The isolation of Shiga toxin-producing Escherichia coli (STEC) other than serogroup O157 from clinical stool samples is problematic due to the lack of differential phenotypic characteristics from non-pathogenic E. coli. The development of molecular reagents capable of identifying both toxin and serogroup-specific genetic determinants holds promise for a more comprehensive characterization of stool samples and isolation of STEC strains. In this study, 876 stool samples from paediatric patients with gastroenteritis were screened for STEC using a cytotoxicity assay, commercial immunoassay and a conventional PCR targeting Shiga-toxin determinants. In addition, routine culture methods for isolating O157 STEC were also performed. The screening assays identified 45 stools presumptively containing STEC, and using non-differential culture techniques a total of 20 O157 and 22 non-O157 strains were isolated. These included STEC serotypes O157 : H7, O26 : H11, O121 : H19, O26 : NM, O103 : H2, O111 : NM, O115 : H18, O121 : NM, O145 : NM, O177 : NM and O5 : NM. Notably, multiple STEC serotypes were isolated from two clinical stool samples (yielding O157 : H7 and O26 : H11, or O157 : H7 and O103 : H2 isolates). These data were compared to molecular serogroup profiles determined directly from the stool enrichment cultures using a LUX real-time PCR assay targeting the O157 fimbrial gene lpfA, a microsphere suspension array targeting allelic variants of espZ and a gnd-based molecular O-antigen serogrouping method. The genetic profile of individual stool cultures indicated that the espZ microsphere array and lpfA real-time PCR assay could accurately predict the presence and provide preliminary typing for the STEC strains present in clinical samples. The gnd-based molecular serogrouping method provided additional corroborative evidence of serogroup identities. This toolbox of molecular methods provided robust detection capabilities for STEC in clinical stool samples, including co-infection of multiple serogroups.
BastianS. N.,
CarleI.,
GrimontF.1998; Comparison of 14 PCR systems for the detection and subtyping of stx genes in Shiga-toxin-producing Escherichia coli
. Res Microbiol 149:457–472[CrossRef]
BelangerS. D.,
BoissinotM.,
MenardC.,
PicardF. J.,
BergeronM. G.2002; Rapid detection of Shiga toxin-producing bacteria in feces by multiplex PCR with molecular beacons on the smart cycler. J Clin Microbiol 40:1436–1440[CrossRef]
BeutinL.,
ZimmermannS.,
GleierK.2002; Evaluation of the VTEC-Screen “Seiken” test for detection of different types of Shiga toxin (verotoxin)-producing Escherichia coli (STEC) in human stool samples. Diagn Microbiol Infect Dis 42:1–8[CrossRef]
ChurchD. L.,
EmsheyD.,
SemeniukH.,
LloydT.,
PitoutJ. D.2007; Evaluation of BBL CHROMagar O157 versus sorbitol-MacConkey medium for routine detection of Escherichia coli O157 in a centralized regional clinical microbiology laboratory. J Clin Microbiol 45:3098–3100[CrossRef]
GannonV. P.,
KingR. K.,
KimJ. Y.,
ThomasE. J.1992; Rapid and sensitive method for detection of Shiga-like toxin-producing Escherichia coli in ground beef using the polymerase chain reaction. Appl Environ Microbiol 58:3809–3815
GilmourM. W.,
TraczD. M.,
AndrysiakA. K.,
ClarkC. G.,
TysonS.,
SeveriniA.,
NgL. K.2006; Use of the espZ gene encoded in the locus of enterocyte effacement for molecular typing of shiga toxin-producing Escherichia coli
. J Clin Microbiol 44:449–458[CrossRef]
GilmourM. W.,
OlsonA. B.,
AndrysiakA. K.,
NgL. K.,
ChuiL.2007a; Sequence-based typing of genetic targets encoded outside of the O-antigen gene cluster is indicative of Shiga toxin-producing Escherichia coli serogroup lineages. J Med Microbiol 56:620–628[CrossRef]
GilmourM. W.,
TaborH.,
WangG.,
ClarkC. G.,
TraczD. M.,
OlsonA. B.,
MascarenhasM.,
KarmaliM. A.,
MailmanT.,
NgL. K.2007b; Isolation and genetic characterization of a coinfection of non-O157 Shiga toxin-producing Escherichia coli
. J Clin Microbiol 45:3771–3773[CrossRef]
KarmaliM. A.,
PetricM.,
LimC.,
FlemingP. C.,
ArbusG. S.,
LiorH.1985; The association between idiopathic hemolytic uremic syndrome and infection by verotoxin-producing Escherichia coli
. J Infect Dis 151:775–782[CrossRef]
LindstedtB. A.,
BrandalL. T.,
AasL.,
VardundT.,
KapperudG.2007; Study of polymorphic variable-number of tandem repeats loci in the ECOR collection and in a set of pathogenic Escherichia coli and Shigella isolates for use in a genotyping assay. J Microbiol Methods 69:197–205[CrossRef]
LouieM.,
ReadS.,
SimorA. E.,
HollandJ.,
LouieL.,
ZiebellK.,
BruntonJ.,
HiiJ.1998; Application of multiplex PCR for detection of non-O157 verocytotoxin-producing Escherichia coli in bloody stools: identification of serogroups O26 and O111. J Clin Microbiol 36:3375–3377
McCarthyT. A.,
BarrettN. L.,
HadlerJ. L.,
SalsburyB.,
HowardR. T.,
DingmanD. W.,
BrinkmanC. D.,
BibbW. F.,
CartterM. L.2001; Hemolytic-uremic syndrome and Escherichia coli O121 at a lake in Connecticut, 1999. Pediatrics 108:E59[CrossRef]
ParkC. H.,
KimH. J.,
HixonD. L.,
BubertA.2003; Evaluation of the duopath verotoxin test for detection of shiga toxins in cultures of human stools. J Clin Microbiol 41:2650–2653[CrossRef]
PatonA. W.,
PatonJ. C.2002; Direct detection and characterization of Shiga toxigenic Escherichia coli by multiplex PCR for stx 1,stx 2, eae , ehxA ,and saa
. J Clin Microbiol 40:271–274[CrossRef]
PerelleS.,
DilasserF.,
GroutJ.,
FachP.2007; Screening food raw materials for the presence of the world's most frequent clinical cases of Shiga toxin-encoding Escherichia coli O26, O103, O111, O145 and O157. Int J Food Microbiol 113:284–288[CrossRef]
ShenS.,
MascarenhasM.,
MorganR.,
RahnK.,
KarmaliM. A.2005; Identification of four fimbria-encoding genomic islands that are highly specific for verocytotoxin-producing Escherichia coli serotype O157 strains. J Clin Microbiol 43:3840–3850[CrossRef]
ThompsonL. H.,
GierckeS.,
BeaudoinC.,
WoodwardD.,
WylieJ. L.2005; Enhanced surveillance of non-O157 verotoxin-producing Escherichia coli in human stool samples from Manitoba. Can J Infect Dis Med Microbiol 16:329–334
WangG.,
ClarkC. G.,
RodgersF. G.2002; Detection in Escherichia coli of the genes encoding the major virulence factors, the genes defining the O157: H7 serotype, and components of the type 2 Shiga toxin family by multiplex PCR. J Clin Microbiol 40:3613–3619[CrossRef]
ZiebellK. A.,
ReadS. C.,
JohnsonR. P.,
GylesC. L.2002; Evaluation of PCR and PCR-RFLP protocols for identifying Shiga toxins. Res Microbiol 153:289–300[CrossRef]