TAXONOMY Back to top
Taxonomically, the Enterobacteriaceae remain
a challenging group to define on a
phylogenetic level, as sequence divergence within
the 16S rRNA gene, the most commonly
used molecular identification tool, is generally
insufficient to accurately discriminate between
closely related members of this family (34,
75, 101). Sequencing of other housekeeping
genes (i.e., dnaJ, rpoB, gyrB, etc.) often
yields different phylogenetic classifications
depending on their discriminatory powers.
Moreover, 16S rRNA and housekeeping gene
sequencing results may not correspond with data
generated by the gold standard, DNA-DNA
hybridization, or with multilocus enzyme
electrophoresis. For instance, Salmonella bongori is
a distinct species by dnaJsequencing,
DNA-DNA hybridization, and multilocus enzyme
electrophoresis, while tuf, atpD, and 23S
rRNA sequences place S. bongori as a subspecies
within Salmonella enterica (101).
Adoption of a cohesive approach to classification would
provide some stability to the taxonomy of this
family. Gevers et al. (47) have suggested
using multilocus sequence analysis with a set of
housekeeping genes selected on the criteria
that (i) they are useful for all strains within
the taxon being studied, (ii) they present as
single copies, and (iii) recombination would not
convey a selective advantage. The
continuous reclassification of organisms,
particularly those with clinical significance, can
confuse clinicians, which could have serious
consequences for patients.
A number of additions or taxonomic changes within
the Enterobacteriaceae have occurred in
the interval since the ninth edition of this Manual;
however, only two are of clinical
importance. A new species of Morganella, M.
psychrotolerans, has been reported from coldsmoked
tuna and has been implicated in an outbreak of histamine
(scombroid) poisoning
(38). Unlike Morganella morganii strains,
which produce histamine from the histidine present
in seafood at temperatures above normal
refrigeration (7 to 10°C), M.
psychrotoleransproduces toxic concentrations of histidine between 0 and 5°C, so
even
properly refrigerated product could cause disease.
The second change of clinical significance
is the move of Enterobacter sakazakii, a
species recognized to be heterogeneous at the DNA
level, to a newly created genus, Cronobacter, with
the subsequent creation of four additional
species and an unnamed genomospecies (69).
Although not common,E. sakazakii is well
recognized by infectious disease clinicians as a
cause of severe disease (meningitis,
necrotizing colitis, and bacteremia) with
relatively high mortality rates in neonates. At this
time, species ofCronobacter cannot be
routinely identified in clinical laboratories due to lack
of discriminatory biochemical tests.E.
sakazakii remains an acceptable, validly published
name, but PubMed queries indicate that, based on
usage,Cronobacter sakazakii is gaining
acceptance in the literature. The best options
available for reporting this organism would be
to continue to use E. sakazakii or use Cronobacter
sakazakii complex until such time as the
species are readily identifiable by routinely used
methodologies. When laboratories adopt
taxonomic changes, the older epithet should be
included in parentheses following the new
name for a minimum of 6 months (11).
For infrequently encountered pathogenic organisms,
6 months may be insufficient for physicians to
become familiar with the taxonomic change. It
is the laboratory’s responsibility to ensure that
clinicians are aware of the significance of the
organism being reported in these cases.
Other genera, including Pantoea (Enterobacter)
agglomerans and Enterobacter
cloacae remain heterogeneous at the DNA level, but because they are not
phenotypically
separable, genomic groups residing within these
species remain unnamed (46, 62). Hafnia
alvei is composed of two distinct DNA hybridization groups, and studies
are in progress to
name DNA hybridization group 2 (78).
All the other new species of Enterobacteriaceae
covered in this chapter that have been added
to or transferred between existing genera are
included in Tables 1 and 2. The genera listed in
the last two columns of Table 2are not isolated from human clinical specimens or are isolated
but may not be significant, and most of these taxa
will be unfamiliar to clinical
microbiologists. Genera belonging to the Enterobacteriaceae
that contain one or more
species isolated exclusively from insects, plants,
fish, marine animals, or birds are not
included in the tables or text of this chapter;
however, information on them is available
elsewhere (72).
DESCRIPTION OF THE GENERA Back to top
Members belonging to the family Enterobacteriaceae
are gram-negative, facultative
anaerobic rods or coccobacilli ranging from 0.3 to
1.0 μm wide to 0.6 to 6.0 μm
long. Serratia marcescens subsp. sakuensis
is the only reportedly spore-forming organism in
this family (1). Prototrophic strains
grow readily on ordinary media. Among these genera,
auxotrophic strains from clinical specimens are
rare. However, cysteine-requiring urinary
isolates of Klebsiella pneumoniae, which grow
as pinpoint colonies on routine media, do
occur. When encountered, these strains require
supplementation of biochemical media or
commercial identification systems with 0.63 mM
cysteine for accurate identification. K.
granulomatis is culturable only by cell culture techniques.
Of the organisms in Tables 1 and 2 isolated
from human specimens, all Klebsiella,
Leminorella, Moellerella, Tatumella, and Enterobacter asburiae strains are
nonmotile,
although any strain of any genus may be nonmotile
and recent data for E. asburiae indicate
some strains may be motile (64).
Some strains of Serratia plymuthicamay not grow at 37°C,
but most other members of the genera discussed in
this chapter grow well between 25°C
and 37°C. Only Klebsiella and Raoultella
spp. are encapsulated, but strains from all genera
may grow as mucoid or rough colonies. Six genera
produce pigment. Some strains of S.
marcescens and most Serratia rubidaea and S. plymuthica strains
produce a red pigment,
prodigiosin, which may appear throughout the
entire colony or only as a red center or
margin. Yellow-pigment-producing organisms include
environmentalEnterobacter species (E.
pulveris and E. turicensis [133, 134])
and most strains of Cronobacter (Enterobacter)
sakazakii, P. agglomerans, Leclercia adecarboxylata, and Photorhabdus
asymbiotica. Yellow
pigment may be enhanced by incubation at 25°C;
weak pigment producers may only be
detected by observing growth placed on a swab or
filter paper. Photorhabdus
luminescens and P. asymbiotica cultures are luminescent, giving a
visible glow in a darkroom
after 5 minutes. Serratia odorifera, as
indicated by its name, and some Cedecea spp.
produce a pungent (potato-like) odor due to the
production of alkyl-methoxypyrazines (50).
Species of Proteusand Providencia oxidatively
deaminate α-amino acids, producing pyruvic
acids. L-Phenylalanine deamination yields a green
color when ferric chloride is added;
however, deamination of dl-tryptophan
produces the deep reddish brown pigment often seen
in media inoculated with these organisms without
the addition of ferric chloride
(114). Proteus species also produce swarmer
cells, i.e., elongated forms created when cells
fail to septate or divide. These cells, which are
profusely covered with flagella, act in concert
to produce swarming motility on solid media (12).
Plesiomonas shigelloides organisms are also gram-negative, facultative
anaerobes growing
as straight rods of sizes similar to those of
other Enterobacteriaceae. However, unlike
other Enterobacteriaceae, P. shigelloidesstrains
are oxidase positive, do not produce gas
from glucose (Enterobacteriaceae are
variable), and are susceptible to O/129 vibriostatic
agent (2,4-diamino-6,7-diisopropylpteridine). Both
P. shigelloides and enterobacteria grow at
similar salt concentrations (0 to 5%) and pH
ranges (4.0 to 8.0).
EPIDEMIOLOGY, TRANSMISSION, AND CLINICAL
SIGNIFICANCE Back to top
The Enterobacteriaceae are widely
distributed throughout the environment (Tables 1 and 2).
Many species of the genera in Table 1 are commonly recognized pathogens, consistently
ranking among the top 10 organisms seen in health
care-associated infections (42, 61, 94).
Between 2002 and 2004, 7 of the 10 most common
gram-negative organisms isolated from
respiratory tract, urinary tract, and bloodstream
infections from intensive care unit patients
in the United States were K. pneumoniae (15%),
E. cloacae (9%), S.
marcescens(6%), Enterobacter aerogenes (4%), Proteus mirabilis (4%),
Klebsiella
oxytoca (3%), and Citrobacter freundii (2%). Between 2006 and 2007,
the National
Healthcare Safety Network (formerly National
Nosocomial Infections Surveillance System)
reported K. pneumoniae, Enterobacter spp.,
and K. oxytoca among the top 10 most
frequently isolated health care-associated
infections, making up 6%, 5%, and 2% of the
isolates, respectively (61).
Notably, Enterobacter was reported as the third most common
isolate from ventilator-associated pneumonia.
European data on infection rates, which are
similar to those above, can be found on the
following
websites: http://ecdc.europa.eu/en/Publications/AER_report.aspx andhttp://www.hpa.org.uk
/web/HPAweb&HPAwebStandard/HPAweb_C/1201767919826. Pediatric patient data collected
in 2004 from three continents (North America,
Latin America, and Europe) indicated
that Klebsiellaspp., Enterobacter spp.,
P. mirabilis, and Serratia spp. ranked 4th, 7th, 11th,
and 12th, respectively, in the top 15 most
frequently isolated organisms (42).
Both Klebsiella and Enterobacter were
more prevalent (3rd to 5th versus 10th) in North and
Latin America than in Europe. In all geographic
areas there was a twofold decrease in
prevalence for both Klebsiella and Enterobacter
species in children older than 1 year.
Klebsiella
and Raoultella
Klebsiella
is carried in the nasopharynx and the bowel; however, feces are
arguably the most
significant
source of patient infections (98). Recent data indicate that
K.
pneumoniae
bloodstream infection isolation rates are 1.5 times greater during
the warmest
months
of the year (7). These rates most likely reflect increased fecal carriage in
humans,
which
in turn is a reflection of increased organisms in the environment during warm
months.
This
has important implications since colonized patients have a fourfold-increased
risk of
infection
over noncarriers. Similar isolation rate increases were not seen
with
Enterobacter or Serratia. K. pneumoniae,primarily strains
with capsular type K1, have
emerged
as an important cause of community-acquired pyogenic liver abscess worldwide
(19, 91, 117, 146).
The majority of patients with Klebsiella pyogenic liver abscess are
Asian
males,
50 to 60 years of age, who present primarily with a right-lobe, solitary,
monomicrobial
abscess. Studies by Brisse et al. (19)
indicate that pyogenic liver abscessassociated
K1
isolates belong to a clonal complex designated CC23K1, whereas K1 strains
associated
with severe pneumonia isolated from the respiratory tract and bloodstream
infections
belong to a different complex, CC82K1. magA (mucoviscosity-associated
gene),
which
resides within the cps (capsular polysaccharide synthesis) operon of all
K1 isolates, is
not
an indicator for primary liver abscess. Conversely, the gene allS (an
activator of the
allantoin
regulon) has been detected, to date, only in K1 strains isolated from primary
liver
abscesses
(19, 148). allS may confer an advantage in these strains because
levels of
allantoin,
which can serve as a carbon source, are elevated in non-insulin-dependent
diabetics
(148). Primary liver abscess K1 strains also appear to be separable by
conventional
biochemical
methods (see “Identification” below). Mouse lethality studies show
that
Klebsiellaliver abscess isolates that possessed both the
hypermucoviscosity phenotype
and
the rmpA (regulator of mucoid phenotype) gene, regardless of the K type,
have a 50%
lethal
dose of <102 CFU versus >5 × 107 CFU for type K1 or K2 urine isolates
that were not
hypermucoviscous
and without rmpA or aerobactin genes (148).Klebsiella
rhinoscleromatis
and Klebsiella ozaenae are clones of K. pneumoniae (not
subspecies) that
have
adapted to cause specific chronic infectious diseases, i.e., rhinoscleroma and
atrophic
rhinitis
(ozena), respectively (19). Neither organism is isolated from the environment or the
intestinal
tract, and both have lost the ability to utilize substrates involved in plant
product
degradation
pathways; however, isolates from bloodstream, urinary tract, and other
infection
sites
indicate that K. ozaenae is more diverse in its ability to cause disease
than K.
rhinoscleromatis.
Atrophic rhinitis is restricted to the nose, but rhinoscleroma may spread to
the
trachea and larynx (73). Both of these tissue-destructive diseases occur more frequently
in
tropical areas of the world and are spread by person-to-person transmission,
although
prolonged
contact with persons producing airborne nasal secretions is required. A recent
retrospective
study has updated the epidemiological and clinical features of rhinoscleroma
(35). Klebsiella
granulomatis, also a clone (not species) ofK. pneumoniae, is the
agent of
donovanosis
or granuloma inguinale, a disease characterized by chronic genital ulcers
(19, 56).
It also occurs predominantly in tropical countries and is thought to be
sexually
transmitted,
with humans as the only known reservoir. K. oxytoca strains carrying a
chromosomally
encoded heat-labile cytotoxin have been increasingly recognized as a cause
of
antibiotic-associated hemorrhagic colitis (49, 150). Antibiotic-associated
hemorrhagic
colitis,
associated with the use of β-lactam antibiotics, is self-limiting and resolves
spontaneously
with the withdrawal of the contributing antibiotic. It differs from Clostridium
difficiledisease
in that there is no pseudomembrane formation and K. oxytoca antibioticassociated
hemorrhagic
colitis stools are bloody. The majority of isolations reported to date
for Klebsiella
variicola are from sterile sites, mainly blood and urine (6, 121).
Recent isolates
of
this organism (three of five from urine), identified by rpoBsequencing
reported from a
Brazilian
study, provide increased evidence that this organism is a human pathogen
(6). Raoultella
planticola and Raoultella terrigena share pathogenicity
characteristics with K.
pneumoniae
and are difficult to distinguish from it biochemically without
special tests. In
European
studies, 3.5% to 19% of clinical strains initially identified as Klebsiella were
R.
planticola,
while in U.S. and Brazilian surveys of 436 and 122 strains,
respectively, only one
isolate
in each was identified as R. planticola, indicating that prevalence of
these species may
differ
geographically (6, 113, 142).
Enterobacter,
Pantoea, and Erwinia
Nosocomial
Enterobacter colonization and infection are frequently associated with
contaminated
medical devices and instrumentation; however, Enterobacter spp. are
commonly
consumed in foods, so endogenous sources should also be considered
(73). Enterobacter
ludwigii and all Enterobacter hormaechei subspecies are isolated
from a
variety
of human sources including blood (63, 65). A
nonhuman source for E. ludwigii has
not
been reported, but plants appear to be the natural habitat of E. hormaechei.
Other
environmental
organisms including a Pantoea dispersa-like organism, Pantoea
ananatis,
and Erwinia persicina have been reported from blood or
urine (33, 105, 126). P.
agglomerans
is the species of Pantoea most commonly isolated from
humans. Sporadic
infections
are associated with penetrating trauma by objects contaminated with soil or
vegetation
resulting in soft tissue infections, septic arthritis, or osteomyelitis,
whereas health
care-associated
infections and outbreaks often involve contaminated intravenous fluid,
parenteral
nutrition, or other administered fluids.P. agglomerans infections in
children, most
of
whom have severe underlying conditions, are predominantly polymicrobic and
therefore of
questionable
significance even when specimens are from sterile sites (30).
Serratia
Serratia
spp. are notorious health care-associated pathogens and
colonizers. Transmission is
predominately
from person to person, but medical apparatuses, intravenous fluids, and other
solutions
are often implicated as well (73). Indwelling catheters,
particularly for urinary tract
infections,
serve as a primary reservoir for transmission via hospital personnel. In
children,
the
gastrointestinal tract is a common source of infections. Outbreaks transmitted
by hand
are
often insidious, occurring over long periods of time, and may subside and peak
a number
of
times before recognition and infection control efforts can contain them.
Pigment
production
in S. marcescens appears to be a marker that the strain is environmental
in origin
and
of low virulence (9). Community-acquired infections are rare except for S.
marcescens
contact lens-induced acute red eye (67).
Most of the other species
of Serratia
have also been isolated from humans, in whom they too are usually
transients or
cause
opportunistic infections.
Citrobacter
Citrobacters
are primarily inhabitants of the intestinal tract, and their presence in the
environment
may reflect fecal excretion by humans and animals; the natural habitat of some
newer
Citrobacter species is unknown. The three species most commonly involved
in hospital
infections
are C. freundii, C. koseri, and C. braakii; patients are usually
older adults (≥65
years),
more often males, and the urinary tract is the most common site of infection
(90, 123).
One-third to one-half of Citrobacter infections, including septicemias,
are
polymicrobic
and are associated with higher mortality rates (18% to 50%) or longer hospital
stays
(73, 90, 123). Meningitis is almost exclusively associated with C. koseri and
involves
children
<2 months of age, with the highest onset rates recorded in neonates with a
mean
age
of 7 days (73). Brain abscesses occur in 75% of infected infants, and
neurological
defects
are common sequelae in surviving infants. The most prominent risk factor is
prior
colonization;
during outbreaks, colonization rates of 27% have been noted versus a normal
rate
of <1% (93,143). Person-to-person spread by hospital personnel and, less often,
from
mother
to offspring is the most likely source of infections. Sampling of inanimate or
environmental
reservoirs in hospitals usually fails to
yieldCitrobacter.
Other Citrobacter species including C. gillenii and C.
murliniae have been
found
in human clinical, animal, food, and environmental specimens (16, 18).
The role of
many
Citrobacter spp. in human infections is unclear because reports in the
literature are
insufficient
to determine either clinical significance in humans or potential reservoirs for
infection.
C. rodentium causes murine colonic hyperplasia, which is self-limiting
in adult mice
but
causes significant morbidity and mortality in infant mice in mouse colony
outbreaks (95).
Proteus,
Providencia, and Morganella
Members
of Proteus, Providencia, and Morganella genera are widespread in
the environment,
are
normal inhabitants of the gut, and are relatively common in clinical
laboratories,
especially
P. mirabilis. In a large six-year population-based survey of Proteeae,
85% (4,290
of
5,047) of isolates were community acquired, although providenciae were more
likely to be
acquired
in nursing homes (89). Females (69%) and the elderly (median age, 70) were at
highest
risk of infection, and the most common specimen sources were urine (86%), soft
tissues
(7%), blood (3%), miscellaneous fluids (2%), and the respiratory tract (2%). P.
mirabilis,predominantly
from urine, was the most frequently isolated agent (77%). Non-P.
mirabilis
species (primarilyProteus vulgaris) were isolated from
wounds/soft tissue more
often
than urine. Of the Providencia species, P. stuartii was isolated
twice as often as other
species.
P. mirabilis, Proteus penneri, Morganella, and Providencia
alcalifaciens are seen in
diarrheal
stools with greater frequency than in normal stools, leading to speculation
that they
may
cause diarrhea. Some strains of P. alcalifaciens are invasive in HEp-2
cell assays and
elicit
diarrhea in the RITARD (reversible intestinal-tie adult rabbit diarrhea) model,
while
other
strains isolated in pure culture or in large numbers from diarrheal stools fail
to invade
cell
lines (4, 76). However, a number of noninvasive P. alcalifaciens strains
have been shown
to
be nonadherent to cell lines in vitro, which may provide insight to their
inability to invade
(86).
Yoh et al. (147) used a specialized medium to isolate nine strains ofProvidencia
rettgeri
from 130 persons with traveler’s diarrhea; eight of these strains
were invasive in
Caco-2
cells, indicating their potential for virulence in humans. Notably, vomiting
was
present
in five P. rettgeri cases but was not seen in patients from whom other
providenciae
were
isolated.
Hafnia
Few
systematic investigations regarding the ecological distribution of Hafnia have
been
published,
although it is a common inhabitant of the gastrointestinal tract of mammals,
birds,
cold-blooded animals, fish, and insects (74). Isolation
from consumables, especially
meats,
is not uncommon, and presumably its presence indicates prior contact of the
item
with
feces. Hafnia alvei has been linked to gastrointestinal disease,
although putative
virulence
characteristics have not been demonstrated (3, 120).
However, a toxigenic strain
producing
a cytopathic effect on Vero cells indistinguishable from Shiga toxin, but not
neutralized
by anti-Shiga toxin antibody, has been reported (29).
Although seen infrequently
in
extraintestinal disease, such infections occur both in healthy and in
immunocompromised
patients
with monomicrobial infection rates varying from 12 to 75%; the correlation with
disease
increases when the organism is isolated in pure culture and in high numbers
(74). Hafnia
appears to have a predilection for the biliary tree and may produce
abscesses at
the
site of infection (118).
Plesiomonas
shigelloides and Edwardsiella
tarda
Plesiomonas
is isolated from a wide range of mammals, birds, fish,
water-dwelling reptiles,
and
amphibians, but with the possible exception of cats, there is no evidence it
plays a role
in
diarrheal disease in any of these species (70).
Human P. shigelloides infections are
associated
with living in or travel to tropical countries and/or a history of seafood
consumption;
both acute diarrhea and chronic diarrhea episodes of >2 weeks have been
reported
(77, 84). Most infections are self-limited with hospitalization required
only for
severe
infections and/or in patients with underlying conditions. Plesiomonas typically
presents
as a secretory (watery) diarrhea, and although it can also manifest as a
dysenteric
(bloody)
diarrhea, the secretory form is seen three times more frequently (144).
Possible
diarrheal
virulence determinants in some isolates include cholera-like, heat-stable, and
heatlabile
toxins,
while other strains have been shown to invade and multiply within human
gastrointestinal
cells (71, 139). The somatic antigen of Plesiomonas may also play a role
in
pathogenicity,
since the gene encoding the most common type, O17, shares almost complete
identity
with the form 1 (smooth) antigen gene of Shigella sonnei (25).
Wound infections
associated
with water contact are not encountered with plesiomonads despite their aquatic
reservoir.
Plesiomonas bacteremia, which is rare and usually polymicrobic, is
generally
community
acquired, and major risk factors include biliary tract disease and advanced age
(>75
years) (145). Edwardsiella tarda is typically associated with water
and animals that
inhabit
water; it is an infrequent cause of gastroenteritis in humans, with most
infections
linked
to contact with fish or turtles. A low carriage rate in humans, except in
tropical areas
of
the world, and the ability to produce a cell-associated hemolysin and invade
HEp-2 cells
suggest
that E. tarda is a diarrheal agent (77).
Serious wound infections, including
myonecrosis,
are reported in immunocompetent individuals with aquatic exposure, but
systemic
infections usually occur in patients with liver disease or iron overload conditions
(129).
Miscellaneous Enterobacteriaceae
Miscellaneous
members of Enterobacteriaceae that are infrequently encountered in
clinical
laboratories
are primarily opportunistic pathogens in compromised patients or are present as
transients
or commensals in clinical specimens (73).
For some,
like
Cedecea spp., Leminorella spp., Moellerella, and Tatumella,
a reservoir has not been
determined
because they are rarely isolated from nonhuman sources, while Ewingella,
Leclercia,
andKluyvera spp. are found in a variety of foods, water, or
animals (snails and
slugs)
and like many enterics appear to be ubiquitous in the environment
(40, 54, 55, 59, 60, 66, 73, 137).
Other genera isolated from humans have more specific
natural
habitats such as Rahnella (water) or Yokenella and P.
asymbiotica (insects and
infections
from insect bites) (2, 41, 73, 88). There have been increased reports of clinically
significant
isolations of L. adecarboxylata and Kluyvera species, all of
which cannot be
covered
here. Kluyvera infections occur in competent and immunosuppressed
patients of all
age
groups (23, 122). The organism is isolated from a broad spectrum of sources
including
blood,
tissue, urine, cerebrospinal fluid, and peritoneal fluid, although urinary
tract and
bloodstream
infections each account for approximately one-third of the infections. When
strains
are determined to species level, Kluyvera ascorbata accounts for more
than twice the
number
ofKluyvera cryocrescens infections. Originally considered to be an
opportunistic
pathogen,
L. adecarboxylata is quite often isolated from polymicrobial infections
in healthy
patients
without underlying disease (58). This suggests that the
coinfecting agent(s) might
alter
the local tissue environment, allowing growth of Leclercia, or that a
transfer of genetic
factors
occurs, enhancing its virulence. The isolation of Leclercia in pure
culture from
previously
healthy persons (from a foot abscess and blood and wound caused by a
hydrofluoric
acid chemical injury) in two recent reports would indicate that at least some
strains
may be pathogenic (31, 58). Moellerella wisconsensis, a rarely reported organism
first
isolated from stools of diarrheal patients, has been reported from an
additional series of
five
patients with disease ranging from self-limited acute watery diarrhea without
mucus to
protracted
diarrhea, lasting several weeks. No other common diarrheal bacterial pathogens
or
parasites were present in the stools, and specimens taken after clinical
recovery were
negative
(116).
COLLECTION, TRANSPORT, AND STORAGE OF
SPECIMENS Back to top
The
organisms covered in this chapter are, in general, readily isolated from
clinical material,
and
the principles in chapter 16 of this Manual on specimen collection, handling, and
processing
are applicable. Most of these organisms will survive in culture deeps for
approximately
a year. Long-term storage methods as recommended in chapter
9 work well
for
these organisms. Plesiomonas cells do not survive for more than 1 to 2
months when
held
at room temperature and should be maintained at -70°C. In our laboratory,
strains
of Tatumella
have failed to survive longer than a year even at -70°C.
ISOLATION PROCEDURES Back
to top
Few
of the clinically relevant strains covered in this chapter present difficulties
in isolation
from
sterile body sites. Isolation from nonsterile body or environmental sites may
require
specialized
media such as CHROMagar Orientation (Becton Dickinson, Sparks, MD) and
chromID
CPS (bioMerieux, Hazelwood, MO), which perform similarly for the detection of
urinary
tract pathogens covered in this chapter and can reliably replace MacConkey and
blood
agars (20, 125). These media prevent swarming of Proteus and limit the
spread of
mucoid
colonies, which reduces overgrowth of pathogenic colonies. Additionally,
colonies on
CHROMagar
can be used to inoculate antimicrobial susceptibility tests directly without
subculture.
CHROMagar media can also be used for specimens from other nonsterile sites;
when
colony color was combined with indole, lysine, and ornithine decarboxylase
tests and
serology,
98.7% (466 of 472) of the above organisms were correctly identified from
nonurine
samples
(109).
Both
of the diarrheal pathogens covered in this chapter are easily isolated. E.
tarda is a
lactose-negative,
H2S- positive organism, indistinguishable from Salmonella on enteric
plating
media (opaque or opaque with black centers). A positive indole reaction and
failure to
agglutinate
in specific Salmonella antisera separate the two
organisms.
Plesiomonas produces non-lactose-, non-sucrose-fermenting colonies on
enteric
plating
media. It does not grow on thiosulfate-citrate-bile salts-sucrose medium, but
on
cefsulodin-Irgasan-novobiocin
medium, opaque colonies without a pink center (mannitol not
fermented)
are suspicious for plesiomonads. Two other oxidase-positive
organisms,
Pseudomonas and Aeromonas, grow on cefsulodin-Irgasan-novobiocin
as well,
although
Aeromonas colonies have a pink center with an opaque apron. Inositol
fermentation
and
a positive reaction in Moeller’s lysine, arginine, and ornithine tests will
differentiate
Plesiomonas from these agents as well as other organisms.
Other
Enterobacteriaceae involved in opportunistic infections and that may be
isolated from a
variety
of specimen types generally grow well on commonly used laboratory media (80).
Some
genera are lactose or sucrose fermenters and give the appearance of normal
biota on
enteric
plating media, while others may produce H2S and appear Salmonellalike.
Rahnella, Ewingella,
and Tatumella may require 48 hours for growth.Tatumella also
grows
poorly on Mueller-Hinton agar, and a broth dilution method may be required for
susceptibility
testing. K. granulomatis does not grow on conventional laboratory media
but
has
been grown in HEp-2 monolayers (22).
Detection of Donovan bodies from tissue smears
using
Giemsa or Wright stains is the method most commonly used to detect this
organism.
However,
these pleomorphic, bipolar staining bodies shaped like a closed safety pin are
not
always
present and are not reliable for diagnosis.
IDENTIFICATION Back to top
The
biochemical tests most useful for separating members covered in this chapter
are given
in Tables
3through 13. A table for the identification of Plesiomonas can be
found in chapter
39, which
describes vibrios, since these organisms most closely resemble one another
biochemically.
Correct identification to species level is increasingly important in
recognizing
strains
that are of high risk for carrying extended-spectrum beta-lactamases (ESBLs),
cephalosporinases,
or carbapenemases.
Enterobacter
and Pantoea
Because
of the genetic heterogeneity in several species, members of the
genera
Enterobacter and Pantoeaappear to confound commercial systems
more often than
other
genera (73). E. ludwigii and E. hormaechei,species previously
residing within the E.
cloacae
complex, can be separated from E. cloacae by growth on
3-0-methyl-Dglucopyranose
and
putrescine and 3-hydroxybutyrate, respectively (63, 65).
By using
commercially
available tests, the three subspecies of E. hormaechei can be separated
with
adonitol,
dulcitol, and D-sorbitol (subsp. hormaechei tests negative, positive,
and negative,
respectively;
subsp. steigerwaltii tests positive, negative, and positive,
respectively; and
subsp.
oharae tests negative, negative, and positive, respectively) (63). P.
agglomerans is
very
difficult to identify with either commercial systems or conventional
biochemicals
(21, 103).
Yellow-pigmented and lysine-, arginine,- and ornithine-negative organisms
should
raise
suspicion that the strain is P. agglomerans; Leclercia adecarboxylata and
P.
asymbiotica
also share these characteristics, but they can be separated from P.
agglomerans
by positive indole and negative D-mannitol reactions, respectively.
P. ananatis,
P.
dispersa, and E. persicina most closely resemble P. agglomerans, but
P. dispersa can be
separated
from P. agglomerans by negative reactions for raffinose, salicin, and
sucrose
and E.
persicina can be distinguished by negative reactions for maltose and
D-xylose,
respectively.
P. ananatis is more difficult to differentiate, and all suspected
isolates of this
organism,
as with other rare Enterobacteriaceae, should be sent to a reference
laboratory for
confirmation.
Serratia,
Citrobacter, and Proteeae
Serratia
spp. are generally easily identified except for the S.
liquefaciens group; separation
of
members within this group can be achieved using a combination of API 50 CH
(carbohydrate)
and API ZYM (enzymatic) (bioMerieux, Hazelwood, MO) strips
(50, 73). Citrobacter
spp. may be included in databases individually or by subgroups (C.
braakii-C.
freundii-C. sedlakii, C. werkmanii-C. youngae, or C.
koseri-C. amalonaticus);
however,
subgroup identification requires further biochemical testing by standard
methodologies,
and final species identification is delayed (73). A
PYR (L-pyroglutamic acid,
Oxoid
PYR) disk, which detects pyrrolidonyl peptidase, may be useful for separating
biochemically
atypical strains of Citrobacter (positive) and Salmonella (negative)
(13). Gramnegative,
oxidase-
negative organisms that swarm on blood agar and appear flat with
tapered
edges on MacConkey agar may be reported as Proteus. Spot indole-negative
and
ampicillin-susceptible
strains may be reported as P. mirabilis, while spot indole-positive,
ampicillin-resistant
strains are reported as P. vulgaris (10). P.
hauseri, previously a subgroup
of P.
vulgaris, can be differentiated from P. vulgaris by negative
salicin/esculin and trehalose
reactions
(108). Proteus organisms that do not fit the above criteria
must be fully identified
by
commercial or conventional biochemical methods (10). Proteus
species are identified with
95%
to 100% accuracy by commercial systems, but Providencia identification
rates vary
from
79% to 100% (108). When Providencia spp. are misidentified, they are
usually
called
Morganella or Proteus. Urea-positive P. stuartiimay be
misidentified as P. rettgeri, or
the
system may require additional tests for identification. Two-hour identification
methods
misidentify
M. morganii subsp. morganii about 66% of the time.
Other Enterobacteriaceae
Averyella
dalhousiensis is not in commercial system databases. It may be confused with K.
ascorbata
or misidentified as S. enterica in commercial systems. It
shares biochemical traits
(positive
for ONPG [o-nitrophenyl-β-D-galactopyranoside], malonate, potassium
cyanide, and
fermentation
of dulcitol and salicin) with Salmonella subspecies 2, 3, and
4. Ewingella
and Tatumella are biochemically inactive, and the latter organism
grows poorly
in
vitro. Kluyvera can be identified only to genus level by commercial
systems, but species
determination
requires an ascorbate test and Irgasan susceptibility and/or gas liquid
chromatography
profiles (73). P. asymbiotica is not in most commercial databases.
Molecular Identification
Information
on molecular identification techniques is available elsewhere (chapter
4). For
laboratories
using partial 16S rRNA (~500 bp) sequencing to identify members of
the Enterobacteriaceae,
the Clinical and Laboratory Standards Institute (CLSI) provides
extremely
useful information, including guidelines with suggested cutoff values for
percent
identity
scores and identification algorithms (27).
Specifically, Table 3 of the MM18-A
guideline
provides information on the usefulness of 16S rRNA for various enteric groups,
comments
regarding relatedness within groups, alternative DNA targets, and indications
for
identification
to species level and recommendations for resolving species identification.
Suggested
cutoff values help the laboratory provide clinicians with practical,
recognizable
identifications
of clinically significant organisms and allow the same organisms to be
identified
with consistency between laboratories. Appropriate cutoff values notwithstanding,
accurate
organism identification is ultimately dependent upon the availability of a
reliable
database
of known sequences for comparison of the sequences generated for the unknown
isolate.
Both public and private databases are available, and each offers advantages (128).
An
evaluation of two commercially available reference sequence libraries, MicroSeq
(Applied
Biosystems,
Foster City, CA) and SmartGene IDNS (SmartGene, Inc., Raleigh, NC), found
that
the second had a greater diversity of sequences for comparison and provided
userfriendly
software
with enhancements such as the ability to add alternative gene target
databases
and to store and compare previous clinical sequences (128).
TYPING SYSTEMS Back to top
The
ability to trace the spread of nosocomial pathogens in outbreaks caused by
the Enterobacteriaceae
has become a major responsibility for the laboratory.
Chapters
7 and 8 provide useful information on the molecular epidemiology of
enteric
outbreaks.
Molecular techniques, including plasmid analysis, ribotyping, pulsed-field gel
electrophoresis
(PFGE), and various PCR methodologies all appear to be satisfactorily
discriminatory,
with some working better for a specific genus or species than others. PCR
techniques,
particularly repetitive element-PCR methods for the Enterobacteriaceae, have
proliferated
at an astonishing pace and cannot be covered here more fully. To date, because
economic
constraints dictate the need for a single method that is applicable for a
variety of
organisms,
PFGE remains the most universally accepted standardized technique for
epidemiological
studies. The disadvantage of a long turnaround time (usually 4 days) has
been
partially overcome by a rapid PFGE protocol that is suitable for most enteric
bacteria as
well
as other common clinical strains (45).
ANTIMICROBIAL SUSCEPTIBILITY Back
to top
Increasing
resistance in members of the Enterobacteriaceae (Table
13) has culminated with
the
emergence of panresistant strains of K. pneumoniae for which there are
no therapeutic
options
(37, 110, 135, 136). While K. pneumoniae presents the most serious threat, K.
oxytoca,
S. marcescens, Enterobacter spp., Proteus spp.,
andMorganella and Citrobacter spp.
have
all been reported to possess one or more Ambler Class A ESBLs or
carbapenemases,
Class
C cephalosporinase, and/or class B metallo-β-lactamases. Frequently, plasmids
encoding
these enzymes also carry resistance genes for aminoglycosides, quinolones, and
other
antimicrobials (37, 102, 140). Although their clinical significance and the need to
control
intra- and interhospital transmission make detection of these enzymes a
priority for
the
clinical laboratory, they are often unrecognized by routine susceptibility
testing because
of
the difficulty in detecting these enzymes and the lack of standard techniques
(110, 135, 136, 140).
Both phenotypic and genotypic methods are available for detection of
ESBLs,
cephalosporinases, and carbapenemases, and while the latter tests are limited
to
large
institutions and research laboratories, they identify specific genes and can
detect lowlevel
resistance
missed by phenotypic tests. General antimicrobial susceptibility and
specialized
phenotypic testing procedures are discussed elsewhere in
this
Manual (chapters 67 to 70) and by Patel et al. (110)
and Sundin (136).
ESBL-Producing Enterobacteriaceae
In
addition to screening and confirmatory methods recommended by CLSI (28), a
chromogenic
agar, chromID ESBL (bioMerieux, Marcy l’Etoile, France), has been developed
for
detection of ESBLs. Several studies evaluating this medium report chromID ESBL
sensitivity
equal to or better than that obtained by automated or agar-based methods
(39, 48, 119).
The specificity for chromID ESBL was similar to that for the comparison
methods
in two studies (89% and 91%) but was only 11% in the third, causing those
authors
to recommend that suspected ESBL-producing isolates on this medium be verified
with
another test (39). The negative predictive value calculated in one study (>99%)
indicated
that this medium can be used as an effective tool to rule out ESBL-producing
strains
in clinical samples (119). chromID ESBL uses cefpodoxime as a substrate rather than
ceftazidime
or cefotaxime, which was noted to increase its sensitivity and was thought to
be
responsible
for the greater recovery of CTX-M type ESBL-producing isolates in one of the
studies
(48, 119). Strains with hyperproducing AmpC
(predominantly
Enterobacter and Citrobacter spp.) and hyperproducing
penicillinase (K.
oxytoca)
enzymes produced false positives (strains with correct colony color but ESBL
negative
on chromID ESBL) in all three studies from both the chromID ESBL agar and the
comparison
methods (39, 48,119). In the two studies testing clinical samples, the recovery
rate
for ESBL-producing strains was 7% and 4%, respectively (48, 119).
In addition to
chromID
ESBL media, Farber et al. (39) tested three Vitek 2
(bioMerieux, Durham, NC)
panels
and two Phoenix (Becton Dickinson, Sparks, MD) panels with their corresponding
expert
interpretation software for ESBL detection. Of the two Phoenix panels, the
NMIC/ID-
70
panel slightly outperformed the best Vitek 2 panel result (AST-N041) with a
sensitivity
and
specificity of 84% and 75% versus 84% and 50%, respectively. In another study
comparing
the Phoenix NMIC/ID-108 panel with the MicroScan (Siemens Healthcare
Systems,
West Sacramento, CA) Neg BP combo panel type 30, both systems did well in
detecting
ESBL-positive K. pneumoniae in clinical strains but MicroScan performed
better
with
various Enterobacteriaceae challenge strains (130).
When ESBL-producing strains are
detected
by automated systems, it is generally recommended that they be confirmed using
a
manual
method (39, 130,135).
Expanded-Spectrum Cephalosporin Resistance
The
clinical significance of strains with chromosomal, inducible AmpC enzymes
(transient
high-level
production induced only in the presence of a beta-lactam) is still unclear and
the
need
for treatment controversial. However, the use of a beta-lactam in patients with
strains
exhibiting
permanent AmpC hyperproduction (due to mutations in the regulatory genes that
result
in more efficient production of the enzyme) can result in treatment failure (135).
The
fact
that AmpC genes are now plasmid mediated in a variety of Enterobacteriaceaeadds
to
the
need for laboratories to recognize AmpC-positive strains, as they are an even
greater
risk
for transmission within the hospital. Their recognition is also complicated by
the fact that
reduced
susceptibility to cefoxitin, which may be used as a screen for AmpC activity,
may
reflect
a loss of permeability through the outer membranes as well. Incorrect reporting
of a
strain
as AmpC positive may result in the unnecessary use of carbapenems and the
concomitant
risk of developing resistance to these drugs (136, 138). Nonetheless,
intermediate
or reduced susceptibility to cefoxitin is still a useful indicator of AmpC
presence,
signaling
the need for further testing (136). Tan et al. (138)
evaluated three methods for the
detection
of AmpC-producing strains of Escherichia coli, Klebsiella, and Proteus
spp. This
study
found that the disk approximation test using imipenem, cefoxitin, and
amoxicillinclavulanic
acid
as inducing agents against ceftazidime had the least sensitivity (25%) when
compared
with PCR (94%), agar dilution using cefoxitin } cloxacillin (90%), or a
disk-based
test
using cefpodoxime or cefoxitin } cloxacillin or boronic acid (an inhibitor of
AmpC and
KPC
enzymes). In the disk-based inhibitor assay cefoxitin } cloxacillin, using a
zone increase
cutoff
of ≥4 mm, gave the best overall sensitivity (95%). For the induction test,
imipenem
performed
the best of the agents tested. Overall AmpC activity was detected in 50% (127
of
255)
of strains, and AmpC was found to be plasmid-borne in 94% of the 27 strains.
Carbapenem Resistance
Carbapenem
resistance genes (which are defined in Table 13)
may be plasmid-mediated
Class
A carbapenemases, most commonly the KPC (K. pneumoniae carbapenemase)
types,
Class
A chromosomally encoded SME (S. marcescens enzyme), and IMI/NMC
(imipenemhydrolyzing
β-lactamase/not
metalloenzyme carbapenemase) types; Class B metallo-β-
lactamases
(MBL), primarily the IMP (active on meropenem) type found in Asia; and Class D
OXA
(oxacillin-hydrolyzing) carbapenemases (110, 136).
Because carbapenem MICs, even
when
elevated, appear susceptible by CLSI breakpoints, carbapenem-resistant strains
are
difficult
to detect. In testing for carbapenemases, the use of ertapenem as an indicator
substrate
appears to be more sensitive for the detection of KPCs than imipenem or
meropenem
(96, 110, 136). McGettigan et al. (96)
screened 2,696 Enterobacteriaceae by
using
an ertapenem disk diffusion test or Vitek 2 GN-20 card and obtained equivalent
results,
detecting
85 ertapenem-intermediate or -resistant strains, of which 63 were KPC-positive K.
pneumoniae.
While all of the K. pneumoniae KPC-positive isolates were confirmed by
the
modified
Hodge test and PCR, the four Enterobacter spp. isolates were found to be
false
positives.
Anderson et al. (8) found that the broth microdilution test was more sensitive than
disk
diffusion, Etest, Vitek 2, and MicroScan assays for the detection of
carbapenemase
resistance
regardless of the carbapenem used. Ertapenem was the most sensitive substrate
for
the disk test, Etest, and automated methods. While sensitive, ertapenem may be
less
specific
than imipenem or meropenem because it detects resistance caused by mechanisms
other
than carbapenemases (8, 141). Tsakris et al. (141)
detected KPC-producing K.
pneumoniae
with 100% sensitivity using disks with imipenem, meropenem, or
cefepime
alone
and with 400 μg of boronic acid, although ertapenem gave false-positive results
with
five
KPC-negative, AmpC-positive isolates. An elevated MIC (≥1 or 2 μg/ml) to
meropenem
or
imipenem in automated assays or a zone of ≤19 mm with an imipenem disk can also
be
used
to screen isolates for KPCs or MBLs (8, 110).
Isolates should then be tested by a
phenotypic
method such as the modified Hodge assay (see chapter 70),
which has been
found
to be 100% sensitive and specific for carbapenemases. Unlike an inhibitor test
using a
carbapenem
or cefepime } boronic acid, the modified Hodge assay cannot differentiate
between
KPCs and MBLs, information that is useful for infection control (110, 141).
EDTA
inhibition
assays can also differentiate between KPC and MBL production; Etest markets a
double-sided
IP/IPI strip with imipenem on one end and imipenem-EDTA on the other end to
detect
MBLs (110). CHROMagar KPC (CHROMagar, Paris, France) is another product
available
for
the detection of KPC-producing K. pneumoniae. When used on rectal swabs,
its sensitivity
and
specificity relative to PCR were 100% and 98%, respectively, compared to 93%
and
96%
for MacConkey agar with 10-μg carbapenem disks versus PCR (124).
Overall, 41 of the
122
(34%) swabs yielded KPC-producing K. pneumoniae.
Resistance in Miscellaneous Enterobacteriaceae
Strains
of P. mirabilis are resistant to nitrofurantoin but susceptible to
trimethoprimsulfamethoxazole
(SXT),
ampicillin, amoxicillin, piperacillin, cephalosporins, aminoglycosides,
and
imipenem. Although most strains are susceptible to ciprofloxacin, resistance
occurs with
unrestricted
use of the drug (108). P. penneri and P. vulgarishave a resistance
profile similar
to
that of Morganella, although P. penneri is more resistant to
penicillin than P. vulgaris. All
three
organisms are susceptible to broad-spectrum cephalosporins, cefoxitin,
cefepime,
aztreonam,
aminoglycosides, and imipenem. They are resistant to piperacillin, amoxicillin,
ampicillin,
cefoperazone, cefuroxime, and cefazolin. P. rettgeri and P. stuartii are
resistant to
gentamicin
and tobramycin but susceptible to amikacin. Urine isolates are susceptible to
broad-
and expanded-spectrum cephalosporins, ciprofloxacin, amoxicillin-clavulanic
acid,
imipenem,
and SXT. Providencia heimbachae, although infrequently seen in humans,
is
resistant
to tetracycline, most cephalosporins, gentamicin, and amikacin. Human isolates
of E.
tarda are susceptible to cephalosporins, aminoglycosides, imipenem,
ciprofloxacin,
aztreonam,
and antibiotic-β-lactamase inhibitor combination agents (26).
Isolates from fish
and
fish ponds may be more resistant because of the antibiotics used
prophylactically in fish
farming.
Most strains of E. tarda produce β-lactamases, even though they are
susceptible to
β-lactams.
P. shigelloides is resistant to ampicillin, carbenicillin, piperacillin,
and ticarcillin
and
is variably resistant to most aminoglycosides and tetracycline (71).
Cephalosporins,
quinolones,
carbapenems, and SXT show good activity against P. shigelloides.
Susceptibility
results of uncommonly seen species of Klebsiella,
Enterobacter,
and Serratia are similar to those of conventional species
within these genera
(44).
Susceptibilities for other Enterobacteriaceae vary from isolate to
isolate, so that no
empirical
guidelines are available for therapy prior to susceptibility testing of the
suspected
strain.
EVALUATION, INTREPRETATION, AND REPORTING OF
RESULTS Back to top
When
commercial systems identify species included in this chapter with a high level
of
accuracy
(>90% probability), the identification is probably reliable. However, for
organisms
isolated
from sterile sites that are identified with a probability of <90%, the
isolate should be
confirmed
by conventional or molecular methods or sent to a reference laboratory using
these
techniques. In the interim, the isolate may be reported to the physician with a
presumptive
identification. Rare species that are identified with low probabilities should
always
be sent to a reference laboratory accompanied by a brief history.
An
increasing number of patients are infected or colonized
with
Enterobacteriaceae possessing ESBLs and carbapenemases, and they serve
as
reservoirs
for transmission of these enzymes within and between health care institutions.
The
ability to recognize these strains is critical not only for patient care but
also to prevent
the
emergence of panresistant strains. Reliable, cost-effective methodologies for
the
detection
and reporting of these enzymes that are manageable for all institutions
regardless
of
size and capability are urgently needed. At the very least, all strains of K.
pneumoniae
should be tested for ESBLs, since automated testing methods seem
to be
reliable
with this agent (39). Any strain of Enterobacteriaceae that has been shown by
susceptibility
testing to have an ESBL or AmpC cephalosporinase should be reported as
resistant
to all penicillins, expanded-spectrum cephalosporins, and aztreonam (136, 140).
Data
provided by a CDC database of PFGE patterns used to monitor KPC-producing
strains
isolated
in the United States and other areas worldwide indicate that infection control
(http://www.cdc.gov/ncidod/dhqp/gl_isolation.html)
interventions will be necessary to halt
the
spread of these organisms (110).
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