SECTION 8 Clinical Microbiology: Bacteria

180 
Enterobacteriaceae
CLAIRE JENKINS  |  ROB J. RENTENAAR  |  LUCE LANDRAUD  | 
SYLVAIN BRISSE

KEY CONCEPTS The family Enterobacteriaceae currently includes (as of June 2014)
more than 210 species and 53 genera, and these numbers continue to
• Impact of multilocus sequence typing and whole genome increase. A history of taxonomic changes and synonyms of species is
sequencing on informing the taxonomy of the Enterobacteria- maintained at http://www.bacterio.cict.fr. Some recent taxonomic
ceae family. changes for taxa of medical relevance include the creation of the
• Molecular approach to detection and typing of pathogenic species Escherichia albertii for eae-positive diarrhea-causing strains
Enterobacteriaceae species. that are closely related to Shigella boydii serotype 13,6 the transfer of
Calymmatobacterium granulomatis, the agent of granuloma inguinale
• Incidence and prevalence of disease.
(donovanosis), to the genus Klebsiella,7 the definition of novel Klebsi-
• Modes of transmission. ella species closely related to K. pneumoniae8 and an extensive revision
of the genus Enterobacter.9 The review by Janda3 provides detailed
• Clinical features and symptoms.
information on some of the recent changes to Enterobacteriaceae taxa,
• Pathogenicity mechanisms. including a review of the evidence for their medical significance.
A precise phylogeny of Enterobacteriaceae genera and species
• Laboratory procedures.
would be useful for taxonomic purposes and would allow understand-
• Emerging resistance to the extended-spectrum cephalosporins ing of the evolution of characteristics, such as biochemical capabilities,
and carbapenems.

The phylogenetic relationships determined based on 16S rRNA

Introduction gene sequences for some members of class Gammaproteobacteria

The family Enterobacteriaceae are ubiquitous and members are found

worldwide in various ecological sources such as soil, water, vegetation
and animals.1–3 Some species are important pathogens of plants and
have economic importance in crop production.4 Certain species are
part of the normal flora of animals including humans, although many
are frequently associated with diarrheal disease and extraintestinal
infections.2,5 Some of the most important pathogens in human history, Enterobacteriaceae
such as the agent of plague Yersinia pestis, belong to the Enterobacte-
riaceae family and other members currently represent a huge public > 210 species
health concern (e.g. Salmonella enterica serotype Typhi, Shigella, Esch- > 50 genera
erichia coli).
Two main types of infectious disease are associated with Entero-
bacteriaceae: intestinal and extraintestinal diseases. The transmission
route of intestinal infection is classically fecal–oral either by person-
to-person, direct contact with animals or their environment, or by
consumption of contaminated food or water. An endogenous pathway
of infection is also possible (e.g. bacterial translocation from the gut Pasteurella
to blood), resulting in extraintestinal disease, and is more often Haemophilus
observed in immunocompromised hosts or persons with underlying
conditions such as cirrhosis or those undergoing chemotherapy. Vibrio
Members of the Enterobacteriaceae should not be confused with
the term ‘enteric bacteria’, which refers specifically to species of the Aeromonas
gut and includes all species found in that habitat. This chapter focuses
Shewanella
on aspects of taxonomy, pathogenesis, clinical diagnosis and manage-
ment of human Enterobacteriaceae infections. Alteromonas

Taxonomy, Phylogeny and Pseudomonas

Clonal Relationships Legionella
In the prokaryotic taxonomy, the Enterobacteriaceae represent the Francisella
only family within the order Enterobacteriales, one of 15 orders Xanthomonas
within class Gammaproteobacteria, which belongs to phylum Proteo-
bacteria. Based on 16S rRNA gene sequences, the closest phylogenetic Figure 180-1  The phylogenetic relationships determined based on 16S rRNA
gene sequences for some members of class Gammaproteobacteria. Bacterial
relatives of Enterobacteriaceae are the Pasteurellaceae, the Vibriona- groups most closely related to Enterobacteriaceae are families Pasteurellaceae
ceae, and members of the orders Aeromonadales and Alteromonadales and Vibrionaceae, order Aeromonadales, genus Shewanella and order Altero-
(Figure 180-1). monadales. (Data from the Ribosomal Database Project, http://rdp.cme.msu.edu.)

1565
1566 SECTION 8  Clinical Microbiology: Bacteria

The genus Salmonella

Salmonella 6 Number of Natural

Genus Salmonella
enterica subspecies: described hosts
2 species: serovars

Salmonella enterica (formerly subspecies I) 1531 Warm-blooded

bongori salamae (formerly subspecies II) 505 (mammals, birds)
arizonae (formerly subspecies IIIa) 99
diarizonae (formerly subspecies IIIb) 336 Cold-blooded
(S. bongori as well)
houtenae (formerly subspecies IV) 73
indica (formerly subspecies VI) 13

• Represents 99% of clinical isolates

• Some serovars are highly prevalent (e.g. Typhimurium, Enteriditis, Newport)
• Some serovars cause typhoid and other enteric fevers (Typhi, Paratyphi A, B, C)
• Formerly called Salmonella choleraesuis (this name is now abandoned)
• Serovars were formerly given species status; this practice is now abandoned
• Only serovars of subspecies enterica are given names
• These serovars can be designated in the form either Salmonella ser. Typhimurium
or Salmonella Typhimurium (but neither S. Typhimurium nor S. typhimurium)

Figure 180-2  The genus Salmonella includes two species, S. bongori and S. enterica (formerly S. choleraesuis), with the latter including six subspecies. Nomenclature
has evolved from a system where all serovars (the combination of O antigen and the two phases of the H antigen) were considered as species (e.g. Salmonella typhi)
into the current system, which gives names to serovars of subspecies S. enterica subsp. enterica (e.g. S. enterica subsp. enterica serotype Enteritidis), but designates
the serovars of other subspecies and of S. bongori simply by their antigenic formula. (More details can be found at http://www.bacterio.cict.fr/s/salmonella.html.)

host range, ecology and virulence. Although the 16S rRNA molecule
is a good phylogenetic marker for most bacterial species, it is poorly
informative within this family.10 Analyses of whole genome sequences
of a wide range of bacteria are currently being performed in academic,
clinical and public health settings and data will be used to establish a
robust phylogeny of Enterobacteriaceae species.11,12
All Salmonella strains are currently classified into two species: S.
enterica and S. bongori. Salmonella enterica itself is subdivided into six
subspecies: enterica, arizonae, diarizonae, houtenae, indica and salamae.
Salmonella enterica subsp. enterica is by far the most important from
a medical standpoint, representing 99% of clinical infections. For
details of Salmonella nomenclature, including serovar naming, see
Figure 180-2 and http://www.serotest-thailand.com/upload/news/ aerobic or anaerobic conditions. Growth is generally optimal at 99°F
download/9-8310-0.pdf.
The genus Escherichia currently includes the species E. coli, E. fer-
gusonii, E. albertii, E. hermanii, E. vulneris and E. blattae. Clinically,
E. coli is by far the most important species. Sequence comparisons
indicate that E. fergusonii and E. albertii are closely related to E.
coli, while the three remaining species may be evolutionarily more
distant.
The clonal diversity of E. coli strains was initially described based
on multilocus enzyme electrophoresis (MLEE) and large-scale multi-
locus sequence typing (MLST) studies.13 These studies indicated that
recombination events were a major influence on evolutionary relation-
ships in E. coli.14 Genome-wide sequence data of multiple strains has
confirmed the important contribution of horizontal gene transfer,
demonstrating that the core genome of E. coli comprises ∼2200 genes
but each strain has a large accessory genome that renders the total gene
pool across the species effectively infinite.14
Shigella species were distinguished historically based on clinical
and biochemical characteristics, but whole genome sequencing biochemical activity (enzyme profiles, carbon source utilization,
(WGS) has confirmed that Shigella strains are phylogenetically more
closely related to some E. coli strains than some E. coli strains are
among themselves.15 In addition, the three taxonomic Shigella species
S. flexneri, S. dysenteriae and S. boydii do not correspond to three
phylogenetic clusters within the E. coli species and evolved in parallel
on multiple occasions.16 In contrast, both S. sonnei and S. dysenteriae
serotype 1 form unique and genetically homogeneous clusters. WGS
has confirmed that other pathotypes of E. coli, such as enteropatho-
genic E. coli, enterotoxigenic E. coli, enteroinvasive E. coli, enteroag-
gregative E. coli and enterohemorrahgic E. coli, also show multiple
independent origins and parallel evolution (see Figure 180-3).12,15
Diagnostic Microbiology
All Enterobacteriaceae are gram-negative, nonspore-forming bacilli.
They are either motile by peritrichous flagella (except Tatumella
ptyseos) or nonmotile. With a few exceptions (e.g. Klebsiella granulo-
matis), they grow rapidly on ordinary laboratory media, under either
(37°C). Some specific properties are common: all species utilize glucose
fermentatively (often with gas production), are oxidase-negative (with
the exception of Plesiomonas shigelloides) and catalase-positive, and
reduce nitrates to nitrites (except Photorhabdus and Xenorhabdus).
Some useful features for identification of Enterobacteriaceae species
that are common in clinical samples are given in Table 180-1; more
details on identification methods can be found in other reference
texts.1,17
In classic nonselective media, colonies are circular and convex, with
a smooth surface and a diameter of 1–3 mm after 24 hours. Selective
media are used to recover Enterobacteriaceae from fecal samples or
other specimens containing complex flora. These media enhance the
growth of specific Enterobacteriaceae species, while inhibiting non-
desired species. Classically, these media (e.g. MacConkey lactose- or
sorbitol-containing media) contain substrates selectively used by one
or a few species and facilitate distinction between colonies. More
precise distinction of genera and species is obtained by differences in
pH-based reactions). Several miniaturized tests are available commer-
cially (e.g. Api 20E or Biotype-100 strips from BioMerieux). Typically,
isolates are identified by comparison of their biochemical profile with
a reference database that contains the percentages of positive results
for each substrate/species pair. More automated identification systems
based on the same approach are classically used in larger microbiology
 Chapter 180  Enterobacteriaceae 1567

The polyphyletic origin of Shigella and Escherichia coli pathotypes, which are each found on
several branches that are nested inside the diversity of E. coli

EAEC ON2011
STEC LB226692
EPEC 01–09591
AIEC 55989
ON2010
Shigella
IAI1
ETEC B1
SE11
KO11
W
O103 12009
E24377A
O26 11368
O111 11128
Sb CDC 3083–94
Sb 227 S1
Ss 046
SS
Sf 2002017
Sf 2a 2457T
Sf 2a 301 S3
Sf 5 8401
K– 12 MG1655
K– 12 W3110
DH1
K– 12 DH10B
BW2952
UMNF18
ETEC H10407
UMNK88 A
BL21 DE3
B REL606
BL21–Gold DE3
HS
ATCC 8739
O157 TW14359
O157 EC4115
O157 Sakai E
O157 EDL933
O55 CB9615
Sd 197
SD1
042
UMN026
IAI39
D
SMS–3–5
UTI89
UM146
IHE3034
APEC O1
S88
536
LF82
O83 NRG 857C B2
CFT073
ABU 83972
ED1a
NA114
SE15
O127 E2348/69

Figure 180-3  The polyphyletic origin of Shigella and Escherichia coli pathotypes, which are each found on several branches that are nested inside the diversity of E.
coli. Shigella (black squares), enterotoxigenic E. coli (ETEC; black circles) and shiga toxin-producing E. coli (STEC; thin black circles) pathotypes have several independent
origins from ancestral strains that have incorporated virulence genes by horizontal transfer. EAEC (enteroaggregative E. coli; thin open squares; EPEC-(enteropathogenic
E. coli; bold open squares); AIEC-adherent invasive E. coli; thick open circles) hybrid EAEC and STEC stains denoted as open thin squares and open thin circles (Reprinted
from Croxen M.A., et al. Clin Microbiol Rev 2013; 26:822–80.)

laboratories18,19 although there are limitations to this approach. For increasing levels of antibiotic resistance detected in clinically impor-
example, isolates that are biochemically atypical or correspond to rare tant strains belonging to the Enterobacteriaceae family.
or novel species (thus not incorporated in the reference databases) Matrix-assisted laser desorption/ionization time-of-flight mass
cannot be identified or can even be misidentified. Furthermore, bio- spectrometry (MALDI-TOF-MS) is the first step in the identification
chemically inactive or closely related bacteria may also be misidentified of Enterobacteriaceae in many clinical microbiology laboratories
by certain automated systems. Automated methods can also test (see also Chapter 161). Sample preparation can be fast and simple. A
antimicrobial susceptibility, which is important with respect to the typical sample preparation method used in the identification of
1568 SECTION 8  Clinical Microbiology: Bacteria

TABLE
180-1  Major Properties Used to Identify the Most Common Enterobacteriaceae Implicated in Human Infections
BIOCHEMICAL TESTS
Species TDA VP ONPG Indole Production Citrate (Simmons) Remarks

Escherichia coli (−) (−) (+) (+) (−) Utilization of inositol (−)

Citrobacter spp. (−) (−) (+) Variable (+) Urease variable, H2S (−) except C. freundii

Shigella spp. (−) (−) (−) except S. sonnei (+)* (−) Utilization of xylose (−)

Salmonella enterica (−) (−) (−) except S. arizonae (−) (+) except Typhi and H2S (+)
Paratyphi A

Yersinia spp. (−) (−) Variable Variable (−) H2S (−), urease (+) except Y. pestis

Enterobacter spp. (−) (+) (+) (−) (+) LDC (−) except E. aerogenes and E.
gergoviae, ODC (+)

Klebsiella spp. (−) (+)†,‡ (+)† (−) except K. oxytoca (+)† ODC (−) except K. ornithinolytica

Serratia spp. (−) (+) (+) (−) (+) Gelatin hydrolysis (+) except S. fonticola

Proteus spp. (+) (−) (−) (−) except P. vulgaris Variable H2S (+), urease (+)

Morganella morganii (+) (−) (–) (+) (–) H2S (−), urease (+)

Providencia spp. (+) (−) (−) (+) (+) H2S (−)

*30–70% positive reaction for S. dysenteriae or S. flexneri, and negative for S. sonnei.
†
Except K. pneumoniae subsp. rhinoscleromatis.
‡
Except K. pneumoniae subsp. ozaenae.
(−), 70–100% negative reaction; (+), 70–100% positive reaction; variable, different reactions between different species or <70% positive/negative reaction for strains of
the species. H2S, hydrogen sulfide production; LDC, lysine decarboxylase; ODC, ornithine decarboxylase; ONPG, O-nitrophenyl-β-D-galactopyranoside; TDA,
phenylalanine deaminase; VP, Voges–Proskauer.

Enterobacteriaceae is the ‘direct transfer method’: a thin film of
biomass from a colony is smeared on a target plate and overlaid with
a small volume of matrix solution. Fresh colonies from solid media are
used, colonies from selective media are generally acceptable. Sample
preparation from blood culture bottles is more complicated. However,
in comparison with biochemical identification of Enterobacteriaceae,
MALDI-TOF-MS is much faster. For many extraintestinal infections
with Enterobacteriaceae, MALDI-TOF-MS may be sufficiently accu-
rate to be used for final identification. Thereby, MALDI-TOF-MS-
based identification of Enterobacteriaceae may lead to early instalment
of appropriate antibiotic treatment.20 In contrast, routine MALDI-
TOF-MS analysis is insufficiently accurate for most species or serotype
identifications of pathogenic Enterobacteriaceae from human intesti-
nal infections. If MALDI-TOF-MS is employed in such infections,
identification results typically require additional biochemical, nucleic
acid amplification tests and/or serotyping. One exception may be the
MALDI-TOF-MS identification of Plesiomonas shigelloides, which
seems reliable in at least one of the commercial systems. In contrast,
Shigella is identified as E. coli by commercial MALDI-TOF-MS systems.
Therefore, a lactose-negative colony isolated from a fecal sample from
a patient with diarrheal illness, identified as E. coli by MALDI-TOF-
MS, requires additional biochemical and serological testing for defini-
tive identification. Differentiation of E. coli pathotypes is currently not
feasible in routine MALDI-TOF-MS analyses with commercial data-
bases. MALDI-TOF-MS identification of Salmonella is highly reliable
at the ‘genus level’, but incapable of serotype differentiation. Similarly,
MALDI-TOF-MS identification of Yersinia genus is accurate, but
species differentiation of pathogenic Yersinia spp. may be problematic
and may at least require additional ‘security relevant’ databases of mass
spectra. MALDI-TOF-MS-based detection and identification of
Enterobacteriaceae directly from urine from patients with urinary tract
infections, detection of β-lactam hydrolyzing enzymes in Enterobac-
teriaceae and/or typing of Enterobacteriaceae in outbreak settings may
reveal relevant and relatively fast and accurate information. However,
these techniques are not widely adopted in clinical microbiology
laboratories.
In some species, serotyping remains the predominant means by
which routine identification is performed beyond the species level.
Serotyping is important because of the high epidemiologic and medical
significance of serotype characterization for strains of Salmonella, Shi-
gella and E. coli. In fact, biochemical profiling is not sufficient for
diagnosis of the serotypes causing typhoid or typhoid-like fevers
(Typhi, Paratyphi A, B and C). Moreover, Shigella and E. coli are
notoriously difficult to distinguish using biochemistry alone and sero-
typing is often essential to differentiate the two groups. Serotyping of
Enterobacteriaceae is based on antigenic variation of the O (somatic,
corresponding to the polysaccharide side chain of the lipopolysaccha-
ride), H (flagellar) and K (capsular) surface antigens. In some species,
most or all strains do not have a capsule (E. coli, Salmonella), whereas
the capsule is the predominant and most discriminatory antigen of
Klebsiella. Serotyping is typically performed by slide agglutination
using sets of O, H and K antisera, and the resulting combination of
antigens defines the serotype (also called serovar). The most familiar
serotyping scheme is the Kauffman–White scheme used for Salmo-
nella, which distinguishes more than 2540 serotypes.21 A re-evaluation
of serotypes in the light of multilocus sequence typing22 has shown that
many serotypes are actually polyphyletic, meaning that the same sero-
type can be found in isolates belonging to unrelated lineages of S.
enterica subsp. enterica. In E. coli, serotyping is important for identifi-
cation of particular pathovars, such as the enterohemorrahgic E. coli
O157:H7 or O26:H11 strains associated with hemolytic–uremic syn-
drome, or for identification of E. coli strains that possess the K1 cap-
sular type implicated in neonatal infections.
In the clinical microbiology laboratory, where the focus is on the
detection of pathogenic members of the Enterobacteriaceae family,
knowledge of the mechanisms of infection has facilitated the develop-
ment of rapid molecular diagnostic methods targeting specific viru-
lence genes, especially those associated with gastrointestinal bacterial
pathogens.23,24 In-house and commercial polymerase chain reaction
(PCR) assays for the direct detection from fecal specimens of Salmo-
nella, Shigella and the pathogenic strains of E. coli are routinely used
in many clinical microbiology laboratories (e.g. http://www.iss
.it/binary/vtec/cont/EU_RL_VTEC_Method_02_Rev_0.pdf). Target
genes are typically associated with either invasion, toxin production
or adherence to the host gut mucosa. These genotypic methods
are important in differentiating between diarrheagenic E. coli and
 Chapter 180  Enterobacteriaceae 1569

Human infections caused by Enterobacteriaceae, and the species of this family that are most often implicated

Neurologic infections Pneumonia

Neonatal meningitis Abscesses – Klebsiella pneumoniae

– E. coli K1 – Citrobacter spp. – E. coli
– all others (immunodepression)
Immunodepressive conditions Disrupt hematologic barrier
– Klebsiella pneumoniae – all Enterobacteriaceae

Pyogenic liver abscess

Urinary tract infections – Klebsiella pneumoniae

Community-acquired Hospital-acquired/neonatal
– E. coli same as community-acquired
– Proteus mirabilis – Enterobacter spp. Digestive infections
– Klebsiella pneumoniae – Citrobacter spp.
– all others Community-acquired
– Salmonella
– Shigella, E. coli (EIEC)
– Yersinia
Bacteremia – E. coli (EPEC, ETEC, EHEC,
EAEC)
Community-acquired Hospital-acquired/neonatal
– E. coli – Enterobacteriaceae spp. Hospital-acquired/neonatal
– Klebsiella pneumoniae – Proteus spp. same as community-acquired
– Salmonella – Edwardsiella – K. oxytoca
– Shigella dysenteriae – all others

Figure 180-4  Human infections caused by Enterobacteriaceae and the species of this family that are most often implicated.

commensal strains or rapidly detecting strains associated with life-
threatening disease, such as the enterohemorrahgic E. coli group, asso-
ciated with hemolytic–uremic syndrome.
General Pathophysiologic
Considerations
Enterobacteriaceae are associated with both gastrointestinal and
extraintestinal infections including urinary tract infection, bacteremia,
pneumonia, abdominal or pelvic infection, surgical site infections,
meningitis and various abscesses including wound infections (Figure
180-4). The balance between host defenses and virulence factors of
Enterobacteriaceae members is a key factor that determines commen-
salism or disease (Figure 180-5).25,26
Extraintestinal pathogenic E. coli (ExPEC) are facultative pathogens
that belong to the normal gut flora of a certain fraction of the healthy
population where they live as commensals.5 Infections occur through
microbial colonization of normally sterile sites. Each anatomic site
presents specific molecular structures and defenses against infection,
and bacteria must therefore express specific colonization factors to
adhere to these structures and specific virulence factors to counter
these defenses.
Commensal bacteria represent an important barrier against infec-
tion, as colonization by harmless commensals protects the host from
invading pathogens. The host ‘tolerates’ the commensal gut flora and
homeostasis is maintained by 1) the physical barrier of the mucus and
antibacterial molecules that keeps commensal bacteria separate from
the epithelial surface; 2) specific features of commensal species enabling
them to escape or alter the inflammatory response; 3) particular char-
acteristics of epithelial cells, such as the reduced expression of Toll-like
receptor 4 (TLR4) at the gut surface epithelium that reduce the effects
of bacterial stimuli, thus avoiding inflammation that would be detri-
mental for the host.25 In contrast, enteric pathogens modulate inflam-
mation leading to host responses that facilitate their survival and
restrain other commensal flora.25 Genomic approaches to the human
microbiota of the gut will further elucidate the interactions between
commensal and pathogen.24 The mechanisms by which the well-
known gastrointestinal (GI) pathogens, E. coli, S. enterica, Shigella spp.
and Yersinia, breach the intestinal barrier and cause disease are
described below.
Many pathogenicity factors have been described in Enterobacteria-
ceae. These factors are often phage-encoded or clustered in chromo-
somal regions called pathogenicity islands (PAIs), which were first
described in uropathogenic and diarrheagenic E. coli.27,28 Most patho-
genic Enterobacteriaceae are characterized by specific sets of PAIs
(Table 180-2) whereas these PAIs are absent in nonpathogenic strains.
PAIs are acquired by horizontal transfer and are typically associated
with tRNA genes, flanked by repeated sequences, and may differ from
the core genome in guanine and cytosine (G+C) content and in codon
usage.
Clinical Manifestations
INTESTINAL INFECTIONS
The most important enteric pathogens are S. enterica, some strains of
E. coli, Shigella and Y. enterocolitica. Although other Enterobacteria-
ceae are occasionally implicated in gastrointestinal infections, clinical
significance is sometimes controversial (e.g. for Plesiomonas shigelloi-
des or diarrhea-associated K. pneumoniae). Indeed, Enterobacteriaceae
isolated from stool specimens during acute diarrhea could reflect the
drastic change in stool flora, rather than being the cause of the
symptoms.
Escherichia coli
Six distinct pathotypes of diarrheagenic E. coli are classically dis­
tinguished: enterotoxigenic, enteropathogenic, enterohemorrhagic,
enteroinvasive, enteroaggregative and diffusely adherent E. coli. Iden-
tification of diarrheagenic E. coli strains requires their distinction from
commensal E. coli strains, which is rendered possible by their specific
sets of virulence factors, sometimes in combination with serotyping.29
1570 SECTION 8  Clinical Microbiology: Bacteria

Mechanisms of interaction between gut mucosa, commensal and pathogenic intestinal strains

Mucosal physical barrier:

Impermeability
Gut lumen

IgA Mucus

Defensins
+ Defensins
TLR 2 + TLR 2 Cathelicidins +
M cells
Tight
junctions

NOD/CARD MyD88/COX 2

Sub-mucosa Lymphocytes TLR 2

Repair

–
T regulatory cells, «Treg»
NOD/CARD
NFκB
Macrophages, dendritic cells

Adaptive immune:
Th1 immune response control IL10 ++ Innate immune:
IL1β Balance between anti and pro-inflammatory
Bacteria

Figure 180-5  Mechanisms of interaction between gut mucosa, commensal and pathogenic intestinal strains. At the proximity of the intestinal mucosa, physical barriers
exclude bacteria by the impermeability of the epithelium with tight junctions, and by production of factors excreted by epithelial cells themselves such as defensins or
cathelicidins. The mucus is composed of mucin glycoproteins that interplay with bacteria, which are trapped and maintained at a distance from the epithelium. When
the intestinal mucosa is exposed to bacteria (pathogens or commensal in hosts with underlying conditions), an effective immune activation is essentially based on three
major regulated events. 1) Activation of TLR2/MyD88-dependent signaling is essential for effective intestinal repair in response to epithelial damage in the presence of
commensal bacteria. 2) Innate immune mechanisms with activation of macrophages, dendritic cells or neutrophils are central in the control of local inflammation (NOD2/
CARD15 pathway). A central role of the NOD/CARD system implicated in the controlled production of NF-κB is demonstrated. 3) Induction of regulatory T cells by
commensal bacteria, with low induction of Th1 differentiation, is observed and explains at least in part the associated tolerance.

Table 180-3 summarizes clinical, epidemiologic and biologic features
of the distinct pathotypes.
Enterotoxigenic Escherichia coli (ETEC).  First described in
Calcutta in 1956, ETEC is one of the top four etiologic agents of severe
diarrhea in infants younger than 5 years of age in low- and middle-
income countries (LMIC)30 responsible for approximately 280 million
cases in this age group and 840 million cases in total. ETEC are also a
common cause of travelers’ diarrhea associated with ∼30% of cases in
North American and European travelers.31 The illness caused by ETEC
has a short incubation period and symptoms of watery diarrhea,
similar to Vibrio cholerae infection. ETEC is endemic all year round in
many countries in Africa, Asia and Latin America but more common
in warm and wet months. Outbreaks occur and are associated with
food or water contaminated with human feces.
ETEC are defined by the presence of plasmid-encoded LT-I and
LT-II heat-labile oligomeric toxins and STa and STb heat-stable
monomeric toxins and the expression of fimbrial antigens, also plasmid
encoded. The fimbrial antigens were known previously as colonization
factor antigens (CFAs) but are now referred to as coli surface (CS)
antigens suffixed with a number e.g. CS2.32 CFA/I are still referred to
as such. More than 25 different CS antigens are known. ETEC cause
disease by adhering to the host gut mucosa via CS antigens (and pos-
sible other colonizing factors) and producing either one or both of the
LT/ST enterotoxins.33
Serotyping has limited use in identifying strains of E. coli belonging
to the ETEC group as there are a large number of associated serotypes
(at least 78 O antigens and 34 H antigens) and common serotypes
change over time. ETEC can be detected by PCR targeting the LT and
ST genes in isolated colonies or directly from fecal specimens but are
rarely sought in diagnostic laboratories. PCR methods are also avail-
able to detect ETEC colonization factors.
Enteropathogenic Escherichia coli (EPEC).  EPEC was the first
group of diarrheagenic E. coli to be identified and was associated with
morbidity and mortality in infants with diarrhea in the 1940s and 50s
in the UK and USA. EPEC is still a global cause of diarrhea in infants
younger than 2 years today and is especially common in LMIC where
mortality rates are high. It is more rarely associated with diarrhea in
adults where symptoms are attributed to ingestion of large inocula
and/or travel. Clinical manifestations include profuse, persistent diar-
rhea, vomiting and fever, all of which contribute to severe dehydration,
which can be life-threatening in very young and/or malnourished
children.
Initially strains of E. coli belonging to the EPEC group were defined
by serotype and then by their localized adherence (LA) pattern on
HEp-2 cells. Currently EPEC are defined as having the intmin or eae
(for E. coli attaching and effacing) gene, encoding a 97 kDa bacterial
outer-membrane protein. During infection, the bacterium intimately
attaches to the host gut mucosa disrupting the apical cytoskeleton of

TABLE
180-2  Classification of Virulence Factors Described in Enterobacteriaceae Species
Type of Virulence Factor (VF)
Colonization factors, adhesins
Component of secretion system
Flagella
Capsular polysaccharide
Iron capture system
Toxins, cytotoxic effectors
T3SS EFFECTORS
Effectors targeting or mimicking
Rho GTPase family members
Effectors targeting innate
immune signaling pathways
Effectors targeting actin
polymerization directly
Effectors promoting intracellular
survival
cAMP, cyclic adenosine monophosphate; DAEC, diffusely adherent E. coli; EAEC, enteroaggregative E. coli; EHEC, enterohemorrhagic E. coli; EIEC, enteroinvasive E.
coli; EPEC, enteropathogenic E. coli; ETEC, enterotoxigenic E. coli; IL, interleukin; NF-κB, nuclear factor kappa B; SPATEs, serine protease autotransporters of
Enterobacteriaceae.
Chapter 180  Enterobacteriaceae 1571
DESCRIPTION
Bacterial Source VF-Specific Nomenclature Major Function
Escherichia coli P-fimbriae: type 1 common pili; sfa Adherence to target epithelial cells via
fimbriae: CFA/I, CS2-25 in ETEC; specific receptors
bundle-forming pilus (BFP) in EPEC;
AAF/I-V in EAEC; Afa adhesins
in DAEC
Shigella –
Salmonella Type1 fimbriae; Long polar fimbriae;
Curli fimbriae
Yersinia Invasin
Proteus MR/P fimbriae; PMF (P. mirabilis
fimbriae), ATF (ambient temperature
fimbriae), NAF (nonagglutinating
fimbriae)
Klebsiella Type 1 common fimbriae, type 3 and
type 6 fimbriae
Escherichia coli T3SS sep/esp locus in EPEC and EHEC Molecular needle that permits export
Salmonella T3SS inv/spa locus of secreted bacterial proteins across
Shigella T3SS mxi/spa locus (also described in bacterial membranes and injection
EIEC) directly into target eukaryotic cells
Yersinia T3SS ysc and ysa loci
Many members of – Mobility, pro-inflammatory activity
Enterobacteriaceae except
Shigella
Escherichia coli K1 antigen Resistance to antimicrobial
Salmonella serotype Typhi Vi antigen complement activity and prevention
Klebsiella K capsular antigens: K1, K2, K4 and of antibacterial serum resistance
K57 emergent capsule
Yersinia YadA, Ail (attachment invasion locus)
Escherichia coli Aerobactin, chu protein, yersiniabactin Iron acquisition
in EAEC
Shigella foc, fet, iuc and iut loci
Yersinia Yersiniabactin
Shigella spp. SPATEs Mucolytic activity; modification of 28S
Shigella dysenteriae Shiga toxin (Stx1) (ADP ribosomal subunit leading to
ribosyltransferase toxins) apoptosis of target cells
Enterohemorrhagic Escherichia coli Shiga-like toxins (Stx1 and Stx2)
Enterotoxigenic Escherichia coli Thermolabile and thermostable Increase of intracellular cAMP causes
enterotoxins (LT, ST) modification of apical ion channel
activity in target cells
Enteroaggregative Escherichia coli EAST-1, plasmid-encoded toxin (Pet), Unknown
SPATEs
Uropathogenic Escherichia coli Hemolysin alpha (RTX toxins), CNF1 Pro-inflammatory activity; modification
of Rho GTPase (for CNF1)
Salmonella SopE2, SopE, SptP Modification of cytoskeletal actin and
Shigella IpaA, IpaC, IpgB1, IpgB2 macropinocytosis
Yersinia YopT, YopE, YpkA/YopO
Salmonella SipB (SspB), AvrA, SspH1, SpvC Apoptosis in macrophages and
Shigella OspG, OspF, IpaB dendritic cells; inhibition of NF-κB
Yersinia YopJ signaling and IL-8 production
Enteropathogenic Escherichia coli Tir Actin nucleation and pedestal
formation
Salmonella SipA (SspA), SipC (SspC) Induction of actin polymerization
Shigella IcsA (VirG), IcsP, VirA Actin nucleation
Yersinia YopH Antiphagocytic activity targeting a
major focal adhesin
Salmonella SseF, SseG, SseJ, SopD2, SifA, PipB2, Contribution of Sif formation and
SspH2, SptP microtubule formation
Shigella IcsB Host cell survival and prevention of
autophagic recognition of IcsA/VirG
1572 SECTION 8  Clinical Microbiology: Bacteria

TABLE
180-3  Escherichia coli and Intestinal Infections
Pathotype of E. coli Clinical Manifestations Histologic Effects Specific Virulence Factors Diagnostic Methods

Enterotoxigenic E. coli Watery diarrhea; travelers’
(ETEC) diarrhea; endemic
cholera-like illness in
children; low- and middle-
income countries (LMIC)
LT-B subunit binding LT (enterotoxin, heat labile) and ST PCR targeting the LT and/or
GM1 and LT-A subunit (enterotoxin, heat stable); various ST gene
increase cyclic AMP colonization factors (CFs)
production, leading to including CS1, CS7, CFA/I and
chloride and neutral CFA/II
sodium chloride
secretion

Enteropathogenic E. Watery diarrhea with
coli (EPEC) vomiting and fever;
infantile diarrhea in
children <2 years old;
LMIC
A/E adhesion; actin-rich
pedestal
Outer-membrane intimin adhesin
(eae gene); LEE pathogen (T3SS,
esc genes, Esp effectors); EAF
plasmid (bundle-forming pilus);
intimin receptor Hp90 or Tir;
EAST-1 (astA gene)
PCR targeting the eae gene
encoded on the LEE PAI
and/or bfp gene encoded
on the EAF plasmid

Enterohemorrhagic E. Bloody diarrhea, hemolytic–
coli (EHEC) uremic syndrome triad
(thrombocytopenia,
hemolytic anemia, renal
failure); children and
young adults;
industrialized countries
A/E adhesion; actin-rich Major virulence determinant – stx PCR targeting the phage-
pedestal (identical to genes; intimin adhesin (eae encoded stx genes and the
EPEC) gene); LEE pathogen (T3SS, esc eae gene encoded on the
genes, Esp effectors); pO157 LEE
plasmid encoding
enterohemolysin gene (in
O157:H7 strains); EAST-1 (astA
gene)

Enteroinvasive E. coli Classic watery diarrhea;
(EIEC) occasionally dysentery
syndrome
Shigella-like invasion of
epithelium
Large virulence plasmid (220 kb,
100 genes); TTSA type secretion
(mxi/spa locus); Ipa, Ipg and Osp
effectors; Vir regulators; IcsA/
VirG
PCR targeting the ipaH gene
encoded in the pINV;
guinea pig
keratoconjunctivity or
Sereny test for
differentiation of EIEC from
Shigella

Enteroaggregative E. Watery diarrhea; travelers’
coli (EAEC) diarrhea; children and
adults; HIV patients; LMIC
and industrialized world
Adherence to epithelial Large plasmid pAA (typical strains); PCR targeting the aggR gene
cells with typical EAST-1; transcriptional activator encoded in the pINV and
‘stacked-brick’ pattern; gene; she patho-island; the aaiC gene encoded on
mucoid biofilm AAF/I-AAF/V the chromosome
formation; toxic effects

Diffusely adherent E. Watery diarrhea persistent – Afa, dra operon PCR targeting the daaC gene
coli (DAEC) in young children; LMIC
and industrialized world

A/E adhesion, ‘attaching and effacing’ adhesion; PCR, polymerase chain reaction.
From Croxen M.A., Law R.J., Scholz R., et al.: Recent advances in understanding enteric pathogenic Escherichia coli. Clin Microbiol Rev 2013; 26:822-80.29

the epithelial cells lining the gut mucosa, effacing the microvilli and
forming pedestal-like structures.34 The process is coordinated by a type
III secretion system (T3SS) and multiple EPEC-secreted protein (Esp)
effectors on a 35 kb chromosomal pathogenicity island called the locus
of enterocyte effacement (LEE). One of the effectors, the translocated
intimin receptor (Tir), acts as a receptor for intimin. Symptoms of
diarrhea are not toxin-mediated and are likely to be caused by the
cellular changes in the host’s gut mucosa; for example, changes in etiologic agent of infectious HUS in children and is an emerging
the electrolyte balance within the epithelial cells and destruction of the
microvilli, resulting in the reduction in the absorptive capacity of (10–100 organisms) and transmission is via contaminated food or
the intestinal epithelium.34
Certain strains of EPEC also carry an adherence factor (EAF)
plasmid encoding a cluster of 14 genes coding for the expression and
assembly of the bundle-forming pilus (BFP) and are known as typical
(tEPEC) or classical EPEC. Like ETEC, transmission of tEPEC is
human-to-human or from food or water contaminated by human
feces. EPEC without the EAF plasmid are designated atypical EPEC
(aEPEC) – although epidemiological studies show they are more
common than tEPEC. aEPEC is associated with a varied animal reser-
voir, including domesticated and wild animals, and these animal iso-
lates share many characteristics with strains known to cause diarrhea
in humans.35
Historically, EPEC was identified at diagnostic laboratories by sero-
typing, as tEPEC comprised relatively few serotypes and subsequently
confirmed at reference laboratories by the technically-demanding
fluorescent actin staining (FAS) test. This approach has now been
replaced by PCR targeting the intmin gene and bfp genes.
Enterohemorrhagic Escherichia coli (EHEC).  First reported in
1983, EHEC strains are responsible for gastrointestinal illnesses includ-
ing severe abdominal pain and grossly bloody diarrhea that can
develop into hemolytic–uremic syndrome (HUS) between 4 and 15
days after the onset of diarrhea.36 HUS is specifically associated with
thrombocytopenia and hemolytic anemia, as well as acute renal failure
and cases often require dialysis treatment. EHEC is the most common
pathogen in industrialized countries. The infectious dose is low
water, direct contact with animals, especially ruminants, indirect
contact with a contaminated environment or person-to person.
The EHEC group is defined by the production of two phage-
encoded Shiga toxins, Stx1 (identical to Stx1 of S. dysenteriae type
1) and Stx2. Both toxin types have a number of variants and Stx2,
specifically the stx2a subtype, is most commonly associated with
HUS.37 Stx, circulating via the bloodstream, is thought to cause vascu-
lar damage in the colon and kidneys, specifically by adhering to cell
surface globotriaosylceramide (gb3) and the induction of apoptosis.29
Many EHEC associated with causing severe bloody diarrhea or HUS
also have a LEE pathogenicity island, and adhere to the host gut
mucosa in a similar way to EPEC. More recently, stx-positive, LEE-
negative strains causing HUS were found to harbor the aggR and aai
genes, previously associated with EAEC group (see below). EHEC
negative for both the LEE and aggR/aai genes have also been associated
with HUS and it is likely that other, as yet unknown, attachment
mechanisms exist.38
 Chapter 180  Enterobacteriaceae 1573

Microbiological diagnosis is difficult as the onset of HUS often
occurs several days after the diarrheal prodrome, when patients no
longer have detectable levels of EHEC in their stools. The recom-
mended diagnostic method is the detection of stx genes directly from
fecal specimens using PCR and the subsequent culture and identifica-
tion of stx-positive colonies using the same PCR. The most common
EHEC serotype, EHEC O157:H7, does not ferment d-sorbitol (as
opposed to most other E. coli strains), and can be clearly identified as
colorless, sorbitol non-fermenting colonies on sorbitol MacConkey
agar containing cefixime and tellurite (CT-SMAC). HUS can be caused
by other serogroups of stx-producing E. coli (e.g. O26, O103, O111 and
O145), that (unlike EHEC O157:H7) do not have specific biochemical
characteristics that aid identification and this further complicates the
bacterial diagnosis of HUS. Immunomagnetic separation techniques,
involving magnetic beads coated in antibodies to the most commonly
detected serogroups, may facilitate detection when EHEC is present in
the fecal specimen at low levels.
Enteroinvasive Escherichia coli (EIEC).  First described in 1971
as being capable of causing diarrhea in volunteers, EIEC has similar
pathogenic, biochemical and genetic properties to Shigella. EIEC infec-
tions occur most commonly in LMIC and are associated with travelers
recently returned from these regions. Like Shigella, EIEC is transmitted
mainly through contaminated water and food or direct person-to-
person spread.
The major virulence factor is a 220 kb plasmid, designated the
invasion plasmid (pINV). The pathogenic mechanism of EIEC is virtu-
ally identical to that of Shigella (Figure 180-6). Although EIEC can
be responsible for dysentery with fecal blood, mucus and leukocytes,
this clinical presentation is less common than with Shigella infection
and volunteer studies indicate that the infectious dose is higher.
Watery diarrhea is common and indistinguishable from Shigella
infections.
Differential diagnosis from Shigella is difficult even using the PCR
approach as the ipaH gene, the target for detecting Shigella is on the
pINV plasmid and is also carried by EIEC. EIEC and Shigella can only
be clearly differentiated by the guinea pig keratoconjunctivity or
Sereny test (now rarely used) or by extended biochemical tests and
serology.
Enteroaggregative Escherichia coli (EAEC).  Nataro et al.39 first
described the aggregative adherence (AA) or stacked-brick formation
of bacterial cells attached to HEp-2 cells that characterizes the EAEC
group. EAEC is the most common bacterial cause of diarrhea in studies
in the USA and UK40,41 and an emerging cause of travelers’ diarrhea.
It makes a significant contribution to morbidity in children younger
than 2 years of age in LMIC and is also an important cause of diarrhea
in HIV-infected patients. The typical illness is characterized by a per-
sistent watery, mucoid, secretory diarrhea with low-grade fever and
symptoms may continue for more than 10 days. Transmission of
EAEC is thought to be similar to that of EIEC and Shigella, via food
and water contaminated with human feces or person-to-person spread.
EAEC strains have been reported in animals but there is, as yet, little
evidence to suggest a significant animal reservoir.
Certain EAEC harbor a plasmid, designated pAA, encoding a
number of putative virulence factors including AggR (involved in the
regulation of many of the virulence genes involved in both the aggrega-
tion and toxin production stages of EAEC pathogenesis) and are
known as typical EAEC. Atypical strains also adhere to HEp-2 cells in
a stacked-brick formation but do not have the pAA plasmid. Although
the exact mechanism of pathogenesis is not fully understood, a model
in three stages has been proposed:29
1. Adherence to host gut mucosa by fimbrial and afimbrial adhes-
ins. To date, five types of aggregative adherence fimbriae have
been described (AAFI-V) all encoded on pAA and regulated by
aggR.

A view of the Shigella invasion process

Orofecal contamination
Gut lumen

Invasion and survival

Epithelial cells M cells

IpaA NOD1
IcsA/VirH alarm
system
IpgD
Transcytosis NFκB IpaH family
IpaB/C/D Osp family

IpaA
VirA
IpaB/C/D
Submucosa
IpaB/C IL-8
Apoptosis
(caspase-1 dependent) Migration
Macrophages

IL-1β
IL-18
Epithelial destruction, innate
NK cells immune reaction, inflammation Polymorphonuclear cell
INF-γ

Figure 180-6  A view of the Shigella invasion process. Shigella transmitted by the fecal–oral route reach the large intestine, transcytose across the epithelial layer
through M cells, and encounter resident macrophages. Shigella flexneri is capable of survival in macrophages, inducing apoptosis and release of proinflammatory cyto-
kines such as IL-1β or IL-8. IL-8 induces a massive infiltration of neutrophils, which destroy the integrity of the epithelial layer. Once released from macrophages, bacteria
invade the epithelial cells from the basolateral side, escape to phagosomal vesicles, and move in the intracellular cytoplasm by polymerization of actin, ultimately invad-
ing neighboring cells. (Adapted from Schroeder & Hilbi Clin Microbiol Rev 2008; 21:134–156.)
1574 SECTION 8  Clinical Microbiology: Bacteria
2. Production of enterotoxins and cytotoxins including a
cytoskeleton-altering plasmid-encoded toxin (Pet), SHet, a
toxin also produced by Shigella flexneri, heat-stable enterotoxin
(EAST-1) and Pic, a mucinase, both encoded by many other
pathogens.42
3. Mucosal inflammation induced by both the pathogen and the
host’s own immune system.
Recent studies suggest that the aggR gene also regulates
chromosomally-encoded genes located on the Aai operon.43
EAEC strains are phenotypically and genetically diverse and, there-
fore, comprehensive diagnostic assays based on biochemical, serologi-
cal or molecular approaches all have limitations. The gold standard
method for the identification of EAEC is the HEp-2 adherence assay
with the characteristic aggregative or ‘stacked-brick’ adherence pattern
but this is technically demanding and unsuitable as a routine diagnos-
tic tool. PCR targeting the aggR and aaiC genes is the most commonly
used method.
In 2011, 4321 previously healthy people from 16 countries were
associated with an outbreak of food-borne illness caused by an EAEC
Shiga toxin-producing strain identified as E. coli O104:H4. Over 900
cases developed HUS and more than 50 people died.44 This hybrid
pathogen carried virulence genes found in both typical EAEC strains
(aggA, aggR, set1, pic, and aap) and EHEC (stx2). Other examples of
the acquisition of Shiga toxin genes by EAEC isolated from patients
with HUS have been reported in Japan, France, Northern Ireland and
Central African Republic.
Diffusely Adherent Escherichia coli (DAEC).  Diffusely adher-
ent E. coli defined by a diffuse adherence (DA) pattern to HEp-2 or
HeLa culture cells were originally described as being associated with
watery diarrhea in children older than 1 year of age.45 The adherence
phenotype is due to the production of adhesins encoded by a family
of operons related to afa/dra/daa genes encoding both afimbrial (such
as Afa) or fimbrial (such as F1845 encoded by daaC or Dr) adhesins.
Afa/Dr DAEC was reported initially in extraintestinal E. coli infection
by a uropathogenic strain29 and does not appear specific to strains
associated with diarrheal diseases (DAEC). Moreover, these structures
were also described among commensal E. coli from normal digestive
flora. Although the interaction between Afa/Dr adhesins and receptors
leads to cell signaling resulting in actin modification, the implication
of Afa/Dr DAEC strains in diarrhea remains controversial. Epidemio-
logical studies are hampered by the fact there are no universally agreed
methods for the detection of DAEC in the clinical setting.
Shigella
The four Shigella species (S. dysenteriae, S. flexneri, S. sonnei and S.
boydii), a genus first described by Kiyoshi Shiga in 1897, are part of
the genomic species E. coli (with the exception of S. boydii serotype
13). The Shigellae can be divided into 49 serotypes and additional
subtypes. Symptoms of shigellosis are mild watery diarrhea or more
severe inflammatory bacillary dysentery, characterized by fever,
abdominal cramps, and blood and mucus in stools. S. dysenteriae 1
produces Stx1 and has been associated with HUS similar to the syn-
drome caused by EHEC.46 A large proportion of all diarrheal episodes
worldwide are attributed to Shigella, with 1.1 million fatal cases annu-
ally; approximately 99% of cases occur in LMIC with the majority
occurring in children under the age of 5 years.
Shigella dysenteriae and S. boydii are rare, the latter being largely
restricted to the Indian subcontinent. S. flexneri is the most commonly
isolated species, endemic in LMIC and isolated in industrialized coun-
tries, mainly in travelers returning from abroad. Recently, outbreaks
of S. flexneri have been associated with transmission between men who
have sex with men (MSM) in Europe, North America and Australia.
S. sonnei are the most common species isolated in industrialized coun-
tries, often linked to outbreaks in nurseries and schools.
The Shigellae are facultative intracellular pathogens derived from
E. coli by a combination of gene loss and acquisition. Acquisition
genes are associated with bacterial invasion and intracellular survival
(e.g. virB and mxiE), iron acquisition (e.g. iuc and fuc) and toxin
production (e.g. set1A and set1B) encoded on the invasion plasmid
pINV and the chromosomally located Shigella PAIs (SHIs) SHI-1 and
SHI-2, SHI-O, and SRL. The Shigella genome is also characterized by
deletions associated with the loss of metabolic pathways (e.g. cadA) no
longer required for an intracellular lifestyle and antivirulence factors
(e.g. nadA and nadB) that would suppress pathogenicity.14,29,47 In the
large intestine, during the invasion process, the bacteria cross the
epithelial layer through M-cells and are engulfed by macrophages on
reaching the submucosa. They survive in the macrophage, eventually
inducing apoptosis and the release of cytokines that destroy the integ-
rity of the epithelial layer. On escaping from the macrophage, they
invade the epithelial cells from the basolateral side and traverse the
intracellular cytoplasm of the invaded cell and neighboring cells by
formation of an actin tail on the bacterial surface that provides the
propulsive force required for directed motility (Figure 180-6). Diar-
rhea results from the breakdown of the integrity of the epithelium and
subsequent reduction in its capacity to resorb fluid.
Detection of Shigella from feces relies on the use of differential and
selective media (e.g. deoxycholate agar (DCA)) and xylose-lysine
deoxycholate (XLD). Biochemically, Shigella are unable to ferment
lactose or utilize citrate, urea or produce lysine decarboxylase, are
nonmotile and produce acid from glucose but no gas. The PCR targets
to invasion plasmid antigen H (ipaH) gene. Strains are serotyped (S.
dysenteriae, S. boydii and S. flexneri) or phage typed (S. sonnei) to
facilitate outbreak investigations. Molecular typing methods include
PFGE, MLST, MLVA and WGS.
Salmonella
There are two clinical manifestations of salmonellosis: gastroenteritis
and typhoid (enteric) fever. Salmonella enterica serovars Typhi and
Paratyphi A, B and C are responsible for typhoid and typhoid-like
fevers. These pathogens only infect humans and are transmitted via
the fecal–oral route by contaminated food or water, or by person-to-
person spread. This systemic illness leads to an estimated 20 million
cases and 200 000 deaths worldwide each year. Typhoid fever is char-
acterized by a systemic infection where bacteria colonize the liver,
spleen and bone marrow. Cases often report a short bout of nausea
and diarrhea prior to malaise, headache, fever, myalgia and minor
upper respiratory symptoms. Complications include persistent infec-
tion resulting in relapse or chronic carriage where the bacterium may
persist in the gallbladder. WGS data has shed light in the population
structure and evolution of Typhi and highlights the role of asymptom-
atic carriers as the main reservoir of this pathogen, and the need to
identify and treat carriers.48 Clinical diagnosis can be confirmed by
isolating the bacterium from blood culture, urine or feces. Serological
assays for the detection of antibodies to the Salmonella Typhi O and
H antigens (e.g. Widal test) are no longer recommended for diagnosis
as cross-reactions make results difficult to interpret.
The global burden of nontyphoid Salmonella gastroenteritis
remains high at 93.8 million cases, with 155 000 deaths and an average
incidence of 1.14 episodes/100 person-years each year. The incubation
period is from 4 hours to 72 hours after the ingestion of contaminated
food or water. Symptoms include fever and chills, nausea and vomit-
ing, abdominal cramping and diarrhea. Gastroenteritis can be caused
by a large number of serovars, most commonly Typhimurium and
Enteritidis. Infection is localized to the intestine, without systemic
diffusion in most cases. Some serovars cause disease in both humans
and animals and have a wide host range (e.g. Typhimurium associated
with humans, cattle, swine, horses, sheep, poultry, and wild rodents),
whereas others are host restricted (e.g. S. gallinarum associated with
poultry).49
Salmonella pathogenicity factors are encoded on five Salmonella
PAIs, SPI1-SPI5. In the small bowel, salmonellae cross the intestinal
mucus layer and adhere to intestinal epithelial cells using a variety of
adhesins including type I fimbriae, curli fimbriae, Pef fimbriae, and
Std fimbriae. The invasion of the host epithelial cells by Salmonella is
mediated by T3SS1 effectors (SipA, SipC, SopB, SopE, SopE2) that
trigger the rapid appearance of membrane ruffles on the surface of the

host cell, and subsequent formation of spacious phagosomes or vacu-
oles.50,51 Alternatively, at least two non-fimbrial outer-membrane pro-
teins, Rck and PagN, can mediate invasion of nonphagocytic cells.52
Following invasion, the bacteria remain within a modified phagosome
known as the Salmonella-containing vacuole (SCV), where their sur-
vival and replication is controlled by the down regulation of T3SS1
and up-regulation of T3SS2 (SseF, SseG, SifA and SseJ).53 The SCVs
cross the basolateral membrane and release the bacteria into the sub-
mucosa, where they are internalized within phagocytes and dissemi-
nated through the lymph and the bloodstream.51
Like Shigella, detection of Salmonella from feces relies on the use
of differential and selective media (e.g. deoxycholate agar (DCA) or
XLD). There are exceptions but generally Salmonella are unable to
ferment lactose or urea but do utilize citrate and hydrogen sulfide.
They produce lysine decarboxylase and acid from glucose and gas and
are motile. A variety of published PCR assays exist; generic assays
targeting the ttr genes required for tetrathionate respiration and assays
specific for subspecies 1 targeting in invasion genes such as hilA.
Molecular typing methods include PFGE, MLST, MLVA and WGS.
MLST correlates well with the traditional serotyping scheme.22
Yersinia
Of the 11 established Yersinia species, only Y. enterocolitica, Y. pseudo-
tuberculosis and Y. pestis are clinically important. Yersinia pestis, the
agent of plague, has species status in the nomenclature for obvious
clinical and historic reasons54 and is discussed in Chapter 126. Yersinia
enterocolitica and Y. pseudotuberculosis can cause acute gastroenteritis
with fever, abdominal pains and diarrhea, more commonly in chil-
dren, and acute terminal ileitis and mesenteric lymphadenitis with
abdominal pain mimicking appendicitis in adults, erythema nodosum,
reactive polyarthritis, and, occasionally, bloodstream infections.55 Yer-
siniosis represents the third most frequently reported food-borne gas-
troenteritis in Europe; the most important source of infection is
believed to be contaminated porcine products.
Pathogenic Y. enterocolitica colonize the terminal ileum, invade
Peyer’s patches and disseminate to lymphoid tissues, including liver
and spleen, where they replicate, suppress and reorient the immune
system. Virulence factors are located on the chromosome and on a
70 kb plasmid. Key invasion proteins are encoded by inv, yadA and ail.
Pathogenesis involves many effectors (named Yop for ‘Yersinia outer
proteins’), which translocate via a secretory mechanism designated Ysc
T3SS (encoded by the virulence plasmid) directly into eukaryotic cells
and inhibit phagocytosis and down-regulate the host immune response
by hijacking the host’s intracellular machinery. The most pathogenic
Y. enterocolitca biotype 1B carries an additional recently described
T3SS, Ysa T3SS.56
On Yersinia selective agar, CIN (cefsulodin, irgasan, novobiocin),
Yersinia enterocolitica and Y. pseudotuberculosis appear as translucent,
well-defined colonies with a deep red center (bull’s eye colonies) due
to mannitol fermentation. Y. enterocolitica is a heterogeneous group of
strains classified into six biotypes, of which 1A is regarded as non-
pathogenic and 1B and 2-5 are pathogenic. Strains belonging to sero-
groups O3 (biotype 4), O5, 27 (biotypes 2 and 3), O8 (biotype 1B) and
O9 (biotype 2) are most frequently isolated worldwide. MLST and
WGS have been used to investigate the population structure and evo-
lution of the genus.57
EXTRAINTESTINAL INFECTIONS
Urinary Tract Infections
Urinary tract infections (UTIs) are among the most common bacterial
infections in humans and account for significant morbidity and high
medical costs (see Chapters 53, 55 and 56). UPEC infections account
for roughly 80% of all UTIs, causing cystitis in the bladder and acute
pyelonephritis in the kidneys.
The ability to cause UTI requires a combination of highly regulated
virulence factors, including multiple pili, secreted toxins (for example
Sat and vacuolating autotransporter toxin (Vat)), multiple iron acqui-
sition systems and polysaccharide capsules, often encoded on PAIs.
Chapter 180  Enterobacteriaceae 1575
Attachment is mediated by fimbrial adhesin H (FimH), which is found
at the tip of the phase-variable type 1 pili and binds to proteins called
uroplakins that coat the uroepithelium, a specialized stratified transi-
tional epithelium composed of three cell layers. Uroplakin Ia is the
receptor for one important adhesive appendage of UPEC, the type 1
pili. FimH also mediates UPEC invasion and once internalized, UPEC
can rapidly replicate and certain strains produce hemolysin A (HlyA)
toxin causing tissue damage and apoptosis. Adhesins include the fim-
brial adhesins such as the type 1 pili, P-fimbriae and S-fimbriae, and
the afimbrial adhesins such as AfaE. Crosstalk between P fimbriae, type
1 pili and other adhesion clusters prevents co-expression of multiple
surface structures.28,58,59
Other common Enterobacteriaceae associated with UTIs are Kleb-
siella pneumoniae and Proteus mirabilis.60 These species infect specific
groups of patients, such as those with diabetes mellitus or dysfunc-
tional bladders and urinary catheters. A common pathogen in both
community and hospitalized UTIs, K. pneumoniae possesses a number
of virulence determinants, including adhesins, urease activity, capsule
and iron-scavenging systems.61 Proteus mirabilis is an important agent
of UTI, particularly in hospitalized patients and notably those who are
catheterized. A hallmark of infection with this species is stone forma-
tion, resulting from active urease which hydrolyzes urea to ammonia,
raising urinary pH and subsequently leading to precipitation of mag-
nesium ammonium phosphate and calcium phosphate. Another spe-
cific characteristic of Proteus infections is the ability to colonize the
surfaces of catheters forming biofilms.62
Respiratory Tract Infections
Enterobacteriaceae may cause community-acquired pneumonia in the
elderly and are implicated in ventilator-associated pneumonia.63 In
particular, K. pneumoniae and E. cloacae are among the most frequent
bacteria responsible for ventilator-associated pneumonia, after Staphy-
lococcus aureus and Pseudomonas aeruginosa. Klebsiella pneumoniae
was originally described as the agent of Friedländer’s pneumonia, a
severe lobar pneumonia, and remains an important cause of hospital-
and community-acquired pneumonia, even though the incidence of
community-acquired pneumonia has decreased in North America and
Europe61 (see ‘Other infections and rare or emerging Enterobacteria-
ceae pathogens’ below and Chapter 28).
Meningitis and Neurologic Infections
Certain types of E. coli can cause gram-negative-associated meningitis
(neonatal meningitis E. coli or NMEC) in newborns. Fatality rates
can approach 40%, and survivors are usually burdened with severe
neurological sequelae.64 Survival in the blood is facilitated by an anti-
phagocytic polysialic acid capsule and manipulation of the classical
complement pathway by the bacterial outer-membrane protein A
(OmpA).65 Invasion of macrophages and monocytes prevents apopto-
sis and chemokine release, providing a niche for replication before
dissemination back into the blood. A lambdoid phage that encodes O
acetyltransferase acetylates the O antigen to provide phase variation
and diversity to the capsule, hiding the bacteria from host defenses.
Attachment to the endothelial cells of the blood–brain barrier is medi-
ated by FimH of the type 1 pili binding to CD48 and by OmpA binding
to its receptor, ECGP96. Invasion occurs through the actions of Ibe
proteins, FimH, OmpA and cytotoxic necrotizing factor 1 (CNF1).28
The K1 capsule – which is found in approximately 80% of NMEC
isolates – also has a role in invasion by preventing lysosomal fusion
and thus allowing delivery of live bacteria across the blood–brain
barrier.66 In the central nervous system the bacterium can induce
edema, inflammation and neural damage.
Citrobacter koseri is another classic bacterium described in neonatal
neurologic infections, in particular as a cause of devastating neonatal
meningitis characterized by the formation of multiple brain abscesses.67
There is some evidence that C. koseri is able to resist phagocytosis
and the presence of a 32 kDa protein in the external membrane of
the bacteria may be linked to meningeal tropism and abscess
formation.
1576 SECTION 8  Clinical Microbiology: Bacteria
Bacteremia
Bacteremia is a major complication of infection by Enterobacteriaceae
as it can lead to severe sepsis with acute organ failure and septic shock
(see Chapter 47).
In recent years E. coli bacteremia has increased and in the UK the
species now accounts for more than 30% of bacteremia in those aged
over 75 years.68 There have also been significant increases in bactere-
mia caused by other gram-negative pathogens. The primary source of
bacteremia is UTIs. Other classic sources of blood infections include
the digestive tract, implicating both specific intestinal pathogens and
opportunistic enteric bacteria, which can translocate from the intesti-
nal lumen to blood in hosts with underlying conditions (e.g. digestive
solid tumor, cholecystitis or immunosuppressive treatment). Salmo-
nella serovars that cause typhoid fever69 and, more rarely, nontyphoi-
dal Salmonella can cause sepsis.
OTHER INFECTIONS AND RARE
OR EMERGING ENTEROBACTERIACEAE
PATHOGENS
Many, if not most, Enterobacteriaceae can cause infection, even if
rarely, and those species that are considered medically most important
are described in Table 180-4. Klebsiella spp., principally K. pneumoniae
and to a lesser extent K. oxytoca, are second only to E. coli as a noso-
comial cause of urinary tract, respiratory tract or blood infections.61 K.
pneumoniae also cause serious community infections such as pneumo-
nia, bacteremia and meningitis. Klebsiella pneumoniae owes its name
to Friedländer’s pneumonia, a severe form of lobar pneumonia that
used to devastate chronic alcoholics. A recent population study by
MLST61 has defined a number of virulent clones of K. pneumoniae, one
of which is associated with pyogenic liver abscess and two of which
correspond to K. pneumoniae subsp. rhinoscleromatis and K. pneu-
moniae subsp. ozaenae, respectively. The latter two are regarded as a
cause of chronic upper respiratory disease. Klebsiella planticola and K.
terrigena are often misidentified as K. pneumoniae or K. oxytoca; as a
result, knowledge of their clinical importance remains poor (note that
the validity of genus Raoultella, which was proposed for the two former
species, has been questioned). Cytotoxin-producing K. oxytoca were
recently shown to be a cause of antibiotic-associated hemorrhagic
colitis.74
Klebsiella (Calymmatobacterium) granulomatis is the causative
agent of donovanosis (granuloma inguinale), a chronic genital ulcer-
ation (see Chapter 64). Specific diagnosis is possible by detection of
large mononuclear cells with intracytoplasmic cysts filled with deeply
stained gram-negative Donovan bodies in biopsy samples or PCR tar-
geting two unique base changes in the phoE gene.73 In fact, comparison
of phoE sequences of K. granulomatis and K. pneumoniae suggests that
the former may represent particular strains of K. pneumoniae.61
Serious infections by Cronobacter (formerly Enterobacter) sakazakii
with fatal outcome have been described, particularly in children. This
species has been associated with small outbreaks or sporadic cases of
sepsis, meningitis and necrotizing enterocolitis reported in infants
younger than 1 month, especially in premature babies and neonatal
intensive care units (ICUs). High morbidity and mortality were associ-
ated with neurologic forms of this infection. Powdered infant formulas
were incriminated as a source of C. sakazakii infections.71
Other species such as Enterobacter cloacae (itself a complex of
several species), Citrobacter freundii, Citrobacter koseri, Proteus mira-
bilis, Hafnia alvei, Morganella morganii, Serratia marcescens62,67,76,81 and
others can cause disease in humans. These are generally considered as
opportunistic pathogens, as they ordinarily occur as commensals in
the gastrointestinal tract. The clinical significance of most unusual
Enterobacteriaceae species remains unclear, given limited data and that
specific virulence factors remain unknown in most.
Rare pathogenic species are more difficult to identify with classic
methods than more frequent pathogens. Molecular methods, such as
the sequencing of 16S rRNA or rpoB genes, are especially helpful in
these cases and may also lead to the discovery of novel species. For
example, some strains that were misidentified as Hafnia alvei express-
ing the eae gene have now been reclassified as Escherichia albertii,
leading to reconsideration of the implication of H. alvei in gastroin-
testinal disease.6,70
Prevention, Management and
Control of Infections
SYMPTOMATIC TREATMENT
Rapid oral rehydration using intravenous fluids is the first recom-
mended therapy in digestive Enterobacteriaceae infections, specifically
during EPEC infection affecting very young children or ETEC-related
diarrhea and shigellosis.29 Symptomatic treatment of EHEC infections
is critical for management of HUS, which requires dialysis in more
than half of cases36 (see Chapters 37 and 38 for a discussion on the
management of these conditions).
ANTIMICROBIAL RESISTANCE AND
EMERGENCE OF MULTIDRUG RESISTANCE
Resistance mechanisms to antimicrobial agents can be intrinsic and
acquired. The latter mechanisms result from mutational events (espe-
cially for quinolone resistance) or, more often, acquisition of various
mobile genetic elements such as transposons and plasmids.
First reported in the early 1980s in Germany and France, a major
problem in the medical field in recent times is the increasing numbers
of Enterobacteriaceae strains found to produce extended-spectrum
β-lactamases (ESBLs).82 ESBLs are defined as enzymes produced by
certain bacteria that are able to hydrolyze extended-spectrum cepha-
losporins. The ‘workhorse hospital antibiotics’, such as ceftazidime,
ceftriaxone and cefotaxime, are rendered ineffective against ESBL-
producing bacteria. There are three important types of ESBLs: TEM,
SHV and CTX-M.82 The numbers of TEM and SHV type antibiotics
each currently exceeds 100. The most common types are carried by E.
coli and K. pneumoniae83 but they are also found in Enterobacter,
Proteus, Morganella and Salmonella species. TEM and SHV enzymes,
widespread in E. coli and Klebsiella, have evolved by mutation of
common plasmid-encoded penicillinases, whereas CTX-M enzymes
represent examples of plasmid acquisition of beta-lactamase genes
normally found on the chromosome in Kluyvera species. CTX-M
family, first described in Japan in 1986, hydrolyze cefotaxime in prefer-
ence to ceftazidime and are prevalent among community-acquired
infections. In E. coli, one predominant CTX-M clone, CTX-M-15, rep-
resented two-thirds of all extended-spectrum β-lactamase (EBSLs)
isolated in 2004 in the UK.84 CTX-M is also a common ESBL identified
in Enterobacteriaceae isolated in fecal flora.
A recent study showed that community ESBL fecal carriage, which
was unknown before the turn of the millennium, has since increased
significantly everywhere, with LMIC being the most affected. Intercon-
tinental travel may have emphasized and globalized the issue and
CTX-M enzymes, especially CTX-M-15, are the dominant type of
ESBL. The authors of the study suggest that CTX-M carriage is evolv-
ing toward a global pandemic.85
Most outbreaks reported in ICUs, involve unrelated strains of the
same species but may involve the dissemination of the ESBL plasmid
to different species. Risk factors for infection by ESBL-producing
Enterobacteriaceae included increased length of hospital stay, admis-
sion to the ICU or pre-existing co-morbidities and specifically prior
use of third-generation cephalosporins.81
Developed in the 1980s, carbapenems (the most widely used is
imipenem, followed by meropenem and ertapenem) are derivatives of
thienamycin and have broad-spectrum activities and are used to treat
infections caused by ESBL-producing strains. Three distinct mecha-
nisms have been described for carbapenem resistance:86
• outer-membrane protein mutation causing decreased
permeability;

TABLE
180-4  Unusual, Emerging or Recently Reclassified Enterobacteriaceae Species Causing Human Infections
Species Human Infections
Escherichia albertii Diarrhea
Enterobacter Bacteremia, meningitis and
(Cronobacter) cerebrolysis, necrotizing
sakazakii enterocolitis
Klebsiella pneumoniae Rhinoscleroma, a chronic
subsp. granulomatous disease of
rhinoscleromatis upper airways
Klebsiella pneumoniae Ozena, a chronic rhinitis
subsp. ozaenae with atrophic nasal
mucosa
Klebsiella granulomatis Donovanosis (granuloma
inguinale)
Enterobacter Intra-abdominal abscesses,
hormaechei urinary tract infections
(UTIs), bacteremia
Edwardsiella tarda Gastrointestinal disease
(controversial),
bacteremia, wound
infections with abscesses
following trauma in
aquatic environment
Hafnia alvei Bacteremia, respiratory tract
infections associated with
abscesses, gastroenteritis
(controversial)
Kluyvera ascorbata UTIs, soft tissue infections,
intra-abdominal abscesses
Leclercia Bacteremia, wound
adecarboxylata infections, pneumonia
Pantoea agglomerans Bacteremia associated with
venous catheter, bone or
joint infections, soft tissue
infections with abscesses
Plesiomonas Water-borne gastrointestinal
shigelloides infections
ADH, antidiuretic hormone; H2S, hydrogen sulfide production; LDC, lysine decarboxylase; ODC, ornithine decarboxylase.
• production of carbapenemases (four classes encoded either on
chromosomes or plasmids are known; classes A and B are mostly
identified in Enterobacteriaceae); and
• altered affinity of the penicillin-binding proteins for
carbapenems.
IMI-1 was the first acquired carbapenemase identified in Serratia
marcescens isolated in Japan in 1991. KPC-1 was reported from a K.
pneumoniae isolate from the USA in 2001. Both molecules are
β-lactamase enzymes that hydrolyze β-lactams, conferring a high level
Chapter 180  Enterobacteriaceae 1577
Properties Risk Factors Recent References
Eae-positive strains, previously – Hyma et al.,6 Nimri70
misidentified as Hafnia alvei;
closely related to Shigella boydii
serotype 13
Described in 1980, phenotypically Neonatality, low birth Holý & Forsythe71
close to E. cloacae but sorbitol- weight
negative, specific yellow pigment,
α-lucosidase activity detected in
specific agar medium
Related to K. pneumoniae but Nutritional deficiency, Brisse et al.61
72
ONPG-negative; no acid production tropical and subtropical Botelho-Nevers et al.
with lactose; urease-, LDC- and areas
citrate-negative; K3 capsular type
Closely related to K. pneumoniae but LMIC Brisse et al.61
72
always urease-negative; K4 or rarely Botelho-Nevers et al.
K5
Previously named Southern Africa or Asia, O’Farrell et al.73
Calymmatobacterium granulomatis; Papua New Guinea,
uncultured in defined medium northern and central
Australia
Often misidentified as E. cloacae; a – Mezzatesta et al.74
frequent cause of nosocomial
infections
Described in 1965, resides in intestine Immunocompromised Leung et al.75
of cold-blooded animals and water.
LDC-, ODC-, H2S- and indole
reaction-positive; absence of
fermentation of most sugars except
glucose and maltose
Voges–Proskauer reaction-positive, Immunocompromised Janda & Abbott76
indole production-negative,
absence of fermentation of lactose,
motility at 22°C positive
Described in 1936, including four Neutropenia, underlying Isozaki et al.77
species (ascorbata, cryocrescens, malignancy
cochleae, georgiana) closely related
to E. coli but esculin-negative,
citrate- and malonate-positive,
growth in KCN
Described in 1962, closely related to Immunocompromised Keren et al.78
E. coli but lysine decarboxylase- status, neutropenia,
negative, malonate-positive; acid cirrhosis
production from arabitol and
cellobiose but not from adonitol
and sorbitol
Includes strains originally named Blood products, Cruz et al.79
Enterobacter agglomerans or intravenous fluids;
Erwinia herbicola; presents a wounds from thorns or
characteristic LDC-, ADH- and knives
ODC-negative profile
Oxidase-positive Consumption of seafood Salerno et al.80
or untreated water;
biliary tract diseases or
acute cholangitis
of resistance even to carbapenems and thus compromising treatment.87
Their dissemination has significantly increased over the last decade
among various Enterobacteriaceae, as the corresponding genes are
often located on transferable plasmids.
VACCINATION AND PREVENTION
Prevention of digestive infections depends on adequate treatment of
drinking water and food contamination control, which remains very
difficult in many places in the world.
1578 SECTION 8  Clinical Microbiology: Bacteria

Vaccination constitutes a major challenge for the control of fre-
quent Enterobacteriaceae infections specifically against digestive ill-
nesses affecting several millions of people globally.29 The emergence
and rapid dissemination of multidrug resistance and the challenges
faced by the development of novel agents makes vaccination an
attractive option. The key Enterobacteriaceae vaccination programs
currently being developed include vaccines against Salmonella Typhi88
and Shigellae,89,90 the rCTB-CF ETEC vaccine consisting of a combina-
tion of recombinant cholera toxin B subunit and formalin-inactive
ETEC bacteria,91 and EHEC vaccines to prevent human disease as well
as carriage of EHEC in animals.92
References available online at expertconsult.com.

KEY REFERENCES
Chaudhuri R.R., Henderson I.R.: The evolution of the Hazen T.H., Sahl J.W., Fraser C.M., et al.: Refining the
Escherichia coli phylogeny. Infect Genet Evol 2012;
12:214-226.
Croxen M.A., Finlay B.B.: Molecular mechanisms of Esch-
erichia coli pathogenicity. Nat Rev Microbiol 2010;
8:26-38.
Croxen M.A., Law R.J., Scholz R., et al.: Recent advances in
understanding enteric pathogenic Escherichia coli. Clin
Microbiol Rev 2013; 26:822-880.
Fàbrega A., Vila J.: Salmonella enterica serovar Typhimurium
skills to succeed in the host: virulence and regulation.
Clin Microbiol Rev 2013; 26:308-341.
Fleckenstein J.M., Munson G.M., Rasko D.A.: Enterotoxi-
genic Escherichia coli: orchestrated host engagement. Gut
Microbes 2013; 4:392-396.
Grad Y.H., Lipsitch M., Feldgarden M., et al.: Genomic epi-
demiology of the Escherichia coli O104:H4 outbreaks in
Europe, 2011. Proc Natl Acad Sci USA 2012; 109:
3065-3070.
pathovar paradigm via phylogenomics of the attaching
and effacing Escherichia coli. Proc Natl Acad Sci USA
2013; 110:12810-12815.
Holt K.E., Parkhill J., Mazzoni C.J., et al.: High-throughput
sequencing provides insights into genome variation and
evolution in Salmonella Typhi. Nat Genet 2008;
40:987-993.
Karmali M.A.: Host and pathogen determinants of
verocytotoxin-producing Escherichia coli-associated
hemolytic uremic syndrome. Kidney Int Suppl 2009;
112:S4-S7.
Lai Y., Rosenshine I., Leong J.M., et al.: Intimate host
attachment: enteropathogenic and enterohaemorrhagic
Escherichia coli. Cell Microbiol 2013; 15:1796-1808.
Papp-Wallace K.M., Endimiani A., Taracila M.A., et al.:
Carbapenems: past, present, and future. Antimicrob
Agents Chemother 2011; 55:4943-4960.
Reuter S., Connor T.R., Barquist L., et al.: Parallel indepen-
dent evolution of pathogenicity within the genus Yer-
sinia. Proc Natl Acad Sci USA 2014; 111(18):6768-6773.
Schroeder G.N., Hilbi H.: Molecular pathogenesis of Shi-
gella spp.: controlling host cell signaling, invasion, and
death by type III secretion. Clin Microbiol Rev 2008;
21:134-156.
Steyert S.R., Sahl J.W., Fraser C.M., et al.: Comparative
genomics and stx phage characterization of LEE-negative
Shiga toxin-producing Escherichia coli. Front Cell Infect
Microbiol 2012; 2:133.
Woerther P.L., Burdet C., Chachaty E., et al.: Trends in
human fecal carriage of extended-spectrum β-lactamases
in the community: toward the globalization of CTX-M.
Clin Microbiol Rev 2013; 26:744-758.

REFERENCES
1. Brenner D.J.: Enterobacteriaceae. In: Garrity G., ed.
Bergey’s manual of systematic bacteriology, vol. 2. New
York: Springer; 2006:587-607.
2. Janda J.M., Abbott S.L.: The Enterobacteria, 2nd ed.
Washington, DC: ASM Press; 2006.
3. Janda J.M.: New members of the family Enterobacte-
riaceae. In: Dworkin M., Falkow S., Rosenberg E., et al.,
eds. The prokaryotes: a handbook on the biology of bac-
teria, vol. 6, 3rd ed. New York: Springer; 2006:5-40
Proteobacteria: gamma subclass.
4. Toth I.K., Pritchard L., Birch P.R.: Comparative
genomics reveals what makes an enterobacterial plant
pathogen. Annu Rev Phytopathol 2006; 44:305-336.
5. Leimbach A., Hacker J., Dobrindt U.: E. coli as an all-
rounder: the thin line between commensalism and
pathogenicity. Curr Topics Microbiol Immunol 2013;
358:3-32.
6. Hyma K.E., Lacher D.W., Nelson A.M., et al.: Evolu-
tionary genetics of a new pathogenic Escherichia species:
Escherichia albertii and related Shigella boydii strains.
J Bacteriol 2005; 187:619-628.
7. Lagergård T., Bölin I., Lindholm L.: On the evolution
of the sexually transmitted bacteria Haemophilus
ducreyi and Klebsiella granulomatis. Ann N Y Acad Sci
2011; 1230:E1-E10.
8. Brisse S., Passet V., Grimont P.A.: Description of Kleb-
siella quasipneumoniae sp. nov., isolated from human
infections, with two subspecies, Klebsiella quasipneu-
moniae subsp. quasipneumoniae subsp. nov. and Kleb-
siella quasipneumoniae subsp. similipneumoniae subsp.
nov., and demonstration that Klebsiella singaporensis is
a junior heterotypic synonym of Klebsiella variicola. Int
J Syst Evol Microbiol 2014; 64:3146-3152.
9. Brady C., Cleenwerck I., Venter S., et al.: Taxonomic
evaluation of the genus Enterobacter based on multilo-
cus sequence analysis (MLSA): proposal to reclassify E.
nimipressuralis and E. amnigenus into Lelliottia gen.
nov. as Lelliottia nimipressuralis comb. nov. and Lelliot-
tia amnigena comb. nov., respectively, E. gergoviae and
E. pyrinus into Pluralibacter gen. nov. as Pluralibacter
gergoviae comb. nov. and Pluralibacter pyrinus comb.
nov., respectively, E. cowanii, E. radicincitans, E. oryzae
and E. arachidis into Kosakonia gen. nov. as Kosakonia
cowanii comb. nov., Kosakonia radicincitans comb.
nov., Kosakonia oryzae comb. nov. and Kosakonia ara-
chidis comb. nov., respectively, and E. turicensis, E.
helveticus and E. pulveris into Cronobacter as Crono-
bacter zurichensis nom. nov., Cronobacter helveticus
comb. nov. and Cronobacter pulveris comb. nov.,
respectively, and emended description of the genera
Enterobacter and Cronobacter. Syst Appl Microbiol 2013;
36:309-319.
10. Naum M., Brown E.W., Mason-Gamer R.J.: Is 16S
rDNA a reliable phylogenetic marker to characterize
relationships below the family level in the Enterobacte-
riaceae? J Mol Evol 2008; 66:630-642.
11. Bertelli C., Greub G.: Rapid bacterial genome sequenc-
ing: methods and applications in clinical microbiology.
Clin Microbiol Infect 2013; 19:803-813.
12. Chaudhuri R.R., Henderson I.R.: The evolution of the
Escherichia coli phylogeny. Infect Genet Evol 2012;
12:214-226.
13. Wirth T., Falush D., Lan R., et al.: Sex and virulence in
Escherichia coli. an evolutionary perspective. Mol
Microbiol 2006; 60:1136-1151.
14. Touchon M., Hoede C., Tenaillon O., et al.: Organised
genome dynamics in the Escherichia coli species results
in highly diverse adaptive paths. PLoS Genet 2009;
5:e1000344.
15. Lukjancenko O., Wassenaar T.M., Ussery D.W.: Com-
parison of 61 sequenced Escherichia coli genomes.
Microb Ecol 2010; 60:708-720.
16. Yang J., Nie H., Chen L., et al.: Revisiting the molecular
evolutionary history of Shigella spp. J Mol Evol 2007;
64:71-79.
17. Barrow G.I., Feltham R.K.A., eds. Cowan & Steel’s
Manual for the identification of medical bacteria, 3rd ed.
Cambridge: Cambridge University Press; 2003.
18. O’Hara C.M.: Manual and automated instrumentation
for identification of Enterobacteriaceae and other
aerobic Gram-negative bacilli. Clin Microbiol Rev 2005;
18:147-162.
19. Dingle T.C., Butler-Wu S.M.: Maldi-tof mass spec-
trometry for microorganism identification. Clin Lab
Med 2013; 33:589-609.
20. Clerc O., Prod’hom G., Vogne C., et al.: Impact of
matrix-assisted laser desorption ionization time-of-
flight mass spectrometry on the clinical management of
patients with Gram-negative bacteremia: a prospective
observational study. Clin Infect Dis 2013; 56:1101-
1107.
21. Guibourdenche M., Roggentin P., Mikoleit M., et al.:
Supplement 2003-2007 (No. 47) to the White-
Kauffmann-Le Minor scheme. Res Microbiol 2010;
161:26-29.
22. Achtman M., Wain J., Weill F.X., et al.: Multilocus
sequence typing as a replacement for serotyping in Sal-
monella enterica. PLoS Pathog 2012; 8(6):e1002776.
23. Persson S., Olsen K.E., Scheutz F., et al.: A method for
fast and simple detection of major diarrhoeagenic Esch-
erichia coli in the routine diagnostic laboratory. Clin
Microbiol Infect 2007; 13:516-524.
24. de Boer R.F., Ott A., Kesztyüs B., et al.: Improved detec-
tion of five major gastrointestinal pathogens by use of
a molecular screening approach. J Clin Microbiol 2010;
48:4140-4146.
25. Sansonetti P.J.: To be or not to be a pathogen: that is
the mucosally relevant question. Mucosal Immunol
2011; 4:8-14.
26. Eloe-Fadrosh E.A., Rasko D.A.: The human microbi-
ome: from symbiosis to pathogenesis. Annu Rev Med
2013; 64:145-163.
27. Dobrindt U., Hochhut B., Hentschel U., et al.: Genomic
islands in pathogenic and environmental microorgan-
isms. Nat Rev Microbiol 2004; 2:414-424.
28. Croxen M.A., Finlay B.B.: Molecular mechanisms of
Escherichia coli pathogenicity. Nat Rev Microbiol 2010;
8:26-38.
29. Croxen M.A., Law R.J., Scholz R., et al.: Recent
advances in understanding enteric pathogenic Esche-
richia coli. Clin Microbiol Rev 2013; 26:822-880.
30. Kotloff K.L., Nataro J.P., Blackwelder W.C., et al.:
Burden and aetiology of diarrhoeal disease in infants
and young children in developing countries (the Global
Enteric Multicenter Study, GEMS): a prospective, case-
control study. Lancet 2013; 382(9888):209-222.
31. Shah N., DuPont H.L., Ramsey D.J.: Global etiology of
travelers’ diarrhea: systematic review from 1973 to the
present. Am J Trop Med Hyg 2009; 80:609-614.
32. Isidean S.D., Riddle M.S., Savarino S.J., et al.: A system-
atic review of ETEC epidemiology focusing on coloni-
zation factor and toxin expression. Vaccine 2011;
29:6167-6178.
33. Fleckenstein J.M., Munson G.M., Rasko D.A.: Entero-
toxigenic Escherichia coli: orchestrated host engage-
ment. Gut Microbes 2013; 4:392-396.
34. Lai Y., Rosenshine I., Leong J.M., et al.: Intimate host
attachment: enteropathogenic and enterohaemorrhagic
Escherichia coli. Cell Microbiol 2013; 15:1796-1808.
35. Hazen T.H., Sahl J.W., Fraser C.M., et al.: Refining the
pathovar paradigm via phylogenomics of the attaching
and effacing Escherichia coli. Proc Natl Acad Sci USA
2013; 110:12810-12815.
36. Karmali M.A.: Host and pathogen determinants of
verocytotoxin-producing Escherichia coli-associated
hemolytic uremic syndrome. Kidney Int Suppl 2009;
112:S4-S7.
37. Scheutz F., Teel L.D., Beutin L., et al.: Multicenter
evaluation of a sequence-based protocol for subtyping
Shiga toxins and standardizing Stx nomenclature. J Clin
Microbiol 2012; 50:2951-2963.
38. Steyert S.R., Sahl J.W., Fraser C.M., et al.: Comparative
genomics and stx phage characterization of LEE-
negative Shiga toxin-producing Escherichia coli. Front
Cell Infect Microbiol 2012; 2:133.
39. Nataro J.P., Kaper J.B., Robins-Browne R., et al.: Pat-
terns of adherence of diarrheagenic Escherichia coli to
HEp-2 cells. Pediatr Infect Dis J 1987; 6:829-831.
40. Nataro J.P., Mai V., Johnson J., et al.: Diarrheagenic
Escherichia coli infection in Baltimore, Maryland, and
New Haven, Connecticut. Clin Infect Dis 2006; 43:402-
407.
41. Wilson A., Evans J., Chart H., et al.: Characterisation of
strains of enteroaggregative Escherichia coli isolated
Chapter 180  Enterobacteriaceae 1578.e1
during the infectious intestinal disease study in
England. Eur J Epidemiol 2001; 17:1125-1130.
42. Ruiz-Perez F., Nataro J.P.: Bacterial serine proteases
secreted by the autotransporter pathway: classification,
specificity, and role in virulence. Cell Mol Life Sci 2014;
71:745-770.
43. Morin N., Santiago A.E., Ernst R.K., et al.: Character-
ization of the AggR regulon in enteroaggregative Esch-
erichia coli. Infect Immun 2013; 81:122-132.
44. Grad Y.H., Lipsitch M., Feldgarden M., et al.: Genomic
epidemiology of the Escherichia coli O104:H4 outbreaks
in Europe, 2011. Proc Natl Acad Sci USA 2012;
109:3065-3070.
45. Labigne-Roussel A.F., Lark D., Schoolnik G., et al.:
Cloning and expression of an afimbrial adhesin (AFA-I)
responsible for P blood group-independent, mannose-
resistant hemagglutination from a pyelonephritic Esch-
erichia coli. strain. Infect Immun 1984; 46:251-259.
46. Butler T.: Haemolytic uraemic syndrome during shigel-
losis. Trans R Soc Trop Med Hyg 2012; 106:395-399.
47. Schroeder G.N., Hilbi H.: Molecular pathogenesis of
Shigella spp.: controlling host cell signaling, invasion,
and death by type III secretion. Clin Microbiol Rev 2008;
21:134-156.
48. Holt K.E., Parkhill J., Mazzoni C.J., et al.: High-
throughput sequencing provides insights into genome
variation and evolution in Salmonella Typhi. Nat Genet
2008; 40:987-993.
49. Majowicz S.E., Musto J., Scallan E., et al.: The global
burden of nontyphoidal Salmonella gastroenteritis. Clin
Infect Dis 2010; 50:882e9.
50. Grassl G.A., Finlay B.B.: Pathogenesis of enteric Salmo-
nella infections. Curr Opin Gastroenterol 2008; 24:22-
26.
51. Fàbrega A., Vila J.: Salmonella enterica serovar
Typhimurium skills to succeed in the host: virulence
and regulation. Clin Microbiol Rev 2013; 26:308-341.
52. Malik-Kale P., Jolly C.E., Lathrop S., et al.: O. Salmo-
nella – at home in the host cell. Front Microbiol 2011;
2:125.
53. Steele-Mortimer O.: The Salmonella-containing
vacuole: moving with the times. Curr Opin Microbiol
2008; 11:38-45.
54. Drancourt M.: Plague in the genomic area. Clin Micro-
biol Infect 2012; 18(3):224-230.
55. Ternhag A., Torner A., Svensson A., et al.: Short- and
long-term effects of bacterial gastrointestinal infections.
Emerg Infect Dis 2008; 14:143-148.
56. Fàbrega A., Vila J.: Yersinia enterocolitica: pathogenesis,
virulence and antimicrobial resistance. Enferm Infecc
Microbiol Clin 2012; 30:24-32.
57. Reuter S., Connor T.R., Barquist L., et al.: Parallel inde-
pendent evolution of pathogenicity within the genus
Yersinia. Proc Natl Acad Sci USA 2014; 111(18):6768-
6773.
58. Nielubowicz G.R., Mobley H.L.: Host-pathogen inter-
actions in urinary tract infection. Nat Rev Urol 2010;
7(8):430-441.
59. Mulvey M.A., Schilling J.D., Martinez J.J., et al.: Bad
bugs and beleaguered bladders: interplay between uro-
pathogenic Escherichia coli and innate host defenses.
Proc Natl Acad Sci USA 2000; 97:8829-8835.
60. Ronald A.: The etiology of urinary tract infection: tra-
ditional and emerging pathogens. Dis Mon 2003; 49:71-
82.
61. Brisse S., Fevre C., Passet S., et al.: Virulent clones of
Klebsiella pneumoniae: identification and evolutionary
scenario based on genomic and phenotypic character-
ization. PLoS ONE 2009; 4:e4982.
62. O’Hara C.M.F., Brenner W., Miller J.M.: Classification,
identification, and clinical significance of Proteus, Prov-
idencia, and Morganella. Clin Microbiol Rev 2000;
13:534-546.
63. Remington L.T., Sligl W.I.: Community-acquired
pneumonia. Curr Opin Pulm Med 2014; 20:215-224.
64. Barichello T., Fagundes G.D., Generoso J.S., et al.:
Pathophysiology of neonatal acute bacterial meningitis.
J Med Microbiol 2013; 62:1781-1789.
65. Houdouin V., Bonacorsi S., Bidet P., et al.: Association
between mortality of Escherichia coli meningitis in
young infants and non-virulent clonal groups of
strains. Clin Microbiol Infect 2008; 14:685-690.
1578.e2 SECTION 8  Clinical Microbiology: Bacteria
66. Corbett D., Roberts I.S.: Capsular polysaccharides in
Escherichia coli. Adv Appl Microbiol 2008; 65:1-26.
67. Rodrigues J., Rocha D., Santos F., et al.: Neonatal Citro-
bacter koseri meningitis: report of four cases. Case Rep
Pediatr 2014; 2014:195204.
68. Wilson J., Elgohari S., Livermore D.M., et al.: Trends
among pathogens reported as causing bacteraemia in
England, 2004-2008. Clin Microbiol Infect 2011; 17:451-
458.
69. Darton T.C., Blohmke C.J., Pollard A.J.: Typhoid epi-
demiology, diagnostics and the human challenge
model. Curr Opin Gastroenterol 2014; 30:7-17.
70. Nimri L.F.: Escherichia albertii, a newly emerging
enteric pathogen with poorly defined properties. Diagn
Microbiol Infect Dis 2013; 77:91-95.
71. Holý O., Forsythe S.: Cronobacter spp. as emerging
causes of healthcare-associated infection. J Hosp Infect
2014; 86:169-177.
72. Botelho-Nevers E., Gouriet F., Lepidi H., et al.: Chronic
nasal infection caused by Klebsiella rhinoscleromatis or
Klebsiella ozaenae: two forgotten infectious diseases. Int
J Infect Dis 2007; 11:423-429.
73. O’Farrell N.: European guideline for the management
of donovanosis, 2010. IUSTI/WHO European STD
guidelines Editorial Board. Int J STD AIDS 2010;
21:609-610.
74. Mezzatesta M.L., Gona F., Stefani S.: Enterobacter
cloacae complex: clinical impact and emerging antibi-
otic resistance. Future Microbiol 2012; 7:887-902.
75. Leung K.Y., Siame B.A., Tenkink B.J., et al.: Edwardsi-
ella tarda – virulence mechanisms of an emerging gas-
troenteritis pathogen. Microbes Infect 2012; 14:26-34.
76. Janda J.M., Abbott S.L.: The genus Hafnia: from soup
to nuts. Clin Microbiol Rev 2006; 19:12-18.
77. Isozaki A., Shirai K., Mimura S., et al.: A case of urinary
tract infection caused by Kluyvera ascorbata in an
infant: case report and review of the literature. J Infect
Chemother 2010; 16:436-438.
78. Keren Y., Keshet D., Eidelman M., et al.: Is Leclercia
adecarboxylata a new and unfamiliar marine pathogen?
J Clin Microbiol 2014; 52:1775-1776.
79. Cruz A.T., Cazacu A.C., Allen C.H.: Pantoea agglomer-
ans, a plant pathogen causing human disease. J Clin
Microbiol 2007; 45:1989-1992.
80. Salerno A., Cižnár I., Krovacek K., et al.: Phenotypic
characterization and putative virulence factors of
human, animal and environmental isolates of Plesiomo-
nas shigelloides. Folia Microbiol 2010; 55:641-647.
81. Mahlen S.D.: Serratia infections: from military experi-
ments to current practice. Clin Microbiol Rev 2011;
24:755-791.
82. Ghafourian S., Sadeghifard N., Soheili S., et al.:
Extended spectrum betalactamases: definition, classifi-
cation and epidemiology. Curr Issues Mol Biol 2014;
17:11-22.
83. Haeggman S., Lofdahl S., Paauw A., et al.: Diversity and
evolution of the class A chromosomal beta-lactamase
gene in Klebsiella pneumoniae. Antimicrob Agents Che-
mother 2004; 48:2400-2408.
84. Nicolas-Chanoine M.H., Blanco J., Leflon-Guibout V.,
et al.: Intercontinental emergence of Escherichia coli
clone O25:H4-ST131 producing CTX-M-15. J Antimi-
crob Chemother 2008; 61:273-281.
85. Woerther P.L., Burdet C., Chachaty E., et al.: Trends
in human fecal carriage of extended-spectrum β-
lactamases in the community: toward the globalization
of CTX-M. Clin Microbiol Rev 2013; 26:744-758.
86. Papp-Wallace K.M., Endimiani A., Taracila M.A., et al.:
Carbapenems: past, present, and future. Antimicrob
Agents Chemother 2011; 55:4943-4960.
87. Perez F., Van Duin D.: Carbapenem-resistant Entero-
bacteriaceae: a menace to our most vulnerable
patients. Cleve Clin J Med 2013; 80:225-233.
88. Anwar E., Goldberg E., Fraser A., et al.: Vaccines for
preventing typhoid fever. Cochrane Database Syst Rev
2014; (1):CD001261.
89. Levine M.M., Kotloff K.L., Barry E.M., et al.: Clinical
trials of Shigella vaccines: two steps forward and one
step back on a long, hard road. Nat Rev Microbiol 2007;
5:540-553.
90. Camacho A.I., Irache J.M., Gamazo C.: Recent progress
towards development of a Shigella vaccine. Expert Rev
Vaccines 2013; 12:43-55.
91. Walker R.I., Steele D., Aguado T.: Analysis of strategies
to successfully vaccinate infants in developing countries
against enterotoxigenic E. coli (ETEC) disease. Vaccine
2007; 25:2545-2566.
92. Matthews L., Reeve R., Gally D.L., et al.: Predicting the
public health benefit of vaccinating cattle against Esch-
erichia coli O157. Proc Natl Acad Sci USA 2013;
110:16265-16270.