach
for detecting these
organisms. Commercial enzyme-linked immunoassays (EIAs) are a
sensitive means of
detecting Shiga toxin (21, 78,
111, 125, 151). Isolation and serotyping of STEC from fecal
specimens that are positive by nonculture assays should always be
attempted because
serotype information is important for public health purposes and
may also help in clinical
decisions.
Enrichment. Although broth enrichment is widely used for the recovery of O157
STEC from
foods, there is little evidence that it enhances isolation from
human fecal specimens.
However, immunomagnetic separation (IMS), a technique shown to
increase the rate of
isolation of O157 STEC from food specimens, has been adapted to
culture of fecal specimens
(109). IMS enhances the detection of O157 STEC from
patients with HUS, patients
presenting an extended time after the onset of illness,
asymptomatic carriers, or specimens
that have been stored or transported improperly. IMS beads for
O157, O111, and O26 are
available commercially (Table 3), or laboratories may
produce beads with other O-specific
antibodies (153).
Plating media. Because O157 STEC strains ferment lactose, they are impossible to
differentiate from other lactose-fermenting organisms on
lactose-containing media. Most
O157 STEC strains do not ferment the carbohydrate D-sorbitol
overnight, in contrast to the
approximately 80% of other E. coli strains that ferment
sorbitol rapidly. Thus, sorbitolcontaining
selective media are often used for isolation of O157 STEC.
Sorbitol-nonfermenting
colonies are suspected (but not definitively known) to be O157:H7
(130). In some areas of
central Europe, sorbitol-fermenting O157 STEC strains are commonly
isolated from patients
with HUS (24); these organisms are very rare in North America
(Strockbine, unpublished
data).
Specific culture media have been developed to exploit phenotypic
and antibiotic resistance
traits that are characteristic of STEC strains. Although
sorbitol-containing MAC (SMAC) is
widely used, cefixime-tellurite-containing SMAC (CT-SMAC) and
CHROMAgar O157 have been
shown to increase the sensitivity of culture for O157 STEC (49,
213). It has been reported
that some O157:NM strains fail to grow on CT-SMAC (109).
Several chromogenic agar media
are available commercially to assist in rapid identification (Table 3); these media generally
perform well for O157:H7 and for some non-O157 STEC strains (19,
127, 144).
Screening procedures for STEC strains. For the isolation of O157 STEC from SMAC,
colorless (nonfermenting) colonies are tested with O157 antiserum
or latex reagent (186)
(Table 3). If the O157 latex reagent is used, it is
important to test positive colonies with the
latex control reagent to rule out nonspecific reactions. The
manufacturers of these kits
recommend that strains reacting with both the antigen-specific and
control latex reagents be
heated and retested. However, in a study that followed this
procedure, none of the
nonspecifically reacting strains were subsequently identified as
O157 STEC (27).
Unlike most other E. coli strains, O157 STEC strains do not
express beta-glucuronidase;
therefore, the MUG reaction
(4-methylumbelliferyl-beta-D-glucuronide for detection of betaglucuronidase
activity) is helpful for screening for O157 STEC (177).
MUG-positive, ureasepositive
O157 STEC strains have been isolated in the United States but are
still rare (93;
Strockbine, unpublished data).
For the recovery of STEC strains from stool specimens which test
positive for Shiga toxin,
either SMAC or MAC should be inoculated. It is advantageous to use
SMAC because O157
STEC can be identified quickly and easily. If
sorbitol-nonfermenting colonies are negative
with O157 latex, then sorbitol-fermenting colonies (because most
non-O157 STEC strains
ferment sorbitol) and a representative sample of
sorbitol-nonfermenting colonies may be
selected for Shiga toxin testing. Latex reagents and antisera (Table 3) for detecting certain
non-O157 STEC serotypes are now available and could also be used
to test colonies from
Shiga toxin-positive specimens or to serogroup Shiga
toxin-positive isolates.
Virtually all O157 STEC strains and 60 to 80% of non-O157 STEC
strains produce a
characteristic E. colihemolysin, referred to as
enterohemolysin (Ehly), which is distinct from
the alpha-hemolysin produced by otherE. coli strains (20).
Washed sheep blood agar
supplemented with calcium (WSBA-Ca) is used as a differential
medium for the detection of
enterohemolytic activity (20). Ehly-producing colonies
can be differentiated from alphahemolysin-
producing colonies on WSBA-Ca because the latter are visible after
3 to 4 h of
incubation. After 3 to 4 h, colonies are marked for the appearance
of alpha-hemolysin, and
the plates are examined again after 18 to 24 h to detect the
enterohemolysin producers.
Incorporation of mitomycin C into WSBA-Ca enhances the appearance
of Ehly hemolysis and
increases the proportion of non-O157 STEC strains that exhibit
this activity (191). Because
many non-O157 STEC strains do not demonstrate the enterohemolytic
phenotype and
because nontoxigenic enterohemolytic strains have been reported,
additional screening
methods should be used in conjunction with WSBA-Ca medium (176).
Presumptive STEC isolates should be sent to a reference laboratory
or a public health
laboratory for further characterization.
Isolation Procedures for Other Diarrheagenic E.
coli
Methods for the isolation of ETEC, EPEC, EIEC, EAEC, and putative
diarrheagenic E.
coli strains are generally available only in reference or research
settings. Public health and
reference laboratories usually examine specimens for these
pathogens only when an
outbreak has occurred and specimens are negative for routine
bacterial pathogens. ETEC and
EAEC should be considered possible etiologic agents of watery
diarrhea for which no other
pathogen has been identified, especially for travelers (53).
EPEC should be considered a
possible pathogen in outbreaks of severe nonbloody diarrhea
occurring in infants or young
toddlers, particularly in nursery or day care settings. EIEC
should be considered a possible
etiologic agent in outbreaks of nonwatery diarrhea (bloody or
nonbloody).
To capture E. coli for further testing, fecal specimens
should be plated on a differential
medium of low selectivity (e.g., MAC). Five to 20 colonies, mostly
lactose fermenting but
with a representative sample of nonfermenting colonies, should be
selected and inoculated
onto nonselective agar slants (such as L agar or nutrient agar).
These colonies are then sent
to a reference laboratory for testing or are screened for
virulence-associated characteristics if
assays are available. Strains can be kept frozen for long periods
in L broth with 15 to 50%
glycerol at -80°C. Arrangements for sending E. coli isolates
from well-characterized
outbreaks to the CDC for testing can be made through local and
state health departments.
Screening procedures for ETEC, EPEC, EAEC, and
EIEC strains. E. coli pathotypes other
than STEC cannot be distinguished from other E. coli strains
by phenotypic screening
techniques. Many EIEC strains are nonmotile and fail to
decarboxylate lysine; however, some
EIEC strains are motile or lysine positive. Use of commercial
antisera to the classical EPEC
somatic (O) and capsular (K) antigens is no longer recommended.
Identification
Phenotypic Identification
With the exception of E. albertii, the commercial
identification systems do a good job of
identifying mostEscherichia strains. Identification of E.
albertii with these systems remains
problematic because representative strains of this species are not
yet included in commercial
databases (1). Abbott and colleagues, who extensively
characterized five strains of E.
albertii by conventional phenotypic methods and by commercial
identification panels,
reported that E. albertii is an indole-negative species
that ferments D-mannitol but not Dxylose
(1). In their study, E. albertii strains were
identified by commercial systems as Hafnia
alvei, Salmonella orSalmonella enterica serotype
Choleraesuis, E. coli (inactive or serotype
O157:H7), or Yersinia ruckeri. Although some strains were
clearly misidentified, the majority
of the strains generated probability scores for the final
identification that were unacceptable,
or the identification was inconsistent with the source of the
specimen (e.g., identification of
the fish pathogen Y. ruckeri from a human specimen), which
should have triggered additional
phenotypic tests to establish a more reliable identification. The
authors found that the most
reliable clue to the possible presence of E. albertii was
an unacceptable first-choice
identification of H. alvei for an isolate that is both L-rhamnose
and D-xylose negative.
Phenotypic tests that can help discriminate E. albertii strains
from selected members of
the Enterobacteriaceaefamily with similar phenotypic traits
are shown in Table 1. Two
biogroups of E. albertii are listed in Table 1. These correlate with two of the distinct clusters
of strains identified in the E. albertii lineage by
phylogenetic studies (101). Biogroup 1 is
comprised of the five strains isolated from Bangladeshi children
with diarrhea, while biogroup
2 is comprised of strains formerly identified as S. boydii 13.
The strains in the two biogroups
differ from each other in the abilities to produce indole from
tryptophan, decarboxylate
lysine, and ferment D-sorbitol. Antigenic relationships between
members of the E.
albertii lineage and other members of theEnterobacteriaceae family
have been observed
(e.g., S. boydii 7 and E. coli O28). A diagnostic
PCR assay using three housekeeping genes
was described by Hyma et al. (101) for detection of E.
albertii; this assay is independent of
phenotypic or antigenic traits and should facilitate studies to
learn about the diversity within
the lineage, the natural habitat of this species, and its role in
enteric disease.
Phenotypic identification of presumptive O157 STEC isolates is
necessary because other
species may cross-react with O157 antiserum or latex reagents,
including Salmonella O
group N (O:30), Yersinia enterocoliticaserotype O9, Citrobacter
freundii, and E. hermannii.
Additional phenotypic tests (cellobiose fermentation and growth in
the presence of KCN) may
be necessary to differentiate E. hermannii from E. coli,
but because E. hermannii is rarely
detected in stool specimens, use of these tests is not
cost-effective for most laboratories.
Serotyping
The serologic classification of E. coli is generally based
on the O antigen (somatic) and the H
antigen (flagellar) (18). The O and H antigens of
E. coli are stable and reliable strain
characteristics, and although 181 O antigens and 56 H antigens
have been described (a few
of which are no longer recognized), the actual number of serotype
combinations associated
with diarrheal disease is limited (Table 2). Determination of the O
and H serotypes of E.
coli strains implicated in diarrheal disease is particularly useful in
epidemiologic
investigations (Table
2). Even though antisera
for the tube agglutination test are available
from several manufacturers, most laboratories do not attempt to
complete E. coli serotyping
because it is costly. For well-characterized outbreaks with no
identified etiologic agent,
arrangements may be made through state health departments to send E.
coli isolates to the
CDC for virulence testing and serotyping.
Serologic Confirmation of O157 STEC
Confirmation of E. coli O157:H7 requires identification of
the H7 flagellar antigen. H7-specific
antisera and latex reagents are commercially available (Table 3), but detection of the H7
flagellar antigen often requires multiple passages (186).
Isolates that are nonmotile or
negative for the H7 antigen should be tested for the production of
Shiga toxins or the
presence of Shiga toxin gene sequences.
Approximately 85% of O157 isolates from humans received by the CDC
are serotype
O157:H7, 12% are nonmotile, and 3% are H types other than H7
(Strockbine, unpublished
data). E. coli O157:NM strains frequently produce Shiga
toxin and are otherwise very similar
to O157:H7, but no O157 strain from human illness with an H type
other than H7 has been
found to produce Shiga toxin (73; Strockbine, unpublished data).
Nucleic Acid-Based Methods
Accurate identification of bacterial isolates is important for
directing patient care and
management. Compared to traditional phenotypic approaches, which
can be influenced by
phenotypic variation or subjective interpretation, 16S rRNA gene
sequencing is a more
objective identification tool and has the potential to reduce
laboratory errors. Some clinical
laboratories have begun using molecular methods to aid in the
identification of organisms
that cannot be cultivated due to unusual growth characteristics or
antibiotic treatment or
cannot be classified by phenotypic methods (76,
134, 155, 166, 181, 213). In one study,
results obtained with 16S rRNA gene sequencing and the SmartGene
IDNS (Zug,
Switzerland) database and software compared favorably to those
obtained by conventional
phenotypic methods and were better than those obtained with a
similar rRNA gene method
employing a smaller database for a collection of 300 clinical
isolates. The performance
differences between the two 16S rRNA gene methods highlight the
importance of the size
and breadth of the database for successful classification.
Difficulties separating E.
coli from Shigella with 16S rRNA gene sequencing should be
expected. These are genetically
the same species and have been maintained as separate taxa for
medical expediency. The
limited findings reported in the studies above and those reported
by others (140) show that
a small region of the 16S rRNA gene alone will not provide
reliable separation of certain
medically relevant members of the Enterobacteriaceae family
(E. coli/Shigella sonnei [181]
and Escherichia/Shigella/Hafnia [134]). The incorporation
of virulence genes that define
the Shigella/EIEC pathotype should help to discriminate it
from noninvasive pathotypes or
communal E. coli. Another approach that has potential to
improve microbial identifcation
involves mass spectroscopy. The Ibis T5000 biosensor system
(Abbott Molecular, Des
Plaines, IL), which is currently used in nonclinical and research
settings, uses multiple
regions of the 16S and 23S rRNA genes plus several housekeeping
genes to discriminate
between species within 6 h (63). Validation studies are
needed to assess the performance of
this technology on clinical specimens.
Virulence Testing
Extraintestinal E. coli
Numerous virulence factors have been identified for
extraintestinal E. coli (108), particularly
the K1 antigen, but these are usually identified only in
epidemiological studies.
Diarrheagenic E. coli
Detection of diarrheagenic pathotypes is typically performed on E.
coli colonies chosen from
selective or nonselective media. If PCR techniques are used, a
sweep of confluent growth
from a MAC plate may be screened; if the PCR assay is positive,
isolated colonies may then
be picked and screened individually. Multiplex PCR assays are
capable of simultaneously
detecting multiple E. coli pathotypes (142).
STEC. Two distinct Shiga toxins, Stx1 and Stx2, also referred to as
verocytotoxins, have
been described. In addition, there are several variant forms of
Stx2, including Stx2c, Stx2d,
Stx2e, and Stx2f, which in one study were identified more
frequently from asymptomatic
carriers than from HUS patients (77). All of these toxins are
similar to the Shiga toxin
expressed by Shigella dysenteriae serotype 1, and the Stx1
toxins produced by O157 STEC
and other STEC serotypes are virtually identical. STEC may produce
either Stx1, Stx2, or
both toxins. The production of Stx or the genes encoding Stx can
be detected by a variety of
biologic, immunologic, or nucleic acid-based assays (139).
Protocols for several of these
tests (e.g., cell culture, DNA probing, and PCR) are available (149).
Stx has also been
detected directly in the blood of HUS patients by use of flow
cytometry, even in the absence
of serologic or microbiologic evidence of STEC infection (198).
STEC strains represent a spectrum of virulence potentials, ranging
from the highly virulent
O157:H7 serotype that has been responsible for the majority of
outbreak cases to apparently
avirulent serotypes that have been isolated only from nonhuman
sources. The presence of
additional virulence factors other than Stx correlates with
disease potential. The most
important of these virulence factors are the intimin adhesin and
the type III secretion system
encoded by the LEE pathogenicity island (108).
The eae gene probe for intimin and the hlyA
(ehxA) gene probe for a plasmid-encoded hemolysin have been the most
frequently
employed methods to determine virulence potential, but probes for
at least 25 different
virulence-associated genes have been employed to characterize STEC
strains (160). STEC
strains have been classified into five “seropathotypes” (A through
E) based on the occurrence
of serotypes in human disease, in outbreaks, and in severe disease
(HUS or hemorrhagic
colitis) and on possession of specific virulence genes (110).
ETEC. The ST and LT enterotoxins produced by ETEC may be detected by a
variety of
biologic, immunologic, and nucleic acid-based assays (139).
Two distinct ST variants (STh
and STp) have been identified in human strains. Strains that
produce ST only or ST in
combination with LT have caused most ETEC outbreaks in the United
States (54).
Immunoassays for the identification of ST or LT in culture supernatants
of ETEC strains are
available from at least two commercial sources (Table 3). The ST EIA assay (Denka Seiken
Co., Ltd., and Oxoid Ltd.) is a competitive EIA for the detection
of ST only (178). A reversed
passive latex agglutination assay (VET-RPLA; Oxoid [a similar kit
is available from Denka
Seiken]) detects both cholera toxin and LT, which are highly
related antigenically. The
effectiveness of VET-RPLA may be optimized by use of a culture
medium designed for LT
production, such as Biken’s medium, rather than the medium
recommended by the
manufacturer (214).
EPEC. EPEC, EAEC, and DAEC can be detected by their characteristic
patterns of adherence
to HEp-2 or HeLa cells in culture (203). These patterns are also
observed on formalin- or
glutaraldehyde-fixed cells, obviating the need to prepare cells
expressly for the assay (135).
EPEC strains are defined on the basis of the A/E histopathology
produced on epithelial cells
and the lack of Stx (reviewed in references 62 and
108). The A/E phenotype can be detected
by tissue culture cell assays or by DNA probe or PCR tests for the
eae gene, encoding
intimin, or the LEE pathogenicity island. The EAF plasmid of
typical EPEC (see above) is
detected by use of fragment or oligonucleotide probes or PCR
primers (139). Atypical EPEC
strains possess only the A/E phenotype (LEE pathogenicity island)
but do not possess the
EAF plasmid.
EAEC. Several simple assays have been described as surrogates for the
cell adherence test
for identification of EAEC. These include a simple biofilm
formation assay on polystyrene
(215) and screening for the presence of a pellicle at
the surface of broth media (6). EAEC
can be identified more definitively by use of a specific DNA probe
(the AA or CVD432 probe)
(16), which is superior to tissue culture adherence
assays for identifying pathogenic strains
of EAEC (53). More recent data suggest that the AA probe
corresponds to a putative
virulence gene called aatA (143), which is under the
control of a regulator termed AggR.
AggR, in turn, controls several other virulence factors (174).
Thus, the aggR gene (which
defines typical EAEC) may represent a superior diagnostic target.
EIEC. EIEC can be identified by various in vivo assays, immunoassays,
and nucleic acidbased
assays for invasiveness, but no commercial kits or reagents are
available. Cell culture
invasion assays or DNA-based assays for the ipaC or ipaH
invasion-related factors are, for
the most part, practical only in research settings (139).
Plasmid DNA electrophoresis may be
used to detect the large 120- to 140-MDa plasmid associated with
invasiveness, but this
plasmid is easily lost when the isolate is subcultured. Because of
shared invasiveness-related
characteristics, these assays also detect Shigella strains.
DAEC. DAEC strains were initially defined on the basis of a diffuse
adherence pattern to
cultured epithelial cells, but this phenotype is not specific for
enteric strains (180). Various
DNA probes and PCR assays have been proposed for DAEC
identification, as reviewed
previously (139).
Typing Systems
Several methods for subtyping have been used for E. coli O157:H7
isolates. In particular,
pulsed-field gel electrophoresis (PFGE) methods and multilocus
variable-number tandemrepeat
analysis methods are useful (102, 139).
A national molecular subtyping network,
PulseNet, was established in 1996 by the CDC to facilitate
subtyping of bacterial food-borne
pathogens, including E. coli O157:H7, Shigella, nontyphoidal
Salmonellaserotypes,
and Listeria monocytogenes (192). Successful detection of
outbreaks by this network of state
and local public health laboratories is dependent upon submission
of isolates by clinical
laboratories for confirmation and subtyping.
Determination of the serotype and the antimicrobial susceptibility
pattern is usually adequate
for defining outbreak strains of ETEC, EPEC, and EIEC. Plasmid
typing or PFGE methods may
also be helpful for distinguishing between sporadic isolates and
outbreak strains, but neither
method has been used widely for these groups of E. coli.
Serodiagnostic Tests
At present, serodiagnostic tests for diarrheagenic E. coli are
valuable only for
seroepidemiology surveys and are not useful for the diagnosis of
sporadic infections. Assays
that measure serum antibody responses to LPS have been used to
detect STEC infection in
culture-negative HUS patients (139). Enzyme-linked
immunosorbent assays have been
described to detect saliva antibodies to LPS (124)
and serum antibodies to the secreted EspB
protein in HUS patients (183).
Antimicrobial Susceptibilities
Extraintestinal E. coli
In E. coli and other Enterobacteriaceae, extended-spectrum
β-lactamases (ESBLs) are an
important cause of antimicrobial resistance. In the past 20 years,
CTX-M β-lactamases and
AmpC β-lactamases have emerged. CTX-M-producing E. coli strains
are often isolated from
urinary tract infections, both health care and community acquired,
and have also been
detected in retail meat samples in the United States (61).
AmpC β-lactamases are
problematic for clinical laboratories because these enzymes can
interfere with β-lactamase
ESBL confirmatory tests, resulting in a false report of
cephalosporin susceptibility.
Carbapenemases are β-lactamases that confer resistance to the
carbapenems, and
the Klebsiella pneumoniae carbapenemase (KPC) is the most
frequently encountered enzyme
of this class in E. coli and other carbapenem-resistantEnterobacteriaceae.
It is important to
detect KPC and other carbapenemases in patients colonized with
carbapenemresistant
Enterobacteriaceae so that isolation precautions may be instituted to
prevent
transmission in health care settings (7).
In January 2010, the Clinical and Laboratory Standards Institute
(CLSI) published new
interpretative criteria for phenotypically assessing the
susceptibility of
the Enterobacteriaceae to the cephalosporins and aztreonam
(51). Under the new guidelines,
lower breakpoints will be used, thereby eliminating the need to
perform routine ESBL tests
and to edit the results on reports from susceptible to resistant
for cephalosporins,
aztreonam, or penicillins. No reduction in breakpoints was
proposed for cefepime and
cefuroxime (parenteral).
The CLSI suggests that routine antimicrobial susceptibility
testing (AST)
for Enterobacteriaceae include ampicillin, cefazolin (MIC
only), gentamicin, and tobramycin
testing. It is noted that E. coli and otherEnterobacteriaceae
strains resistant to certain
cephalosporins may produce a carbapenemase despite having AST
values which fall in the
susceptible range. Screening tests such as the modified Hodge test
should be performed.
Urinary isolates of E. coli may be tested against
fosfomycin and other drugs used only for
urinary tract infections. Current automated susceptibility test
systems may not be able to
accurately detect ESBLs, AmpCs, and KPCs (75,
173).
Diarrheagenic E. coli
STEC
Antimicrobial therapy for O157 STEC diarrhea or HUS is
controversial: some publications
have suggested that antibiotics increase the risk of HUS (84,
212), while a meta-analysis of
published reports found no significantly increased risk (172).
There is a lack of evidence to
support routine antimicrobial susceptibility testing of STEC
strains.
Until recently, E. coli O157:H7 isolates were almost
uniformly susceptible to antimicrobial
agents. However, since the early 1990s, O157 and other STEC
strains have demonstrated
slowly increasing levels of resistance to certain antibiotics,
particularly streptomycin,
sulfonamides, and tetracycline (http://www.cdc.gov/narms/).
ETEC, EPEC, EAEC, and EIEC Strains and Other
Diarrheagenic E. coli Strains
Treatment with an appropriate antibiotic can reduce the severity
and duration of symptoms
of ETEC infection (139). Antimicrobial
resistance, particularly to tetracycline, is common
among ETEC strains isolated from outbreaks in the United States (54).
Antibiotic treatment
may be helpful for diarrhea caused by EPEC (139).
Most EPEC strains associated with
outbreaks are resistant to multiple antimicrobial agents (62).
EAEC strains are commonly
resistant to most antibiotics, though these strains are typically
susceptible to
fluoroquinolones. Clinical studies have demonstrated the
effectiveness of ciprofloxacin for
travelers with diarrhea caused by EAEC (81).
Little information about the efficacy of
antimicrobial treatment or the prevalence of resistance is
available for EIEC or other putative
diarrheagenic E. coli strains, but determination of the
antimicrobial susceptibility pattern may
be helpful in establishing whether the isolates are associated
with an outbreak.
Evaluation, Interpretation, and Reporting of
Results
Extraintestinal E. coli
The final written report should include the final Gram stain
result, the final identification as E.
coli, and the antimicrobial susceptibility test results.
Diarrheagenic E. coli
STEC
A presumptive diagnosis of an O157 STEC (isolate positive for O157
antigen) or a non-O157
STEC (isolate positive for Shiga toxin) infection should be
reported to the clinician as soon as
the laboratory obtains this result. When O157 is not found in a specimen,
it is advisable to
include a comment on reports stating that non-O157 STEC strains
can cause diarrhea and
HUS. Cases of STEC infection and HUS should be reported to public
health authorities.
Presumptive STEC isolates should be confirmed by demonstration of
the O157 and H7
antigens or by assay for Shiga toxin and should be identified
phenotypically as E. coli. STEC
isolates should be forwarded to a local or state public health
laboratory for serotyping and/or
molecular subtyping.
ETEC, EPEC, EAEC, and EIEC Strains
Generally, the ETEC, EPEC, EAEC, and EIEC classes of diarrheagenic
E. coli are identified only
during outbreak investigations. A laboratory reporting these
results, which usually will be a
retrospective diagnosis obtained by a reference laboratory, should
provide an explanation of
the clinical significance of these organisms and may refer the
clinician to the reference
laboratory for further information. All suspected outbreaks should
be reported to public
health authorities.
SHIGELLA Back to top
Taxonomy
Shigella is classified in the family Enterobacteriaceae, which is
addressed in chapter 37 of
this Manual (32, 66).
There are four subgroups of Shigella that historically have been treated
as species, as follows: subgroup A asS. dysenteriae, subgroup
B as Shigella
flexneri, subgroup C as S. boydii, and subgroup D as Shigella
sonnei. From a genetic
standpoint, the four species of Shigella, with the
exception of S. boydii 13, and E.
coli represent a single genomospecies (30, 31,
115). Using a genetic definition for species,
the four species of Shigella would be regarded as
serologically defined anaerogenic biotypes
of E. coli. The current nomenclature for Shigellaorganisms
is maintained largely for medical
purposes because of the useful association of the genus epithet
with the distinctive disease
(shigellosis) caused by these organisms. The G+C content of the
DNA is 49 to 53 mol%, and
the type species for the genus is S. dysenteriae (Shiga
1898) (39).
S. boydii 13 strains were first described in 1952 and then added to the Shigella
scheme in
1958 (69). Early findings from DNA-DNA hybridization
showed that these strains represent a
new species (30); however, it was not until recently that
findings from phylogenetic studies
showed that they cluster in a neighbor-joining tree with E.
albertii, a newly described species
of Escherichia associated with diarrheal disease in
Bangladeshi children (100, 101).
Description of the Genus
The genus Shigella is composed of nonmotile bacteria that conform
to the definition of the
familyEnterobacteriaceae (66, 188).
Species in this genus are gram-negative rods which
grow well on MAC. All strains of Shigella spp. are
nonmotile, do not decarboxylate lysine, do
not utilize citrate, malonate, or sodium acetate (with exceptions
for S. flexneri), and do not
grow in KCN or produce H2S. Compared with Escherichia, Shigellastrains
are less active in
their use of carbohydrates (Table 4). All ferment D-glucose
without the production of gas (a
few exceptions produce gas, e.g., certain strains of S.
flexneri serotype 6 and S.
boydii serotype 14). S. sonnei strains ferment lactose and sucrose
on extended incubation,
but other species generally do not use these substrates in
conventional medium. Salicin,
adonitol, and myo-inositol are not fermented. There are
numerous identical and reciprocal
serologic reactions
between Shigella and E. coli (67).
Epidemiology and Transmission
Humans and other large primates are the only natural reservoirs of
Shigella bacteria. Most
transmission is by person-to-person spread, but infection is also
caused by ingestion of
contaminated food or water. Shigellosis is most common in
situations where hygiene is
compromised (e.g., child care centers and other institutional
settings). In developing
populations without running water and indoor plumbing, shigellosis
can become endemic.
Sexual transmission of Shigella among men who have sex with
men also occurs.
In the United States, an estimated 450,000 cases of shigellosis
occur each year, with 70
deaths (133). Up to 20% of all U.S. cases of shigellosis are
related to international travel.
Most infections in the United States and other developed countries
are caused by S. sonnei;
S. flexneri is the second most common subgroup (http://www.cdc.gov/
nationalsurveillance/shigella_surveillance.html). In the developing world, the majority of
endemic dysentery cases are caused by S. flexneri, with the
balance of cases caused by
subgroups that vary temporally and geographically (35,
114, 205). Epidemic dysentery is
most commonly caused by S. dysenteriae1, whose prevalence
rises dramatically during
outbreak periods and then falls as the epidemic resolves.
Infection with S. dysenteriae 1 is
associated with high rates of morbidity and mortality in
developing countries, particularly
when antimicrobial resistance or misdiagnosis delays appropriate
treatment. In the United
States and other developed countries, S. sonnei is endemic
and causes large, protracted
outbreaks in day care centers (9, 40,
43) and among men who have sex with men (45,
55).
The protracted nature of these outbreaks is attributed to a large
number of asymp
tomatically infected individuals in the population and the
tendency for secondary spread
(88). Most S. sonnei strains in the United
States have developed resistance to ampicillin and
trimethoprim-sulfamethoxazole (55). For most individuals,
antibiotic treatment reduces the
number of symptomatic days and the length of shedding (123).
Resistance to ampicillin and
trimethoprim-sulfamethoxazole is common. If resistance to these
antibiotics is present,
treatment with azithromycin or ciprofloxacin has been effective.
To limit the development of
resistance, some health care providers treat only the most severe
infections.
Clinical Significance
Members of the genus Shigella have been recognized since
the late 19th century as
causative agents of bacillary dysentery. Shigella causes
bloody diarrhea (dysentery) and
nonbloody diarrhea. Shigellosis often begins with watery diarrhea
accompanied by fever and
abdominal cramps but may progress to classic dysentery with scant
stools containing blood,
mucus, and pus. Ulcerations, which are restricted to the large
intestine and rectum, typically
do not penetrate beyond the lamina propria. Bloodstream infections
can occur but are rare.
Appropriate antimicrobial therapy will decrease the duration,
transmission, and severity of
symptoms and should be prescribed based on the severity of illness
or the need to protect
close contacts. Patients in certain occupations (i.e., food
handlers, child care providers, and
health care workers) and children who attend child care often are
required to have a
documented negative stool culture following treatment. The
infectious dose is low (1 to 100
organisms), and the incubation period is 1 to 4 days. Shigellae
are shed in stool for several
days to several weeks after illness, and persons who receive
appropriate antimicrobial
therapy will be culture negative at 72 h (123).
All four subgroups of Shigella are capable of
causing dysentery, but S. dysenteriae serotype 1 has been
associated with a particularly
severe form of illness thought to be related in part to its
production of Shiga toxin. Infection
can occasionally be asymptomatic, particularly infection with S.
sonnei strains. Complications
of shigellosis include HUS, which is associated with S.
dysenteriae 1 infection, and reactive
arthritis or Reiter’s chronic arthritis syndrome, which is
associated with S. flexneri infection
(3). The identification of Shigella species
is important for both clinical and epidemiologic
purposes.
Collection, Transport, and Storage of Specimens
See “Collection, Transport, and Storage of Specimens” in the Escherichia
section.
Direct Examination
Microscopy
Shigella cannot readily be distinguished from other gram-negative rods by
staining or
microscopy methods.
Antigen Detection
Because there is no single somatic antigen common to all Shigella
strains, antigen detection
in clinical specimens is not practical and has not been validated,
and no commercial FDAapproved
kits are available.
Nucleic Acid Detection
There are no FDA-approved nucleic acid detection methods for
clinical diagnosis
of Shigella infections.
Isolation Procedures
Enrichment and Plating Media
There is no reliable enrichment medium for all Shigella isolates,
but gram-negative broth and
Selenite broth (SEL) are frequently used. For the optimal
isolation of Shigella, two different
selective media should be used: a general purpose plating medium
of low selectivity (e.g.,
MAC) and a more selective agar medium (e.g.,
xylose-lysine-deoxycholate agar [XLD]).
Deoxycholate citrate agar and Hektoen enteric agar (HE) are
suitable alternatives to XLD as
media with moderate to high selectivities. Salmonella-shigella
agar should be used with
caution because it inhibits the growth of some strains of S.
dysenteriae 1.
Screening Procedures
Shigella strains appear as lactose- or xylose-nonfermenting colonies on the
isolation media
described above.S. dysenteriae 1 colonies may be smaller on
all of these media, and these
strains generally grow best on media with low selectivities (e.g.,
MAC). S. dysenteriae 1
colonies on XLD agar are frequently very tiny, unlike those of
other Shigella species. S.
sonnei colonies often appear flattened and spread out on blood agar
plates.
Suspect colonies may be screened phenotypically on Klig ler iron
agar (KIA) or triple sugar
iron agar (TSI).Shigella species characteristically produce
an alkaline slant because strains
do not ferment lactose (or sucrose) but do not produce gas or H2S.
A few strains of S.
flexneri 6 and very few strains of S. boydii produce gas in KIA or
TSI. Motility and lysine
decarboxylase tests are characteristically negative for Shigella
and can be used to further
screen isolates before serologic testing (Table 4). Isolates that react appropriately with the
screening tests should then be identified with a complete set of
phenotypic tests, with
automated systems or self-contained commercial kits being
satisfactory, and should be
tested with grouping antisera. Confirmation requires both
phenotypic and serologic
identification, and laboratories that do not perform both types of
tests should
send Shigella isolates to a reference laboratory for
confirmation.
Identification
Phenotypic Identification
Because the somatic antigens of most serotypes of Shigella are
either identical or related to
those of E. coli,suspicious cultures that are serologically
negative should be tested further
phenotypically (66). Shigella and inactive E. coli (anaerogenic
or lactose-nonfermenting)
strains are frequently difficult to distinguish by routine
phenotypic tests. See Table 4 for the
phenotypic reactions characteristic of Shigella spp.
Although S. dysenteriae and S. sonnei are
phenotypically distinct, S. flexneri and S. boydii are
often phenotypically indistinguishable, so
serologic grouping is essential.
Serotyping
Serotyping is essential for the identification of Shigella.
Three of the four subgroups, A (S.
dysenteriae), B (S. flexneri), and C (S. boydii), are made up of
a number of serotypes.
Subgroup A has 15 serotypes; subgroup B has 8 serotypes (with
serotypes 1 to 5 subdivided
into 11 subserotypes); and subgroup C has 19 serotypes, numbered 1
through 20, with S.
boydii 13 reclassified as E. albertii. Subgroup D (S. sonnei) is
made up of a single serotype.
Subgroups A and C are rare. Several provisional Shigella serotypes
have also been
described, which are held sub judice until findings from the
characterization of representative
isolates show them to be unique and of sufficient prevalence to
merit inclusion in
the Shigella scheme. Antisera for the identification of
provisional serotypes are typically
available only at reference laboratories.
Serotyping is typically performed by slide agglutination with
polyvalent somatic (O) antigen
grouping sera, followed, in some cases, by testing with monovalent
antisera for specific
serotype identification. Monovalent antiserum to S. dysenteriae
1 is required to identify this
serotype and is not widely available. Because of the potentially
serious nature of illness
associated with this serotype, isolates that agglutinate in
subgroup A reagent should be sent
to a reference laboratory immediately for further serotyping.
Phenotypically typical Shigella isolates that agglutinate poorly
or that do not agglutinate at
all should be suspended in saline and heated in a water bath at
100°C for 15 to 30 min. After
cooling, the antigen suspension should be tested in normal saline
to determine if it is rough
(agglutinates spontaneously). If the heated and cooled suspension
is not rough, it may then
be retested for agglutination in antisera.
Typing Systems
A variety of methods have been used to subtype Shigella, including
colicin typing
(particularly for S. sonnei), plasmid profiling, restriction
fragment length polymorphism
analysis, PFGE, and ribotyping (189). For an overview of the
epidemiologic use of typing
methods, see chapter
8.
Serodiagnostic Tests
Several serodiagnostic assays based on different antigens
possessed by Shigella have been
described (121,202). These assays are practical only in research
settings for
seroepidemiology surveys and are not currently used for the
diagnosis of infection in
individual patients.
Antimicrobial Susceptibilities
Shigella infections are often treated with antimicrobial agents. Because of
the widespread
antimicrobial resistance among Shigella strains, all
isolates should undergo susceptibility
testing (http://www.cdc.gov/narms/). Because of widespread
resistance in the United States,
ampicillin and trimethoprim-sulfamethoxazole, two safe drugs that
were the most commonly
prescribed for treatment of children with S. sonnei infections,
are no longer options for
empiric treatment. Macrolides, in particular azithromycin, are
being used to treat these
infections, but there are no interpretive criteria for AST for Shigella,making
it problematic to
monitor for development of resistance (9).
Fecal isolates of Shigella should be tested against
ampicillin, a fluoroquinolone, and
trimethoprim-sulfamethoxazole. Strains may produce susceptible AST
results for fluoroquinolones,
but if they are resistant to nalidixic acid, treatment with a
fluoroquinolone may result
in a delayed clinical response or treatment failure. Shigella should
not be reported as
susceptible to narrow-spectrum and expanded-spectrum
cephalosporins and cephamycins or
to aminoglycosides and N1-substituted aminoglycosides because
these drugs are not
effective clinically.
Reporting of susceptibility results to the clinician is
particularly important for S. dysenteriae 1
isolates. Infections caused by these strains are often acquired
during international travel to
areas where most strains are multidrug resistant (197).
In many areas of Africa and Asia, S.
dysenteriae 1 strains are resistant to all locally available antimicrobial
agents, including
nalidixic acid, but are still susceptible to the fluoroquinolones
(35, 171); however,
fluoroquinolone-resistant strains have been reported in Asia (114,
194, 195).
Evaluation, Interpretation, and Reporting of
Results
A preliminary report of suspected Shigella infection may be
issued if phenotypic or serologic
screening tests are positive. If serotyping results are available,
these should also be
reported, particularly if the isolate is S. dysenteriae 1.
All Shigella isolates should be tested
for antimicrobial susceptibility. Before issuing a final report,
isolates should be confirmed by
both serologic and phenotypic methods. Isolates, particularly
those from individuals with
dysentery-like illness, that are phenotypically identified as Shigella
but that are serologically
negative may be new serotypes of Shigella and should be
sent to a reference laboratory for
further characterization. Isolates from sites other than the
gastrointestinal tract which
resemble Shigella should be scrutinized carefully for gas
production and other differentiating
characteristics because extra intestinalShigella infections
are rare. These isolates should be
sent to a reference laboratory for confirmation because they are
more likely to be
anaerogenic E. coli, certain strains of which may
cross-react with Shigella antiserum.
SALMONELLA Back to top
Taxonomy
Members of the genus Salmonella are classified in the
family Enterobacteriaceae (32, 66).
Species of this genus are motile, gram-negative, facultative rods.
Salmonellae are typically
defined by their ability to use citrate as a sole carbon source
and lysine as a nitrogen source
and by the production of H2S on triple sugar agar; exceptions to
these traits are used to
define specific serotypes (66, 158).
The genus Salmonella is composed of two species, Salmonella
enterica and Salmonella
bongori (169). S. entericais subdivided into six
subspecies: S. enterica subsp. enterica, often
called subspecies I; S. enterica subsp.salamae, or
subspecies II; S.
enterica subsp. arizonae, or subspecies IIIa; S. enterica subsp.
diarizonae, or subspecies
IIIb; S. enterica subsp. houtenae, or subspecies IV;
and S. enterica subsp. indica, or
subspecies VI. The type species is S. enterica subsp. enterica.
Subspecies IIIa and IIIb
represent organisms originally described in the genus “Arizona”;
subspecies IIIa contains the
monophasic strains, and subspecies IIIb contains the diphasic
strains of “Arizona” (170).
Despite their common history, subspecies IIIa and IIIb are more
closely related to some of
the other subspecies of S. enterica than they are to each
other and thus should be
considered separate entities (211).
Genome analysis of salmonellae has revealed a high degree of
genetic variability. Serotypes
within S. entericasubspecies I have been shown to differ by
as much as 10% in their gene
content (i.e., presence or absence of whole genes) (64,
159). Recombination, particularly
among strains of S. enterica subspecies I, likely
contributes to this diversity (116). Wholegenome
sequence analysis of many common serotypes, including serotypes
with unique
virulence properties, such as S. enterica serotypes Typhi,
Paratyphi A, and Choleraesuis,
have been reported and continue to expand our understanding of the
pathogenesis and
evolutionary history of Salmonella
(14, 48, 97, 122, 131).
Epidemiology and Transmission
Salmonella organisms are isolated most frequently from the intestines of
humans and
animals. Some serotypes are isolated only from humans (e.g., Salmonella
serotype Typhi),
while others (e.g., Salmonellaserotype Gallinarum and Salmonella
serotype IV 48:g,z51:-
[formerly serotype Marina]) are strongly associated with certain
animal hosts. Members of
this genus can be isolated from feces-contaminated foods or water
but probably do not occur
as free-living organisms in the environment. Historically, Salmonella
has been considered a
pathogen of meat and poultry products but has recently been associated
with other food
vehicles, such as fresh produce and manufactured products (90).
Salmonella Serotypes
Salmonella serotyping is a subtyping method based on the immunologic
characterization of
three surface structures: the O antigen, which is the outermost
portion of the LPS layer that
covers the bacterial cell; the H antigen, which is the filament
portion of the bacterial flagella;
and the Vi antigen, which is a capsular polysaccharide present in
specific serotypes.
Serotyping of Salmonella is commonly performed to
facilitate public health surveillance
of Salmonella infections and to aid in the recognition of
outbreaks. The serotype of an isolate
often correlates with a particular disease syndrome or food
vehicle, making serotype data
particularly useful in identifying cases and defining outbreaks.
For
example, Salmonella serotype Typhi causes typhoid fever, a
more severe disease syndrome
than those caZused by most other Salmonella serotypes.Salmonella
serotype Enteritidis is
often associated with infections acquired from chicken or egg
products (154).
Furthermore, Salmonella serotyping is performed worldwide
and has aided in the recognition
of international outbreaks (126). Salmonella serotypes
Enteritidis and Typhimurium are the
two most common serotypes in the United States, making up
approximately 35 to 40% of all
culture-confirmed infections
(http://www.cdc.gov/nationalsurveillance/shigella_surveillance.html).
Clinical Significance
Strains of Salmonella are categorized as typhoidal and
nontyphoidal, corresponding to the
disease syndrome with which they are associated. Strains of
nontyphoidal Salmonella usually
cause intestinal infections (accompanied by diarrhea, fever, and
abdominal cramps) that
often last 1 week or longer (96). Less commonly,
nontyphoidal Salmonella can cause
extraintestinal infections (e.g., bacteremia, urinary tract
infection, or osteomyelitis),
especially in immunocompromised persons. Persons of all ages are
affected, but the
incidence is highest in infants and young children. Salmonella is
ubiquitous in animal
populations, and human illness is usually linked to foods.
Salmonellosis is also transmitted by
direct contact with animals, by water, and occasionally by human
contact. Each year, an
estimated 1.4 million cases of illness and 600 deaths are caused
by nontyphoidal
salmonellosis in the United States (133).
Typhoid fever, caused by Salmonella serotype Typhi, is a
serious bloodstream infection
common in the developing world. However, it is rare in the United
States, where an
estimated 800 cases, with fewer than 5 deaths, occur each
year;>70% of U.S. cases are
related to foreign travel (133). Typhoid fever typically
presents with a sustained debilitating
high fever and headache. Adults characteristically present without
diarrhea. Illness is milder
in young children, where it may manifest as nonspecific fever.
Humans are the only reservoir
for Salmonella serotype Typhi, indicating that this
serotype is adapted to the human host.
Healthy carriers have been noted. Typhoid fever typically has a
low infectious dose
(<103 organisms) and a long, highly variable incubation period
(1 to 6 weeks). It is
transmitted through person-to-person contact or feces-contaminated
food and water. Fatal
complications of typhoid most commonly occur in the second or
third week of illness.
A syndrome similar to typhoid fever is caused by “paratyphoidal”
strains
of Salmonella, i.e., Salmonellaserotypes Paratyphi
A, Paratyphi B, and Paratyphi
C. Salmonella serotypes Paratyphi A and Paratyphi C are
rare in the United States
(http://www.cdc.gov/nationalsurveillance/shigella_surveillance.html). Salmonellaserotype
Paratyphi B is a diverse serotype that is associated with both
paratyphoid fever and
gastroenteritis (161). The two pathovars are typically differentiated
on the basis of the ability
to ferment tartrate; isolates causing paratyphoid fever, the
systemic pathovar, are tartrate
negative. Isolates associated with gastroenteritis, the enteric
pathovar, are typically tartrate
positive and are referred to as Salmonella serotype
Paratyphi B variant L(+)-tartrate +
or Salmonella serotype Paratyphi B variant Java. The
systemic pathovar
ofSalmonella serotype Paratyphi B is considered rare in the
United States; however, the
tartrate reaction is often not reported, making it impossible to
distinguish between the two
pathovars
(http://www.cdc.gov/nationalsurveillance/salmonella_surveillance.html).
Salmonella serotypes Choleraesuis and Dublin are host adapted to pigs and
cattle,
respectively, causing serious disease in these two animal species.
They rarely cause human
infection, but such infections are typically severe, with spread
to extraintestinal sites
(128, 206). Salmonella serotype Dublin has been
shown to share virulence traits
with Salmonella serotype Typhi, which may contribute to its
invasiveness in humans
(136, 156).
Collection, Transport, and Storage of Specimens
See “Collection, Transport, and Storage of Specimens” in the Escherichia
section.
Direct Examination
Microscopy
Salmonella cannot be distinguished from other gram- negative rods by
microscopy or
staining methods. There are no FDA-approved methods for direct
examination of clinical
specimens for Salmonella.
Antigen Detection
A number of commercial rapid diagnostic tests are available for
the testing of foods, but to
our knowledge, none has been evaluated in the literature for use
with fecal specimens, and
none are FDA approved for clinical specimens.
Nucleic Acid Detection
There are no FDA-approved methods for nucleic acid detection of Salmonella
in clinical
specimens.
Isolation Procedures
Enrichment
Maximal recovery of Salmonella from fecal specimens is
obtained by using an enrichment
broth, although isolation from acutely ill persons is usually possible
by direct plating of
specimens. Enrichment broths forSalmonella are usually
highly selective and inhibit certain
serotypes of Salmonella, particularly Salmonellaserotype
Typhi. The selective enrichment
medium most widely used to isolate Salmonella from fecal
specimens is SEL. SEL may also
be used for the recovery of Salmonella serotype Typhi and
for Shigella, although its value as
enrichment for the latter has not been clearly established.
Specimens which might contain
organisms inhibited by selective enrichment medium should be
plated directly or cultured in
a nonselective enrichment broth (e.g., gram-negative broth).
Plating Media
Many differential plating media, varying from slightly selective
to highly selective, are
available for isolation ofSalmonella from fecal specimens.
Media of low selectivity include
MAC and eosin-methylene blue. Media of intermediate selectivity
include XLD, deoxycholate
citrate agar, salmonella-shigella agar, and HE. Highly selective
media include bismuth sulfite
agar, the preferred medium for the isolation of Salmonella serotype
Typhi, and brilliant green
agar. Bismuth sulfite agar, XLD, and HE all have H2S indicator
systems, which are helpful for
the detection of lactose-fermenting Salmonella strains.
Many laboratories use HE or XLD
because these media may also be used for the isolation of Shigella.
In the developing world, typhoid fever is frequently diagnosed
solely on clinical grounds, but
isolation of the causative organism is necessary for a definitive
diagnosis. Salmonella serotype Typhi is isolated more
frequently from blood cultures than
from fecal specimens. Blood cultures are positive for 80% of
typhoid patients during the first
week of fever but show decreasing positive results thereafter.
Screening Procedures
A latex agglutination kit has been described for Salmonella screening
in SEL enrichment
broth (Wellcolex ColorSalmonella; Remel Inc., Lenexa, KS) (28).
This kit can also be used to
screen individual colonies from primary plates. In using this kit,
it should be kept in mind
that it identifies only those Salmonella isolates belonging
to the more common O serogroups
and does not differentiate between O groups C1 (O:7) and C2 (O:8).
Suspect colonies may be inoculated onto a screening medium such as
KIA or TSI. On KIA or
TSI, mostSalmonella strains produce an alkaline slant,
indicating that only glucose is
fermented, with gas and H2S. On these media, Salmonella serotype
Typhi isolates
characteristically produce an alkaline slant but do not produce gas,
and only a small amount
of H2S will be visible at the site of the stab and in the stab
line. Lysine iron agar is also a
useful screening medium because most Salmonella isolates,
even those which ferment
lactose, decarboxylate lysine and produce H2S. Alternately,
isolates may be identified by a
battery of phenotypic tests or by slide agglutination with
antisera for Salmonella O groups.
Isolates suspected of being Salmonellaserotype Typhi should
be tested serologically
with Salmonella Vi and O group D antisera (see below).
If the phenotypic traits for a particular isolate are not
characteristic
of Salmonella but Salmonella antigens are found, the
cultures should be plated to obtain a
pure culture, tested with a complete set of phenotypic tests, or
forwarded to a reference
laboratory.
Identification
Clinical laboratories may issue a preliminary report of Salmonella
when an isolate is positive
either withSalmonella O group antisera or by phenotypic
identification methods. An isolate is
confirmed as Salmonellawhen the specific O serogroup has
been determined and phenotypic
identification has been completed.
Phenotypic Identification
Suspect colonies from one of the differential plating media
mentioned above can be identified
phenotypically as Salmonella spp. by use of traditional
media in tubes or commercial
biochemical systems. Methods for phenotypic identification and
specific commercial manual
and automated identification systems are covered inchapter 3. The species and subspecies
of Salmonella can be identified phenotypically, as
indicated in Table 5.
However, Salmonella is a diverse group, and phenotypically
atypical strains are not
uncommon. Phenotypic identification is commonly combined with
serogrouping or serotyping
for culture confirmation.
Serogrouping and Serotyping
O serogroup determination is adequate for confirmation of isolates
as Salmonella. Full
serotype determination is useful for public health surveillance
but is beyond the scope of
most routine clinical laboratories. The methods for serotyping
described below are intended
primarily for reference laboratories. Salmonella isolates
are serotyped on the basis of the
antigenic properties of their O (somatic) antigens, H (flagellar)
antigens, and Vi (capsular)
antigens (34, 85). O antigen is a carbohydrate antigen and is the
outermost component of
LPS. It is a polymer of O subunits; each O subunit is typically
composed of four to six sugars,
depending on the O antigen. O antigens are designated by numbers
and are divided into O
serogroups based on antigenic factors associated with the O
subunit. Many of the common O
groups were originally designated by letter and are still commonly
referred to by letter
(e.g., Salmonella serotype Typhimurium belongs to group O:4
or group B,
andSalmonella serotype Enteritidis belongs to group O:9 or
group D1). Additional O antigenic
factors have been identified for specific O groups. They are
typically associated with a side
sugar that is added to the basic O subunit structure, and they are
often variably present or
variably expressed within O groups or within serotypes.
H antigen is a protein antigen called flagellin; multiple
flagellin subunits make up the flagellar
filament. The ends of flagellin are conserved and give the
flagellum its characteristic filament
structure. The antigenically variable portion of flagellin is the
middle region, which is surface
exposed. Salmonellae are unique among the enteric bacteria in that
they commonly express
two different flagellin antigens, although specific serotypes,
such as Salmonella serotypes
Typhi and Enteritidis, possess only one flagellar antigen. The two
flagellar antigens are
referred to as phase 1 and phase 2 antigens; monophasic and
diphasic strains express one
and two flagellar antigens, respectively. Individual flagellar
antigens can be composed of
multiple antigenic factors. For example, the phase 2 flagellar
antigen of Salmonella serotype
Typhimurium is antigen 1,2, which is composed of two antigenic
factors, i.e., 1 and 2.
Serotypes are designated according to the conventions of the
Kauffmann-White scheme
(85). Many of the O and H antigenic types are found
in multiple subspecies, and isolates
from different subspecies can have the same antigenic profile.
Thus, subspecies
determination is an integral component of serotype determination
forSalmonella. The
serotypes for all Salmonella strains can be designated by
antigenic formulae; additionally,
serotypes belonging to subspecies I are given a name, which is
typically related to the
geographical place where the serotype was first isolated. The
antigenic formulae
of Salmonella serotypes are listed in the Kauffmann-White
scheme and are expressed as
follows: O antigen(s),Vi antigen (when present):phase 1 H
antigen(s):phase 2 H antigen(s)
(when present). For example, the antigenic formula for Salmonella
serotype Typhimurium is
4,5,12:i:1,2. Serotype names for subspecies I serotypes are
written in roman (not italicized)
letters, and the first letter is a capital letter (for example, Salmonella
serotype
Typhimurium). Serotypes belonging to other subspecies are
designated by their antigenic
formulae following the subspecies name (for example, S.
enterica subsp. salamae serotype
50:z:e,n,x or Salmonella serotype II 50:z:e,n,x).
The WHO Collaborating Centre for Reference and Research on Salmonella,
which is located at
the Pasteur Institute in Paris, France, maintains the
Kauffmann-White scheme for the
designation of Salmonella serotypes (85).
Most common serotypes belong to O groups A, B,
C1, C2, D1, and E1 (also known as groups O:2, O:4, O:7; O:8; O:9,
and O:3,10,
respectively). Serotypes belonging to subspecies II (505
serotypes), IIIa (99 serotypes), IIIb
(336 serotypes), IV (73 serotypes), and VI (13 serotypes) and to S.
bongori (22 serotypes)
are found primarily in O groups O:11 (F) through O:67 (commonly
referred to as the higher
O groups).
Determination of O Antigens
O (heat-stable, somatic) antigens are typically identified by
first testing the isolate in
antisera that detect one or multiple antigenic factors corresponding
to the O groups (O
grouping antisera). Once the O group is determined, antisera that
recognize single antigenic
factors are used to confirm the O group and to identify any
additional antigenic factors that
are associated with that O group (O single-factor antisera) (34).
In the clinical laboratory,
the approach most commonly used for determining O antigens is to
initially test the isolates
by slide agglutination in antisera against O groups A to E1
because approximately 95%
of Salmonella isolates from human specimens belong to one
of these O groups. If no
agglutination occurs in antisera for these O groups, the isolate
is tested in pools containing
the remaining Salmonella O antisera, for groups O:11
through O:67.
Determination of H Antigens
H (flagellar) antigens are typically determined by tube or slide
agglutination tests. Isolates
are initially tested with H typing antisera, which recognize
individual or multiple antigenic
factors, and then with H single-factor antisera, which recognize
individual antigenic factors.
Typically, the flagellar antigens in a diphasic strain are
coordinately regulated so that only
one is expressed at a time in a single bacterial cell; however,
both phases may be detected
in the whole culture, particularly with a fresh clinical isolate.
When only one phase is
detected (either phase 1 or phase 2), the strain should be
inoculated into a semisolid
medium to which sterile antiserum to the detected flagellar
antigen has been added
aseptically. Growth of the strain in this semisolid agar
immobilizes cells expressing the
detected antigen and allows the movement of bacteria expressing
the antigen in the other
phase through the semisolid medium. Cells are recovered away from
the area of initial
inoculation, and the strain is tested in appropriate H typing and
single-factor antisera to
complete the serotyping. A strain must be actively motile to
ensure the good expression of H
antigens; sometimes a strain must be passed through one or more
tall tubes of semisolid
agar to enhance motility before H antigens can be detected.
Detection of the Vi Antigen and Identification of Salmonella
Serotype
Typhi (9,12,[Vi]:d:–)
The Vi antigen, a heat-labile capsular polysaccharide, is useful
for the identification
of Salmonella serotype Typhi. It is also occasionally
detected in Salmonella serotype
Dublin, Salmonella serotype Paratyphi C, and some Citrobacter
strains, so its detection does
not constitute definitive evidence of Salmonella serotype
Typhi. The Vi antigen is identified
by slide agglutination with a specific antiserum.
If Salmonella serotype Typhi is suspected, the culture is
first tested live (unheated) in O
group D antiserum (which contains antibodies to O antigens 9 and
12) and Vi antiserum on a
slide. The Vi capsular polysaccharide can mask the O antigens,
blocking their reactivity with
the O grouping antiserum. If only the Vi antiserum is positive,
the bacterial suspension is
heated in boiling water for 15 min to remove the capsule, cooled,
and tested again in the
same antisera. After being heated, Salmonella serotype
Typhi isolates will be negative in the
Vi antiserum but positive in the O group D antiserum. Expression
of the Vi antigen
by Salmonella serotype Typhi is variable but tends to occur
more frequently in freshly
isolated cultures than in cultures that have been subcultured. If
the strain is typical
for Salmonella serotype Typhi on TSI or KIA (see “Screening
Procedures” above), is urease
negative, and reacts in O group D or Vi antiserum, a presumptive report
is made. The
identity of the isolate is typically confirmed by phenotypic
testing (Table 5) and
determination of the H (flagellar) antigen. However, because Salmonella
serotype Typhi has
a unique phenotypic profile, it can and should be reported based
on phenotype alone (i.e.,
identification of the O and H antigens is not required in order to
identify an isolate
as Salmonella serotype Typhi).
Identification Problems Back to top
Several potential problems may prevent accurate serotype
determination. The strain may
express the Vi capsular antigen, which can block the binding of
antibodies against the O
antigens. The strain may be rough, i.e., fail to make complete O
antigens. Rough strains
have a tendency to weakly agglutinate in multiple O grouping antisera.
The strain may be
mucoid and not agglutinate in any O antisera, or isolates can be
nonmotile and not express
any flagellar antigens. Among isolates submitted to the National SalmonellaReference
Laboratory at the CDC, isolates from urine are frequently rough,
mucoid, and/or nonmotile.
When O antigen and/or H antigen is not detected, a strain is
confirmed as
a Salmonella species by characterization of any antigens
that are expressed and by
phenotypic testing (Table 5).
Laboratories may overlook Salmonella serotype Paratyphi A
because they do not screen with
O group A antiserum or because its atypical phenotypic profile
(H2S negative, lysine
negative, and citrate negative) can be confused with E. coli.
Salmonella serotypes Paratyphi
B and Paratyphi B variant L(+)-tartrate + (also known as variant
Java) can be confused
because they have the same antigenic formula (4,5,12:b:1,2), but
they are distinguished
phenotypically by their tartrate reactions. Similarly, Salmonella
serotype Choleraesuis
andSalmonella serotype Paratyphi C have the same antigenic
formula (6,7:c:1,5) but are
differentiated phenotypically. Salmonella serotype
Paratyphi C may express the Vi
antigen. Citrobacter and E. coli strains may possess
O, H, or Vi antigens that are related to
those of Salmonella; biochemical identification may be
necessary to confirm that an isolate
is Salmonella (see Table 5 in this chapter and Table 1 in chapter 31).
Typing Systems Back to top
For rarer serotypes, serotype identification may be all that is
necessary to identify clusters of
temporally related isolates. However, additional subtyping methods
are typically required for
more common serotypes (e.g., Salmonella serotypes
Typhimurium, Enteritidis, and
Newport). A variety of phenotypic and genotyping methods have been
developed for
subtyping within serotypes of Salmonella (141,
192). PFGE is the current method of choice
for the subtyping of most Salmonella serotypes, since it is
universally applicable and
provides good strain discrimination for most serotypes. PulseNet,
an international subtyping
network that tracksSalmonella, is based on PFGE (192).
Salmonella serotype Enteritidis has
limited diversity in PFGE analysis; as a result, phage typing is
still necessary to characterize
strains, particularly in an outbreak setting (74,
154).
Serodiagnostic Tests Back to top
The Widal test, which measures agglutinating antibodies to the O
and H antigens
of Salmonella serotype Typhi, produces false-negative and
false-positive reactions and does
not provide a definitive diagnosis of individual cases of
infection. Two other rapid
serodiagnostic tests have proved more useful than the Widal test
for the serodiagnosis of
typhoid fever (148) (Tubex [IDL Biotech, Sollentuna, Sweden] and
TyphiDot [Malaysian Bio-
Diagnostics Research Sdn. Bhd., Kuala Lumpur, Malaysia]). These
tests are most useful in
areas where typhoid fever is endemic and are less useful in the
United States, where typhoid
fever is rare. Neither of these tests is FDA approved, and
TyphiDot is not available in the
United States.
Antimicrobial Susceptibilities Back to top
Antimicrobial therapy is not recommended for uncomplicated Salmonella
gastroenteritis, and
routine susceptibility testing of fecal isolates is not warranted
for treatment purposes.
However, determination of antimicrobial resistance patterns is
often valuable for surveillance
purposes and may be performed periodically to monitor the
development and spread of
antimicrobial resistance among Salmonella isolates.
Salmonella strains may produce susceptible AST results for fluoroquinolones,
but if they are
resistant to nalidixic acid, treatment with a fluoroquinolone may
result in a delayed clinical
response or treatment failure. For this reason, nalidixic acid,
chloramphenicol, and a broadspectrum
cephalosporin should be tested and reported for extraintestinal
isolates
of Salmonella. Salmonella should not be reported as
susceptible to narrow-spectrum and
expanded-spectrum cephalosporins and cephamycins or to
aminoglycosides and N1-
substituted aminoglycosides because these drugs are not effective
clinically.
In contrast to the case for uncomplicated salmonellosis, treatment
with the appropriate
antimicrobial agent can be crucial for patients with invasive Salmonella
and typhoidal
infections, and the susceptibilities of these isolates should be
reported as soon as possible
(117). Testing methods are detailed in chapter 68 in thisManual. The untreated case
mortality rate for typhoid fever is >10%; when patients with
typhoid fever are treated with
appropriate antibiotics, the rate should be <1%. However,
increasing levels of resistance to
one or more antimicrobial agents in Salmonella isolates,
particularly Salmonella serotype
Typhi isolates, make selection of an appropriate antibiotic
problematic. In particular, reduced
susceptibilities to ciprofloxacin amongSalmonella serotype
Typhi isolates and increasing
numbers of treatment failures are of concern (107,
164).
Antimicrobial resistance, particularly multiple-drug resistance,
has been noted in several
nontyphoidal serotypes of Salmonella. A strain of Salmonella
serotype Typhimurium phage
type DT104 which was resistant to five antimicrobials (ampicillin,
chloramphenicol,
streptomycin, sulfonamides, and tetracycline [ACSSuT]) emerged in
the late 1990s and is
now recognized worldwide. In 2002, 21% of Salmonella serotype
Typhimurium isolates in the
United States had the ACSSuT resistance profile (42).
The ACSSuT resistance determinant
has been found in Salmonella serotype Agona strains (50).
The genomic element that carries
this ACSSuT determinant has been found to harbor this and other
resistance determinants in
a variety of serotypes, indicating that the element may spread
horizontally to other
serotypes and acquire additional resistance determinants (120).
The emergence of a clone of Salmonella serotype Newport
which is resistant to at least nine
antimicrobials, including expanded-spectrum cephalosporins, was
first noted in 2000 in the
northeastern United States (89) and has now been found
in many regions of the United
States (17). In 2002, this strain made up 22% of allSalmonella
serotype Newport strains in
the United States. Similarly resistant strains of Salmonella serotype
Newport were recently
reported in Japan, documenting the potential for worldwide spread
of multiply resistant
strains (103). Additional information regarding these and
other antimicrobial-resistant strains
can be found at the CDC’s NARMS website
(http://www.cdc.gov/narms/).
Evaluation, Interpretation, and Reporting of
Results Back to
top
A preliminary report can be issued as soon as a presumptive
identification of Salmonella is
obtained. In most situations, a presumptive identification is
based on phenotypic traits
determined by either traditional or commercial systems or by
reactivity with Salmonella O
grouping antisera. A confirmed identification requires both
phenotypic identification and O
group or serotype determination. Because national surveillance
systems depend on the
receipt of serotype information for Salmonella strains
isolated in the United States,
laboratories should follow the procedures recommended by their
state health departments
for submitting Salmonellaisolates for further
characterization, including complete serotyping.
The antimicrobial susceptibilities of typhoidal Salmonella strains
and strains from normally
sterile sites should be determined, and the strains should be
forwarded to a reference or
public health laboratory
for complete phenotypic identification and serotyping.
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