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the pathogenesis of severe neurological symptoms in these patients (
Greinacher
et al., 2011
). The avoidance of ventilation or reduction of time on the ventilator
in these patients might help to prevent respirator-associated complications.
Diagnosis
Because
E. coli
O104:H4 are considered emerging strains, a combination of
molecular detection, culture and isolation of the pathogen was implemented
to improve both outbreak detection and control and patient management. With
regard to the microbiological detection and isolation, clinical samples should be
streaked on extended-spectrum beta-lactamase (ESBL) plates for growth of the
outbreak strain and inhibition of the majority of other
E. coli
strains (
Scheutz
et al., 2011
). Also, growth is observed as light red colonies on cefiximetellurite
sorbitol MacConkey (CT-SMAC) plates at 37°C. For rapid screening of clinical
samples, slide agglutination with K9 antiserum (O104 O antigen is identical to
the K9 capsular antigen) can also be used. Immediate positive reactions indi-
cating the presence of
E. coli
O104:H4 need to be confirmed by conventional
serotyping of O and H antigen, presence of the
stx2
gene and lack of the
eae
gene (to rule out other STEC strains) (
Scheutz et al., 2011
).
The
E. coli
O104:H4 strain can also be detected by a number of methods
targeting the
stx2
gene such as PCR, RT-PCR, or commercial Stx detection
kits. For example, PCR analysis indicated that
E. coli
O104:H4 lacked the LEE
pathogenicity island, but are positive for a number of virulence genes typical of
EAEC, including
pic
,
aggA
,
aggR
,
aap
,
set1,
and the virulence plasmid pAA
(
Bielaszewska et al., 2011
). Colonies positive for
stx
genes were identified as
O104:H4 through further testing for the O104 antigen-associated gene
wzx
O104
and the gene encoding the H4 flagellar antigen,
fliC
H4
(
Scheutz et al., 2011
;
Auvray et al., 2012
).
In the case of food samples, standard methods (i.e. enrichment in buffered
peptone water with incubation for 18-24 h at 37°C prior to DNA extraction)
have been reported to detect and recover
E. coli
O104:H4 at a level of approxi-
mately 1 CFU/g of sprouts (
Jinneman et al., 2012
). The use of additional sero-
type-specific real-time PCR assays (the incorporation of the O104
wzx
gene to
the standard PCR method) and supplemental chromogenic media selection (i.e.
Hardy CHROM ESBL agar or Rainbow O157 without tellurite or novobiocin)
assisted for the detection and recovery of
E. coli
O104:H4.
In addition to the PCR methods, a pan-genomic analysis approach for the
identification and validation of specific molecular targets for detection of
E. coli
O104:H4 outbreak strain has been recently reported (
Ho et al., 2011
). In this
approach, unannotated genomic contigs of nine outbreak
E. coli
O104:H4 iso-
lates were downloaded and used as target genomic data for in silico subtraction
and thereby, to identify sequences specific to
E. coli
O104:H4. Primers specific
to the selected targets were designed and successfully amplified the representative
sequences from the outbreak strains with no false-positive results (
Ho et al., 2011
).
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