690 FAMILY I.
ENTEROBACTERIACEAE
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Korhonen, 1985). They are capable of binding to endothelial
cells, respiratory tract epithelial cells, and uroepithelial cells. In
the kidney, they mediate bacterial adhesion to tubular basement
membranes, Bowman’ s capsules, and renal vessels (Podschun
and Ullmann, 1998). Three new putative colonization factors
have also been described (Podschun and Ullmann, 1998).
The most important role of the O antigen is to protect K.
pneumoniae from complement-mediated killing (Williams and To-
mas, 1990). For this protection, O antigen chain length seems
to be important (McCallum et al., 1989).
Iron is essential for bacterial growth. Brewer et al. (1982)
observed that virulence was enhanced by hyperferremi. Since in
the human body iron is complexed to carrier molecules such as
transferrin (in the serum) or lactoferrin (in milk and other se-
cretions), or sequestered within cells (in heme proteins), poten-
tially pathogenic Enterobacteriaceae produce high-affinity systems
(siderophores) to solubilize and import the required iron. The
iron-chelating compounds produced are mostly of two sorts,
phenolates (e.g., enterochelin) and hydroxamates (aerobactin)
(Payne, 1988). Almost all strains of Klebsiella produce entero-
chelin whereas only a few produce aerobactin (Williams et al.,
1987). Nassif and Sansonetti (1986) correlated the virulence of
K. pneumoniae serotypes K1 or K2 with the presence of a 180-kb
plasmid encoding the hydroxamate siderophore aerobactin.
Aerobactin is an essential factor of pathogenicity and the 180-kb
plasmid carries additional genes encoding other virulence factors
(Nassif and Sansonetti, 1986). Some strains of K. pneumoniae ex-
press a ferric aerobactin uptake system without making the che-
lator itself. This may confer a selective advantage in mixed in-
fections in competition with other aerobactin-producing bacteria
(Williams and Tomas, 1990).
The production of cytotoxins, enterotoxins, and hemolysins
have been sporadically described (Podschun and Ullmann,
1998).
Ecology Klebsiellae have been recovered from aquatic en-
vironments receiving industrial wastewaters, plant products, fresh
vegetables, food with a high content of sugars and acids, frozen
orange juice concentrate, sugar cane wastes, living trees, plants,
and plant by-products. They are commonly associated with wood,
saw dust, and waters receiving industrial effluents from pulp and
paper mills and textile finishing plants. Isolates have been de-
scribed in forest environments, degrading kraft-lignin, tannic
acid, pine bark, and condensed tannin, or from living or decaying
wood and bark or composted wood.
Klebsiella can frequently be isolated from the root surfaces of
various plants. K. pneumoniae, K. oxytoca, or K. planticola are ca-
pable of fixing nitrogen and are classified as associative nitrogen
fixers.
Strains from plants certainly need to be reidentified in the
light of present taxonomic schemes. Strains of K. pneumoniae
sensu stricto that are associated with plants differ from those as-
sociated with serious human infections. These environmental K.
pneumoniae strains are most often able to utilize 5-ketogluconate
as sole carbon source and never have capsular types K1 to K6.
Strains involved in serious infection do not utilize 5-ketoglucon-
ate and may have capsular types K1 to K6 as well as other capsular
types (Grimont et al., 1991).
Capsular types K1, K2, and K5 were major causes of epidemic
metritis in mares in England, whereas type K7 was associated with
sporadic, opportunistic genital infection. Outbreaks of metritis
law.
in mare were due to type K2 in the United States and in France.
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The stallion plays an important role in the transmission of K.
pneumoniae. Type K7 was found on the preputial skin of stallions
and may be part of the normal bacterial flora in this location.
Thus it is important to type Klebsiella isolated in the genital tract
of horses to detect a stallion carrying an epidemic strain among
other stallions carrying less pathogenic K. pneumoniae (Grimont
et al., 1991).
Klebsiella have been frequently associated with bovine mastitis,
and causes serious infections in other animals including rhesus
monkeys, guinea pigs, or muskrats. Epidemics of fatal generalized
infections among captive squirrel monkeys (Saimiri sciureus) in
French Guyana and lemurs in a French zoo were due to K. pneu-
moniae K5 and K2, respectively. Immunization of the monkeys
with the corresponding capsular polysaccharide is efficient in
stopping the epidemic (Grimont et al., 1991).
ENRICHMENT AND ISOLATION PROCEDURES
The detection, isolation, and enumeration in clinical, industrial,
and natural environments can be facilitated by using a selective
medium.
On agar plates, although colonies develop overnight, the char-
acteristic elevated and mucoid appearance is observed after in-
cubation for 48 h.
The ability to utilize citrate (Cooke et al., 1979) or myo-inositol
(Legakis et al., 1976) has been applied to the formulation of
selective media. Resistance of Klebsiella spp. to methyl violet
(Campbell and Roth, 1975), double violet (Campbell et al.,
1976), potassium tellurite (Tomas et al., 1986), and carbenicillin
(Thom, 1970) has been used in selective media.
Thom (1970) developed a medium based on the MacConkey
agar in which lactose is replaced by inositol (1% w/v), with the
addition of 100 lg of carbenicillin per ml. Bagley and Seidler
(1978) devised a similar medium with only 50 lg/ml carbeni-
cillin. On this medium, about 95% of pink-to-red colonies were
verified to be Klebsiella spp., whereas only 1% of yellow back-
ground colonies were Klebsiella.
Since about 10% of Klebsiella strains are susceptible to 50
lg/ml of carbenicillin, the antibiotic was replaced in the above
formula by tellurite (K2TeO3, 3 lg/ml) (Tomas et al., 1986),
which is a strong inhibitor of phosphate transport in E. coli. Min-
imal inhibitory concentrations of K2TeO3 were 100 or 200 lg/ml
for K. pneumoniae subsp. pneumoniae, K. oxytoca, K. planticola, and
K. terrigena, 10 lg/ml for K. pneumoniae subsp. ozaenae, and 1–3
lg/ml for other Enterobacteriaceae (Tomas et al., 1986). In a field
test, 77% of pink-to-red colonies on MacConkey–inositol–potas-
sium tellurite agar were confirmed as Klebsiella spp.; however, the
efficiency of plating was about 1% (Dutka et al., 1987).
Bruce et al. (1981) devised an agar medium combining Koser
citrate and raffinose (carbon sources) and ornithine and low pH
(for ornithine decarboxylase). On acidic Koser citrate agar with
ornithine and raffinose, Klebsiella strains grow as yellow mucoid
colonies. Other Enterobacteriaceae either do not grow, or produce
small colorless, pink, red, or orange colonies.
For the isolation of K. pneumoniae and K. oxytoca from human
feces, Van Kregten et al. (1984) have developed a medium based
on the presence of two carbon sources, citrate and inositol, with-
out inhibitor. The medium consists of Simmons citrate agar with
1% inositol. Klebsiella spp. appear as yellow, dome-shaped, often
mucoid colonies, whereas E. coli appears as tiny, watery colonies.
Apart from some Enterobacter strains, no other of bacteria grow
on the medium.