Biology Reference
In-Depth Information
Diagnosis
Rapid detection of STEC infection is crucial for effective management, as
well as for prompt detection of outbreaks and concomitant institution of con-
trol measures. The ability to culture STEC from feces is considered to be the
most definitive diagnostic procedure and is generally combined with tests to
detect Stx in fecal extracts or cultures. As mentioned above, unique biochemi-
cal characteristics of
E. coli
O157:H7, such as the inability to ferment sorbitol
and failure to produce β-D-glucuronidase, are utilized for detection in clini-
cal as well as in food samples. For example,
E. coli
O157 grows as colorless
(sorbitol-negative) colonies on Sorbitol MacConkey (SMAC) agar, in contrast
to pink colonies produced by most fecal
E. coli
strains. Supplementation of
SMAC agar with additional compounds that inhibit the growth of other bac-
teria or enrich
E. coli
O157 strains is often utilized (
Chapman et al., 1991
;
Zadik et al., 1993
). Commercial media containing chromogenic substrates for
β-D-glucuronidase are also available for identification of
E. coli
O157 strains.
A combination of two or more methods is often used to confirm the presence
of
E. coli
O157:H7 (
Paton and Paton, 1998
), and positive colonies are typi-
cally further confirmed by slide or tube agglutination tests with O157 and H7
antisera.
A limitation of the culture detection methods described above is that they
are specific to the
E. coli
O157:H7 serotype. Many non-
E. coli
O157 STEC
serotypes associated with serious outbreaks lack distinguishing biochemical
characteristics. However, most STEC strains produce EHEC-Hly, a hemolysin
that can be used to assist in identification (
Paton and Paton, 1998
). In addition,
assays to detect Stx can be used to identify all STEC strains. For example,
Vero cells have a high plasma membrane concentration of Gb
3
and Gb
4
recep-
tors and are therefore exquisitely sensitive to all variants of Stx, a property that
has been used to detect Stx in fecal extracts and cultures (
Konowalchuk et al.,
1977
). ELISAs using monoclonal or polyclonal antibodies to Stx are easier and
less expensive than cell culture methods to detect STEC, but somewhat less
sensitive, an important limitation for diagnosis at late stages of disease when
the number of STEC in feces may be extremely low. Clinicians often resort to
anti-Stx, anti-O Ag, or anti-H Ag serology for diagnosis (
Yamada et al., 1993
;
De Boer and Heuvelink, 2000
).
More recently, molecular techniques for the detection of STEC have
gained popularity. These techniques primarily depend on the detection of
Stx genes using PCR or RT-PCR, which are highly sensitive and specific
(
Nguyen et al., 2004
). These detection techniques are particularly useful in
the analysis of microbiologically complex samples or those containing non-
viable bacteria (
Paton and Paton, 1998
). Multiplex PCR for simultaneous
detection of
stx
,
eae
, and EHEC-
hly
genes is also frequently used for detec-
tion of STEC, especially EHEC strains (
Fratamico et al., 1995
;
Paton and
Paton, 2002
).
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