Incidence and Virulence Determinants of Verocytotoxin-Producing Escherichia coli Infections in the Brussels-Capital Region, Belgium, in 2008 –2010 Glenn Buvens,a Yves De Gheldre,b Anne Dediste,c Anne-Isabelle de Moreau,d Georges Mascart,e Anne Simon,f Daniël Allemeersch,g Flemming Scheutz,h Sabine Lauwers,a and Denis Piérarda National Reference Center for VTEC/STEC, Department for Microbiology and Infection Control, Universitair Ziekenhuis Brussel, Vrije Universiteit Brussel, Brussels, Belgiuma; Centre Hospitalier Interrégional Edith Cavell, Brussels, Belgiumb; Centre Hospitalier Universitaire St-Pierre, Université Libre de Bruxelles, Brussels, Belgiumc; Hôpitaux Iris-Sud site Bracops, Brussels, Belgiumd; Hôpital Universitaire Des Enfants Reine Fabiola, Université Libre de Bruxelles, Brussels, Belgiume; Cliniques Universitaires St-Luc, Université Catholique de Louvain, Brussels, Belgium f; Cliniques de l’Europe site St-Elisabeth, Brussels, Belgiumg; and WHO Collaborating Centre for Reference and Research on Escherichia and Klebsiella, Statens Serum Institute, Copenhagen, Denmarkh
The incidence of verocytotoxin-producing Escherichia coli (VTEC) was investigated by PCR in all human stools from Universitair Ziekenhuis Brussel (UZB) and in selected stools from six other hospital laboratories in the Brussels-Capital Region, Belgium, collected between April 2008 and October 2010. The stools selected to be included in this study were those from patients with hemolytic-uremic syndrome (HUS), patients with a history of bloody diarrhea, patients linked to clusters of diarrhea, children up to the age of 6 years, and stools containing macroscopic blood. Verocytotoxin genes (vtx) were detected significantly more frequently in stools from patients with the selected conditions (2.04%) than in unselected stools from UZB (1.20%) (P ⴝ 0.001). VTEC was detected most frequently in patients with HUS (35.3%), a history of bloody diarrhea (5.15%), or stools containing macroscopic blood (1.85%). Stools from patients up to the age of 17 years were significantly more frequently vtx positive than those from adult patients between the ages of 18 and 65 years (P ⴝ 0.022). Although stools from patients older than 65 years were also more frequently positive for vtx than those from patients between 18 and 65 years, this trend was not significant. VTEC was isolated from 140 (67.9%) vtx-positive stools. One sample yielded two different serotypes; thus, 141 isolates could be characterized. Sixty different O:H serotypes harboring 85 different virulence profiles were identified. Serotypes O157:H7/Hⴚ (n ⴝ 34), O26:H11/Hⴚ (n ⴝ 21), O63:H6 (n ⴝ 8), O111:H8/Hⴚ (n ⴝ 7), and O146:H21/Hⴚ (n ⴝ 6) accounted for 53.9% of isolates. All O157 isolates carried vtx2, eae, and a complete O island 122 (COI-122); 15 also carried vtx1. Non-O157 isolates (n ⴝ 107), however, accounted for the bulk (75.9%) of isolates. Fifty-nine (55.1%) isolates were positive for vtx1, 36 (33.6%) were positive for vtx2, and 12 (11.2%) carried both vtx1 and vtx2. Pulsed-field gel electrophoresis revealed wide genetic diversity; however, small clusters of O157, O26, and O63:H6 VTEC that could have been part of unidentified outbreaks were identified. Antimicrobial resistance was observed in 63 (44.7%) isolates, and 34 (24.1%) showed multidrug resistance. Our data show that VTEC infections were not limited to patients with HUS or bloody diarrhea. Clinical laboratories should, therefore, screen all stools for O157 and non-O157 VTEC using selective media and a method for detecting verocytotoxins or vtx genes.
V
erocytotoxin-producing Escherichia coli (VTEC), also called Shiga toxin-producing E. coli (STEC), is associated with diarrhea, often bloody, that may be complicated with hemorrhagic colitis and the life-threatening hemolytic-uremic syndrome (HUS), especially in children and the elderly (29). VTEC is characterized by its ability to produce one or more phage-encoded verocytotoxins, VT1 and VT2, that show distinct immunogenic and genetic properties (42). Multiple subtypes of VT1 (VT1a, VT1c, and VT1d) and VT2 (VT2a to VT2g), with significant differences in biologic activity, serologic reactivity, and receptor binding, have been described (36). Many pathogenic strains possess intimin (eae) as part of the locus of enterocyte effacement (LEE), which is associated with adhesion to the intestinal epithelium and the formation of attachment and effacement lesions. Most also possess the plasmid-borne enterohemolysin (ehxA), which is cytolytic to human microvascular endothelial cells. Additional plasmid-borne virulence factors, such as an extracellular serine protease (espP), a catalase-peroxidase (katP), and a type II secretion system (etpD), have been described, but their roles in VTEC pathogenesis are unclear. In recent years, new putative virulence factors, such as STEC autoagglutinating adhesin (saa) and
1336
jcm.asm.org
subtilase cytotoxin (subAB), have been reported in LEE-negative VTEC strains (47, 48). While saa may be of greater importance for attachment in the guts of animals than in humans (9, 23), subAB is linked to eukaryotic apoptosis following proteolytic cleavage of the endoplasmic reticulum chaperone BiP (44). VTEC of serotypes O157:H7/H⫺ has been most frequently associated with HUS and outbreaks in the United States and most parts of the world, but recent studies have shown that the frequency and morbidity of non-O157 infections should not be underestimated (1, 6, 19, 25). Global “hot spots,” in which non-O157 serotypes predominate over O157, include France (51), Germany
Received 1 August 2011 Returned for modification 2 November 2011 Accepted 24 December 2011 Published ahead of print 11 January 2012 Address correspondence to Glenn Buvens,
[email protected]. Supplemental material for this article may be found at http://jcm.asm.org/. Copyright © 2012, American Society for Microbiology. All Rights Reserved. doi:10.1128/JCM.05317-11
0095-1137/12/$12.00
Journal of Clinical Microbiology
p. 1336 –1345
Surveillance of VTEC Infections in Brussels
(2), Spain (5), The Netherlands (60), and Belgium (50). In 2010, the European Center for Disease Prevention and Control (ECDC) reported 3,160 confirmed VTEC cases, or 0.66 infection per 100,000 persons (13a). In Belgium, this rate was higher, with 0.97 infection per 100,000 persons. Since 1995, surveillance of VTEC infections in Belgium has been conducted through a network of sentinel laboratories that refer stools from patients with HUS, bloody diarrhea, and/or E. coli isolates to the Belgian Reference Laboratory for VTEC/STEC at Universitair Ziekenhuis Brussel (UZB) for VTEC analysis by culture and PCR. However, since most clinical laboratories in Belgium do not search for VTEC, the burden of this food-borne pathogen remains underestimated. At UZB, in addition to stools referred by sentinel laboratories, all stool samples collected are routinely screened for VTEC by simultaneous culture and PCR aimed at detecting vtx genes, thus enabling the detection and isolation of O157 as well as non-O157 VTEC. Apart from the studies by Piérard et al., in which the prevalences of VTEC and HUS were investigated (49, 50), recent detailed data on human VTEC infection in Belgium are lacking. In order to gain more insight into the incidence and disease burden of VTEC in Belgium, we have expanded the routine screening at UZB with samples from six hospitals located in the Brussels-Capital Region (BCR) during the years 2008 to 2010. We (i) investigated the incidence of VTEC and identified patient groups at risk, (ii) studied the distribution of serotypes and virulence profiles in correlation with the clinical data, and (iii) assessed the molecular relatedness of VTEC isolates. (Part of this research was presented at the 7th International Symposium on Shiga Toxin [Verocytotoxin] Escherichia coli Infections [VTEC2009], Buenos Aires, Argentina, 10 to 13 May 2009, and at the 4th Congress of European Microbiologists [FEMS 2011], Geneva, Switzerland, 26 to 30 June 2011.) MATERIALS AND METHODS Samples. From April 2008 to October 2010, 14,705 unduplicated stools collected by UZB and six external hospital laboratories for microbiology in the BCR were screened for VTEC by PCR. All stools submitted to UZB (n ⫽ 9,348) for routine culture during the study period were included. Except for two HUS patients, no clinical data for these unselected stools were recorded. In order to gain more insight into patient groups at risk for VTEC infection, we expanded the studied population with selected stools from six external hospital laboratories in the BCR (n ⫽ 5,357). Two hospital laboratories in the BCR were not included due to the low number of stools submitted to these laboratories. The six external laboratories were asked to submit as many as 10 selected stools per week. Samples from patients with HUS, from patients with a history of bloody diarrhea, from children up to the age of 6 years, and from epidemiologically linked cases of diarrhea, as well as stools containing macroscopic (gross) blood, were selected. If the number of 10 samples per week was not reached, laboratories were encouraged to complete the batch with stools from patients with uncomplicated diarrhea. Patients hospitalized for more than 48 h were excluded. Demographic information (sex, age, and postal code) was collected for each PCR-positive patient. This study followed the guidelines of, and was approved by, the Ethical Committee of the Vrije Universiteit Brussel. VTEC screening. All stools were routinely cultured at their respective home laboratory using standard methods for Campylobacter spp., Salmonella spp., Shigella spp. and Yersinia enterocolitica. The selected stools from external laboratories were suspended in MacConkey Broth (MB) (Oxoid Ltd., United Kingdom) and were incubated overnight at 37°C before being shipped to UZB. At UZB, all stools and suspended stools in MB were cultured on sorbitol-MacConkey (SMAC) medium and SMAC
April 2012 Volume 50 Number 4
medium with cefixime and tellurite (CT-SMAC). A colony sweep was suspended in nutrient broth and was used as a DNA template in PCR. All stools were investigated for the presence of VTEC by using a multiplex PCR with specific primers aimed at amplifying the vtx1, vtx2, and vtx2f genes (45, 59). PCRs (20 l) contained 2 l of bacterial suspension, 200 M deoxynucleoside triphosphates (dNTPs), 10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl2, 1 M each primer, and 1.25 U of AmpliTaq Gold polymerase (Applied Biosystems, Belgium). After an initial denaturation for 10 min at 94°C, 35 cycles of amplification (25 s at 94°C, 76 s at 65°C, 64 s at 72°C) were performed. For each PCR-positive sample, 10 colonies obtained on SMAC or CT-SMAC (the original plate or a subculture) were analyzed separately by the same PCR protocol in order to obtain the VTEC isolate for further characterization. If no single colony was found positive, at least 10 more colonies were analyzed. When none of the colonies assayed tested positive, the sample was reported as PCR positive without VTEC isolation. Further characterization was performed on a subculture of one single PCR-positive colony. The isolates were confirmed as E. coli by using classical biochemical tests and were serogrouped by an agglutination assay using antisera for O157, O26, O103, O111, O121, and O145 (Statens Serum Institute [SSI], Copenhagen, Denmark). The H7 antiserum sorbitol fermentation medium (15) and PCR-restriction fragment length polymorphism (RFLP) analysis of the fliC locus (33) were used for identification of the flagellar (H) antigen. Nonagglutinating isolates were sent to SSI for O:H serotyping. All isolates were stored in glycerol broth at ⫺80°C at UZB. Characterization of isolated VTEC. The isolates were classified into four seropathotypes, A to D (see Table 2; see also Table S1 in the supplemental material), as proposed by Karmali et al. (30). Seropathotype E, comprising VTEC that does not cause disease in humans, was not taken into account, since all VTEC strains originated from clinical cases. A recently developed PCR-based method was applied for the identification of the VT1 and VT2 subtypes (F. Scheutz, L. D. Teel, L. Beutin, D. Piérard, G. Buvens, H. Karch, A. Mellmann, A. Caprioli, R. Tozzoli, A. D. O’Brien, A. R. Melton-Celsa, S. Persson, and N. A. Strockbine, unpublished data), according to the subtyping nomenclature established at the 7th International Symposium on Shiga Toxin (Verocytotoxin)-Producing Escherichia coli Infections (Buenos Aires, Argentina, 10 to 13 May 2009). Additional virulence genes eae, ehxA, saa, subA, espP, katP, and etpD were searched for as described previously (7, 20, 46, 47, 58). All isolates were screened for the presence of O island 122 (OI-122)-associated genes pagC, sen, nleB, nleE, efa1, and efa2 (30, 61). Isolates with positive PCR results for all six OI-122 genes were defined as carrying a complete OI-122 (COI122); those with a negative PCR result for at least one OI-122 gene were considered to have an incomplete OI-122; and isolates with no OI-122positive result by PCR were labeled “OI-122 absent.” PCR control strains. VTEC strains EDL933 (for vtx1 and vtx2) and H.I.8 (for vtx2f) were used as positive controls in the VTEC-screening multiplex PCR and all virulence factor PCRs, except for saa and subA, for which a clinical VTEC O113:H21 isolate (EH1516) was used. VT subtyping controls were O157:H7 strain EDL933 (VT1a and VT2a), O174:H8 strain DG131/3 (VT1c), O8:K85ab:HR strain MH1813 (VT1d), O174: H21 strain 031 (VT2b and VT2c), O118:H12 strain EH250 (VT2b), O73: H18 strain C165-02 (VT2d), O139:K12:H1 strain S1191 (VT2e), O128:H2 strain T4/97 (VT2f), and O2:H25 strain 7v (VT2g). PCR-grade water was used as a negative control. PFGE. Pulsed-field gel electrophoresis (PFGE) was applied according to the PulseNet protocol for E. coli (available at http://www.cdc.gov /pulsenet/) for genomic typing of VTEC isolates. XbaI (Bio-Rad) macrorestriction patterns were obtained by using a CHEF-DR III apparatus (Bio-Rad) and were analyzed with BioNumerics, version 6.0 (Applied Maths, Belgium), using the Dice coefficient and the unweighted-pair group method using average linkages (UPGMA) (optimization and band tolerance, 1%). Salmonella enterica serovar Braenderup H9812 was used as a size marker in all experiments according to PulseNet recommendations.
jcm.asm.org 1337
Buvens et al.
TABLE 1 Samples testing PCR positive for verocytotoxin-producing Escherichia coli in the Brussels-Capital Region No. of samples testing vtx positive by PCR/total number of samples (%) Patient group or period
All
UZ Brussel
External hospitals
Patient groups All samples Hemolytic-uremic syndrome Bloody diarrhea History of bloody diarrhea Epidemiologically linked cases Uncomplicated diarrhea Patients ⱕ6 yrsb Patients ⬎6 yrsb
206/14,705 (1.40) 6/17 (35.3) 14/754 (1.86) 5/97 (5.15) 0/36 (0.0) 35/2,469 (1.41) 95/5,004 (1.89) 109/9,701 (1.12)
112/9,348 (1.20) 2/2 (100) NRa NR NR NR 51/2,742 (1.86) 59/6,606 (0.89)
94/5,357 (1.75) 4/15 (26.7) 14/754 (1.86) 5/97 (5.15) 0/36 (0.0) 35/2,469 (1.41) 44/2,262 (1.94) 50/3,095 (1.61)
Time distribution June–September October–May
112/5,896 (1.89) 94/8,809 (1.07)
58/3,677 (1.57) 54/5,671 (0.95)
54/2,219 (2.43) 40/3,138 (1.27)
a b
NR, not recorded. The ages of two vtx-positive patients were not known.
Antimicrobial susceptibility testing. In vitro susceptibility tests were performed by the disk diffusion method for the antimicrobials listed in Table 6 by using Neo-Sensitabs tablets (Rosco, Taastrup, Denmark), with interpretation of zones according to CLSI, as described by the manufacturer (Rosco Diagnostica A/S, Neo-Sensitabs user’ guide, document 3.1.0, 2010). Statistical analysis. The data were analyzed with Fisher’s exact test, the chi-square test, or Pearson’s corrected chi-square test where appropriate. A probability value of ⱕ0.05 was considered significant.
RESULTS
Incidence of VTEC infection and patient groups at risk. A total of 14,705 stools were investigated for the presence of the vtx1, vtx2, and vtx2f genes (Table 1). Overall, positive vtx PCR results were obtained for 206 (1.40%) samples. Among the unselected stools collected at UZB, 112 (1.20%) of 9,348 samples were vtx positive. A higher rate of vtx positivity was observed among the selected stools from the external hospital laboratories (94/5,357 [1.75%]). VTEC was the third most frequently detected enteropathogen in all stools after Campylobacter spp. (n ⫽ 718 [5.03%]) and Salmonella spp. (n ⫽ 245 [1.71%]). Shigella spp. were detected in 55 (0.38%) cases and Yersinia enterocolitica in 13 (0.09%). Coinfection with VTEC and either Campylobacter spp. (n ⫽ 10), Salmonella spp. (n ⫽ 2), or Clostridium difficile (n ⫽ 1) occurred in 13 patients. Clinical information was recorded for the selected stools collected by the external laboratories (Table 1), but not for the specimens from UZB, except for two HUS cases (Table 1). Stools from patients with HUS (6/17 [35.3%]) were most frequently positive for vtx genes, followed by those from patients with a history of bloody diarrhea (5/97 [5.15%]), children up to the age of 6 years (44/2,262 [1.94%]), and stools containing macroscopic (gross) blood (14/754 [1.86%]). When data from patients with a history of bloody diarrhea and from stools containing macroscopic blood were combined, 2.23% (19/851) were vtx positive. All stools from epidemiologically linked cases of diarrhea (n ⫽ 36) were negative for vtx genes. The rate of vtx-positive stools (59/2,888 [2.04%]) among patients with selected conditions (HUS, history of bloody diarrhea, epidemiologically linked patients, patients up to the age of 6 years, and stools containing macroscopic blood) was significantly higher than that for unselected stools from UZB (1.20%) (P ⫽ 0.001). The rate among stools from UZB (1.20%) did not differ statistically from the rate among stools from patients with
1338
jcm.asm.org
uncomplicated diarrhea collected by the external laboratories (35/ 2,469 [1.41%]) (Table 1). The distribution of vtx-positive stools among the selected samples from external laboratories was analyzed for different age groups. The rate of vtx-positive samples among young patients (0 to 17 years old) (54/2,550 [2.12%]) was significantly higher than that for adult patients 18 to 65 years old (21/1,763 [1.19%]) (P ⫽ 0.022). Stools from patients older than 65 years (19/1,044 [1.82%]) were more frequently positive for vtx than those from patients between 18 and 65 years, but this trend was not significant (P ⫽ 0.17). A clear seasonal distribution was observed among all stools (n ⫽ 14,705), with VTEC infection occurring more frequently during the summer months (Table 1 and Fig. 1). Stools were significantly more positive for vtx from June to September (112/ 5,896 [1.89%]) than from October to May (94/8,809 [1.07%]) (P ⬍ 0.0001). Twenty-four (70.6%) of 34 VTEC O157 isolates in this study were recovered during the summer months, while no such trend was observed for non-O157 VTEC infections. No difference in the rate of vtx-positive samples was observed between male (102/7,220 [1.41%]) and female (104/7,485 [1.39%]) patients. Phenotypic characteristics of VTEC isolates. VTEC isolates could be recovered from 140 (67.9%) of 206 vtx-positive stools, including two samples from two patients,which yielded two isolates. Two O111:H8 isolates, one positive for both vtx1 and vtx2 and the other positive only for vtx1, were isolated from a 48-yearold female HUS patient. The PFGE profiles of the two isolates differed only in one band. Consequently, the two isolates were considered to be one. Furthermore, two different serotypes, O128ab:H⫺ and O176:H⫺, were recovered from a 1-year-old male patient with uncomplicated diarrhea. Thus, a collection of 141 VTEC isolates was established for phenotypic and genotypic characterization. By use of slide agglutination, the O antigens of 133 (94.3%) out of 141 isolates could be identified (Table 2; see also Table S1 in the supplemental material). A total of 60 different O:H serotypes were identified, with serotypes O157:H7/H⫺ (n ⫽ 34), O26:H11/H⫺ (n ⫽ 21), O63:H6 (n ⫽ 8), O111:H8/H⫺ (n ⫽ 7), and O146: H21/H⫺ (n ⫽ 6) accounting for 53.9% of all isolates. The pathogenic serotypes O103:H2 (n ⫽ 2) and O145:H⫺ (n ⫽ 1) occurred
Journal of Clinical Microbiology
Surveillance of VTEC Infections in Brussels
FIG 1 Distribution of verocytotoxin-producing Escherichia coli PCR-positive stools in the Brussels-Capital Region by month, April 2008 to October 2010 (n ⫽ 206).
less frequently. Twenty (33.3%) O:H serotypes were represented by only one VTEC isolate. Eight isolates were autoagglutinating (Orough), and the O antigens of two isolates could not be typed (Ount:H14). The biochemical properties of all isolates were assessed using classical microbiological techniques. Thirty-three of 34 (97.1%) O157 isolates did not ferment sorbitol and were tellurite resistant. One sorbitol-fermenting O157:H⫺ isolate recovered from a 1-year-old male HUS patient was tellurite sensitive. Most nonO157 isolates were sorbitol fermenting (89/107 [83.2%]), and most of these (75/89 [84.3%]) were also tellurite resistant. Eighteen of 107 (16.8%) non-O157 isolates were sorbitol negative, and 15 of these were tellurite resistant. Genotypic characteristics of VTEC isolates. PCR was used to investigate the presence of the vtx1 and vtx2 subtypes, eae, ehxA, saa, subA, espP, katP, etpD, and the OI-122 genes pagC, sen, nleB, nleE, efa1, and efa2 in all isolates. All O157:H7/H⫺ isolates (n ⫽ 34) were PCR positive for vtx2, eae, and COI-122 (Table 3; see also Table S1 in the supplemental material). Fifteen of 34 isolates (44.1%) also carried vtx1. Most O157 isolates carried the plasmid TABLE 2 List of verocytotoxin-producing Escherichia coli serotypes isolated from humans in the Brussels-Capital Region Seropathotype (no. of isolates) A (34) B (31) C (3) D (73)
Serotypes (no. of isolates) O157:H7 (19), O157:H⫺ (15) O26:H11/H⫺ (21), O103:H2 (2), O111:H8/H⫺ (7), O145:H⫺ (1) O91:H21 (1), O104:H2 (1), O113:H21 (1) O2:H6 (2), O4:H16/H⫺ (2), O5:H⫺ (3), O8:H9/H⫺ (3), O15:H27/H⫺ (2), O20:H4/H45 (2), O24:H10 (1), O45:H⫺ (1), O55:H12 (1), O63:H6 (8), O76:H19 (1), O78:H⫺ (1), O79:H14 (1), O80:H⫺ (2), O84:H28/ H⫺ (3), O91:H14/H⫺ (3), O98:H⫺ (2), O113:H2/H4 (2), O115:H⫺ (1), O117:K1:H7 (1), O118:H16/H⫺ (3), O128ab:H⫺ (1), O128ac:H⫺ (1), O132:H34 (1), O136:H20 (1), O146:H21/H⫺ (6), O153:H⫺ (1), O166:H28 (1), O168:H8/H⫺ (2), O171:H29 (1), O176: H⫺ (1), OX182:H34 (1), OX183:H18 (3), Ount:H14 (2), Orough:H4/H10/H19/H21/H⫺ (6)
April 2012 Volume 50 Number 4
genes ehxA (33/34 [97.1%]), espP (31/34 [91.2%]), katP (31/34 [91.2%]), and etpD (33/34 [97.1%]), but none were positive for saa or subA. Of 107 non-O157 VTEC isolates, 59 (55.1%) were positive for vtx1, 36 (33.6%) were positive for vtx2 (including 18 vtx2f-positive isolates), and 12 (11.2%) carried both vtx1 and vtx2 (Table 3; see also Table S1 in the supplemental material). eae and ehxA were, respectively, detected in 65 (60.7%) and 67 (62.6%) isolates. The plasmid genes saa (5/107 [4.67%]), subA (3/107 [2.80%]), espP (40/107 [37.4%]), katP (20/107 [18.7%]), and etpD (8/107 [7.47%]) showed intraserotype variation. A COI-122 was detected in 12 (11.2%) isolates, comprising O111:H8/H⫺ (n ⫽ 7), O5:H⫺
TABLE 3 Distribution of virulence factors in verocytotoxin-producing Escherichia coli isolated in the Brussels-Capital Region, April 2008 to October 2010
Virulence gene
Total no. (%) of isolates (n ⫽ 141)
vtx1 vtx2 vtx1 ⫹ vtx2 eae ehxA saa subA espP katP etpD pagC sen nleB nleE efa O island 122 Complete Incomplete Absent
No (%) of isolates positive for O157 (n ⫽ 34)
Non-O157 (n ⫽ 107)
59 (41.8) 55 (39.0) 27 (19.1) 99 (70.2) 100 (70.9) 5 (3.5) 3 (2.1) 71 (50.3) 51 (36.2) 41 (29.1) 59 (41.8) 78 (55.3) 79 (56.0) 75 (53.2) 73 (51.8)
0 (0.0) 19 (55.9) 15 (44.1) 34 (100) 33 (97.1) 0 (0.0) 0 (0.0) 31 (91.2) 31 (91.2) 33 (97.1) 34 (100) 34 (100) 34 (100) 34 (100) 34 (100)
59 (55.1) 36 (33.6) 12 (11.2) 65 (60.7) 67 (62.6) 5 (4.6) 3 (2.8) 40 (37.4) 20 (18.7) 8 (7.5) 25 (23.4) 44 (41.1) 45 (42.1) 41 (38.3) 39 (36.4)
46 (32.6) 41 (29.1) 54 (38.3)
34 (100) 0 (0.0) 0 (0.0)
12 (11.2) 41 (38.3) 54 (50.5)
jcm.asm.org 1339
Buvens et al.
TABLE 4 Distribution of vtx subtypes in verocytotoxin-producing Escherichia coli O157 and non-O157 isolates No. of isolates
vtx subtypea
Total no. (%) of isolates (n ⫽ 139)b
vtx1a vtx1a ⫹ vtx2a vtx1a ⫹ vtx2a ⫹ vtx2c vtx1a ⫹ vtx2b vtx1a ⫹ vtx2c vtx1c vtx1c ⫹ vtx2b vtx2a vtx2a ⫹ vtx2b vtx2a ⫹ vtx2c vtx2b vtx2c vtx2c ⫹ vtx2d vtx2d vtx2e vtx2f
45 (32.4) 5 (3.6) 1 (0.7) 2 (1.4) 13 (9.4) 13 (9.4) 5 (3.6) 8 (5.8) 1 (0.7) 3 (2.2) 4 (2.9) 10 (7.2) 1 (0.7) 6 (4.3) 4 (2.9) 18 (12.9)
Non-O157 (n ⫽ 105) O157 (n ⫽ 34) 2 1
eae positive (n ⫽ 63)
eae negative (n ⫽ 42)
40 1
5 2 2 1 13 5
12
7
1 1
3 4 9
1 2
1 4 4
18
a
vtx subypes in boldface were detected in VTEC isolates associated with severe disease (HUS and/or bloody diarrhea) in this study. b Two isolates lost their vtx phage before subtyping was performed.
(n ⫽ 2), O80:H⫺ (n ⫽ 2), and O103:H2 (n ⫽ 1); 41 isolates (38.3%) carried an incomplete OI-122; and OI-122 was absent from 54 isolates (50.5%). Subtyping of vtx genes. The distribution of vtx subtypes
among O157 and eae-positive and -negative non-O157 VTEC isolates is shown in Table 4. Three isolates of serotypes O84:H28 (vtx1), O146:H⫺ (vtx1), and O168:H⫺ (vtx2) lost their vtx phage before typing was performed. Six different vtx profiles were identified in O157 isolates. Twelve of 34 isolates carried the profile vtx1a plus vtx2a (35.3%). vtx2c and vtx2a were detected individually in 9 of 34 (26.5%) and 7 of 34 (20.6%) VTEC O157 isolates, respectively. Three were positive for both vtx2a and vtx2c; two were positive for both vtx1a and vtx2a; and one isolate carried the combination vtx1a plus vtx2a plus vtx2c. VTEC non-O157 isolates showed a wider diversity of vtx genes than did VTEC O157 isolates (Table 4). eae-positive and eae-negative non-O157 VTEC isolates differed in their toxin subtypes. eae-positive isolates were frequently positive for vtx1a (41/63 [65.1%]) and vtx2f (18/63 [28.6%]), whereas subtypes vtx1c (18/42 [42.8%]), vtx2b (12/42 [28.6%]), and vtx2e (4/42 [9.5%]) were found only in eae-negative isolates. Toxin subtypes vtx1a, vtx2a, vtx2c, and vtx2d were associated with both eae-positive and eae-negative non-O157 isolates (Table 4). Typing of VTEC strains by PFGE. The molecular relatedness within the most frequent serogroups, O157 (n ⫽ 34), O26 (n ⫽ 21), O63 (n ⫽ 8), and O111 (n ⫽ 8), was assessed using PFGE. The similarity of O157:H7/H⫺ isolates ranged between 61.9% and 100% (Fig. 2). Three clusters comprising five, two, and two cases, respectively, with undistinguishable or highly similar (⬎95%) patterns were identified. The largest cluster contained five O157:H⫺ isolates positive for vtx1a plus vtx2c from three patients with bloody diarrhea, one patient with nonbloody diarrhea, and
FIG 2 PFGE dendrogram of 34 VTEC O157:H7/H⫺ isolates. The bar indicates the degree of similarity. The text columns show, from left to right, the strain number, serotype, vtx subtype, diagnosis, date of isolation, and age (in years) and sex of the patient. HUS, hemolytic-uremic syndrome; BD, bloody diarrhea; D, diarrhea; NA, not available. *, sorbitol-fermenting O157:H⫺ isolate.
1340
jcm.asm.org
Journal of Clinical Microbiology
Surveillance of VTEC Infections in Brussels
FIG 3 PFGE dendrogram of 21 VTEC O26:H11/H⫺ isolates. The bar indicates the degree of similarity. The text columns show, from left to right, the strain number, serotype, vtx subtype, diagnosis, date of isolation, and age (in years) and sex of the patient. BD, bloody diarrhea; D, diarrhea; AP, abdominal pain. *, ESBL-producing isolate.
one patient whose diagnosis was not recorded. The second cluster consisted of two VTEC O157:H⫺ isolates positive for vtx1a plus vtx2c recovered from two bloody diarrhea patients in August 2009. The isolates in clusters 1 and 2 showed a high degree of similarity and were recovered during a 17-month period; therefore, they could represent clones of O157 strains that had spread in the population. The third cluster contained VTEC O157:H7 isolates carrying vtx1a plus vtx2a isolated in August 2008 from two girls with bloody diarrhea living in the same area. O26:H11/H⫺ isolates showed 64.5% to 97.3% similarity (Fig. 3). Three coupled isolates showed a high degree of similarity (⬎94%), but no epidemiological link was known for these patients. The similarity of O63:H6 patterns ranged from 77.1% to 100% (Fig. 4). The XbaI patterns of six O63:H6 isolates recovered in the period from August 2009 to October 2010 from patients living in different areas showed high genetic similarity (93.1% to 100%), and four of these isolates had indistinguishable patterns. Genetic heterogeneity was noted between O111:H8/H⫺ isolates, except for two isolates recovered from a 48-year-old female HUS patient. The PFGE patterns of these isolates, one positive for vtx1a plus vtx2a and the other positive only for vtx1a, differed in one band, corresponding
to the genetic changes following the loss of the vtx2a phage (Fig. 5). As noted above, these isolates were considered to be one. Associations of VTEC serotypes, virulence factors, and clinical data. Clinical data were available for 136 (97.1%) of 140 patients for whom a VTEC isolate could be recovered (Table 5). Most patients were diagnosed with nonbloody diarrhea (92/136 [67.6%]). Nineteen of 136 patients (13.9%) suffered from bloody diarrhea, and 5/136 (3.67%) progressed to HUS. Abdominal pain was reported for 12/136 (8.82%), and 8/136 patients (5.88%) had diseases other than diarrhea or HUS. VTEC O157 was significantly more associated with HUS and bloody diarrhea (14/24 [58.3%]) than with other symptoms (19/112 [16.9%]) (P ⬍ 0.0001). There was, however, no association of VTEC O157 infection with young children, nor did bloody diarrhea occur more often in this patient group. Other highly pathogenic serogroups, O26, O103, O111, and O145, were isolated from patients with HUS or bloody diarrhea (3/24 [12.5%]), as well as from other patients (29/112 [25.9%]). Seven isolates associated with bloody diarrhea belonged to the less well known serotypes O15:H⫺, O91: H⫺, O118:H16/H⫺, and O146:H21/H⫺, as well as to the new serotype OX183:H18.
FIG 4 PFGE dendrogram of eight VTEC O63:H6 isolates. The bar indicates the degree of similarity. The text columns show, from left to right, the strain number, serotype, vtx subtype, diagnosis, date of isolation, and age (in years) and sex of the patient. D, diarrhea; O, disease other than diarrhea or HUS.
April 2012 Volume 50 Number 4
jcm.asm.org 1341
Buvens et al.
FIG 5 PFGE dendrogram of eight VTEC O111:H8/H⫺ isolates. The bar indicates the degree of similarity. The text columns show, from left to right, the strain number, serotype, vtx subtype, diagnosis, date of isolation, age (in years), and sex of the patient. HUS, hemolytic-uremic syndrome; D, diarrhea; AP, abdominal pain. *, both isolates were recovered from a 48-year-old female HUS patient.
The presence of vtx1 was significantly associated with nonbloody diarrhea and disease other than HUS or diarrhea (P ⫽ 0.003), but the association of vtx2 with HUS and bloody diarrhea was borderline nonsignificant (P ⫽ 0.058). Infection with vtx2fpositive VTEC occurred more frequently in young children with uncomplicated diarrhea than in diarrhea patients older than 6 years (P ⫽ 0.046). No significant associations were observed for eae, ehxA, saa, and subA, but isolates carrying espP (P ⫽ 0.01), katP (P ⫽ 0.005), etpD (P ⫽ 0.0005), and COI-122, as well as the individual genes pagC, sen, nleB, and nleE, and the efa gene cluster (P ⬍ 0.01), were significantly associated with HUS and bloody diarrhea. Antimicrobial susceptibility. The frequency of antimicrobial resistance is shown in Table 6. Sixty-three of 141 isolates (44.7%) showed resistance to at least one antibiotic. In particular, resistance was highest to antibiotics used in both human and veterinary medicine, such as sulfonamide (52/141 [36.9%]), tetracycline (39/141 [27.7%]), and ampicillin (37/141 [26.2%]), as well as to streptomycin (52/141 [36.9%]), which is used for veterinary purposes only. Multidrug resistance to streptomycin, sulfonamide, and tetracycline occurred in 34 (24.1%) of 141 VTEC isolates, of which 73.5% (25/34) were also resistant to ampicillin. All isolates showing resistance to streptomycin (n ⫽ 52) were also sulfonamide resistant. One O26:H⫺ isolate recovered from an afebrile 70-year-old man with nonbloody diarrhea and abdominal cramps produced a TEM-52 extended-spectrum beta-lactamase (ESBL). There were no significant differences in the occurrence of resistance between O157 and non-O157 VTEC isolates, nor were there any associations with virulence factors (data not shown).
DISCUSSION
Since no recent detailed data on the occurrence of VTEC infection in Belgium were available, we investigated the incidence of vtx genes in unselected human stools from UZB and selected stools from six other hospital laboratories in Brussels, Belgium. The incidences of vtx genes in the unselected stools (1.20%) and the selected stools (1.75%) were higher than those in a study of Belgian patients during the mid-1990s by Piérard et al. (50). In that study, 1.02% of 17,296 samples were PCR positive for vtx genes. Several reasons may account for the higher incidence in the present study. First, we used a more sensitive multiplex PCR protocol capable of detecting all known vtx subtypes. The PCR protocol (28) used in the study by Piérard et al. was not able to detect the vtx2f subtype, which accounted for 12.7% of all isolates in the present study, and was less sensitive (by a factor of 10 to 100) than the presently used multiplex protocol (data not shown). Second, many of the selected samples in this study were from patient groups that were found to be more prone to VTEC infection in previous studies (6, 60). VTEC was the third-most commonly detected enteropathogen in this study, after Campylobacter spp. and Salmonella spp., but was more frequent than Shigella spp. and Yersinia enterocolitica. It is recommended that all microbiology laboratories routinely test stools for the presence of Campylobacter spp., Salmonella spp., and Shigella spp. (62); however, routine VTEC screening of stools is not yet universally implemented (34). Because Salmonella and Shigella are often detected on the same differential and selective direct plating media, Shigella screening is done without significant
TABLE 5 Diagnoses of patients from whom VTEC isolates were recovered No. (%) of patients positive for VTEC Non-O157 Diagnosis or age group Diagnoses HUS Bloody diarrhea Diarrhea Abdominal pain Disease other than diarrhea or HUS Not available Age groupa Young children (ⱕ6 yr) Older patients (⬎6 yr)a a
Total (n ⫽ 140)
O157 (n ⫽ 34)
Total (n ⫽ 106)
eae positive (n ⫽ 65)
eae-negative (n ⫽ 41)
5 (3.6) 19 (13.6) 92 (65.7) 12 (8.6) 8 (5.7)
4 (11.8) 10 (29.4) 19 (55.9) 0 (0.0) 0 (0.0)
1 (0.9) 9 (8.5) 73 (68.8) 12 (11.3) 8 (7.5)
1 (1.54) 4 (6.15) 52 (80.0) 4 (6.15) 4 (6.15)
0 (0.0) 5 (12.2) 21 (51.2) 8 (19.5) 4 (9.76)
4 (2.8)
1 (2.9)
3 (2.8)
0 (0.0)
3 (7.31)
70 (50.7) 68 (49.3)
19 (55.9) 15 (44.1)
51 (49.1) 53 (50.9)
34 (35.1) 30 (46.9)
17 (42.5) 23 (57.5)
The ages of two patients with non-O157 infection were not known (n ⫽ 138).
1342
jcm.asm.org
Journal of Clinical Microbiology
Surveillance of VTEC Infections in Brussels
TABLE 6 Antimicrobial resistance in verocytotoxin-producing Escherichia coli isolates No. (%) of resistant isolates Type of medicine and antimicrobial agent
All (n ⫽ 141)
O157 (n ⫽ 34)
Non-O157 (n ⫽ 107)
Human Piperacillin–tazobactam Cefuroxime Cefotaxime Ceftriaxone Ceftazidime Cefepime Aztreonam Meropenem Nalidixic acid Ciprofloxacin Amikacin
2 (1.4) 2 (1.4) 2 (1.4) 0 (0.0) 2 (1.4) 0 (0.0) 1 (0.7) 0 (0.0) 14 (9.9) 0 (0.0) 0 (0.0)
0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0)
2 (1.9) 2 (1.9) 2 (1.9) 0 (0.0) 2 (1.9) 0 (0.0) 1 (0.9) 0 (0.0) 14 (13.1) 0 (0.0) 0 (0.0)
Veterinary Kanamycin Streptomycin Chloramphenicol
17 (12.1) 52 (36.9) 9 (6.4)
4 (11.8) 15 (44.1) 1 (2.9)
13 (12.1) 37 (34.6) 8 (7.5)
37 (26.2) 3 (2.1)
8 (23.5) 0 (0.0)
29 (27.1) 3 (2.8)
2 (1.4) 2 (1.4) 39 (27.7) 52 (36.9) 21 (14.9)
0 (0.0) 0 (0.0) 11 (32.4) 15 (44.1) 5 (14.7)
2 (1.9) 2 (1.9) 28 (26.2) 37 (34.6) 16 (15.0)
Human and veterinary Ampicillin Amoxicillin ⫹ clavulanic acid Cefazolin Gentamicin Tetracycline Sulfonamide Trimethoprim
additional costs. The detection of Yersinia enterocolitica requires specific selective media, and it would be more cost-effective to look for this organism only when requested by the physician (62). In Belgium, however, screening for Yersinia in stools is imposed by the Belgian National Institute for Health and Disability Insurance and was thus performed in our study. We have no data on the cost-effectiveness of routine VTEC screening for public health in our country. In Australia, McPherson et al. estimated the total annual cost of VTEC infections between 2003 and 2006 at AUD 2,633,181, equating to a mean cost of AUD 3,132 per case (35). Moreover, Elbasha et al. have shown that if 15 cases of O157 infection had been averted during the 1997 VTEC O157:H7 outbreak, the costs of startup and 5 years of operation of the molecular subtyping-based surveillance system PulseNet in the state of Colorado would have been recovered (12). Infections with non-O157 VTEC strains were more common than infections with O157 strains in Brussels patients. This observation is in line with findings in neighboring countries, such as The Netherlands (60), Germany (2), France (51), Spain (5), and Switzerland (26, 27). In contrast to the situation in these continental European countries, the proportions of O157 and nonO157 infections are thought to be quite different in North America, Argentina, and the United Kingdom (53, 55). Yet a recent study by Hedican et al. showed that non-O157 infections may account for as many as 53% of human VTEC isolates in the United States (19). As in data from other countries (5, 6, 26), O26:H11/H⫺ was the most frequently isolated non-O157 serogroup in Brussels pa-
April 2012 Volume 50 Number 4
tients. Although O26 strains have been associated with HUS in Belgium previously (10), none were associated with HUS in this study. Most were associated with nonbloody diarrhea and abdominal pain, but two patients with O26 infection presented macroscopic blood in their stools. Apart from serogroups O157 and O26, our study revealed a wide diversity of VTEC serotypes that are associated with human disease in Belgium. In order to identify serotypes that were not previously associated with human infection, we compared our results with a database of human VTEC serotypes (http://www.usc.es/ecoli/SEROTIPOSHUM.htm) and with previous studies in Spain (5) and Germany (2), where active surveillance was performed. As a result, we identified 14 serotypes (O4:H16, O20:H4/H45, O24:H10, O55:H12, O63:H6, O84:H28/ H⫺, O132:H34, O136:H20, O171:H29, O176:H⫺, OX182:H34, and OX183:H18) that have been isolated either rarely or never from humans. Previous studies have indicated that the subtype of verocytotoxin produced may influence the clinical outcome of VTEC infections (3, 17, 43). VTEC harboring vtx2a, vtx2c, or the elastaseactivatable vtx2d gene have frequently been associated with HUS and bloody diarrhea, while strains carrying vtx1c or vtx2b have often been isolated from patients with milder infections. Other variants, such as vtx2e and vtx2f, have been associated with animals and were rarely isolated from humans. In this study, subtypes vtx2a and vtx2c were found only in intimin-positive isolates, some of which were associated with HUS or bloody diarrhea. On the other hand, vtx1c, vtx2b, and vtx2e were detected in intimin-negative VTEC. In contrast to other reports, intimin-negative isolates carrying toxin subtype vtx1c or vtx2b (in combination with vtx1a or vtx1c) were recovered from patients with bloody diarrhea. Surprisingly, vtx2f was the only toxin type in 18 non-O157 VTEC isolates belonging to serotypes O63:H6 (n ⫽ 8), O2:H6 (n ⫽ 2), O4:H16 (n ⫽ 1), O45:H⫺ (n ⫽ 1), O113:H2 (n ⫽ 1), O128ac:H⫺ (n ⫽ 1), O132:H34 (n ⫽ 1), O153:H⫺ (n ⫽ 1), and Ount:H14 (n ⫽ 2). vtx2f-positive VTEC strains of serogroups O15, O18ab, O25, O45, O75, and O152 were first isolated from pigeons (38). The incidence of vtx2f VTEC in our study was higher than that in previous reports in England, The Netherlands, and Germany (24, 52, 60). This unusual subtype was the second-most frequent subtype among non-O157 isolates. All vtx2f VTEC isolates carried intimin but were negative for the other virulence factors investigated. Remarkably, 14/18 (77.7%) of vtx2f VTEC infections occurred in children (age range, 0 to 11 years; mean, 2 years; median, 1 year), suggesting that these VTEC isolates may be emerging as a cause of uncomplicated diarrhea in this patient group. Highly similar PFGE patterns were observed for isolates within the serogroups O157, O26, and O63, which could have been part of unidentified outbreaks. Because part of this analysis was done retrospectively, no active outbreak investigation was performed, and no vehicles of transmission were identified. Nevertheless, our data show that most infections among Brussels patients were sporadic, revealing a diverse set of VTEC serotypes and virulence profiles associated with human disease. Moreover, 24.1% of VTEC isolates in this study showed multidrug resistance to antimicrobials used in human and veterinary medicine. In agreement with previous data (37), we found no significant difference in the occurrence of resistance among O157 and non-O157 VTEC isolates. There was no association between resistance and virulence genes, in contrast to the findings of previous studies that showed enhanced resistance to streptomycin, kanamycin, and tetracycline
jcm.asm.org 1343
Buvens et al.
among non-O157 strains carrying intimin (8, 31, 37). We identified a new plasmid-borne ESBL of type TEM-52 in one O26:H⫺ isolate (8). To our knowledge, only three ESBL-producing VTEC isolates have been described in the literature, two belonging to serogroup O26 (CTX-M-3 and CTX-M-18) and one to O157 (CTXM-2) (21, 32, 54). More recently, a CTX-M-15-producing E. coli O104:H4 strain was associated with the outbreak of HUS and bloody diarrhea in Germany in 2011 (16). In this outbreak, 4,075 patients contracted bloody diarrhea; as many as 908 developed HUS, and 50 died (http://www.euro.who.int/en/what-we-do/health-topics /emergencies/international-health-regulations/news/news/2011/07 /outbreaks-of-e.-coli-o104h4-infection-update-30). The O104:H4 outbreak strain showed an unusual combination of pathogenic features typical of VTEC (vtx2a, iha, lpfAO26, lpfAO113, ter, irp2, fyuA) and enteroaggregative E. coli (EAggEC) (aggR, aggA, sigA, sepA, pic, aatA, aaiC, aap) (4, 57) and was more accurately designated an enteroaggregative VTEC strain (EAggEC-VTEC). The source of infection was thought to be germinated fenugreek seeds imported from Egypt (14). EAggEC-VTEC strains of serotypes O104:H4, O111:H2, and O86:H⫺ have been associated with sporadic cases and outbreaks of HUS and bloody diarrhea (22, 39, 40, 56), and no natural reservoir has been established. However, two EAggEC-VTEC isolates of serotypes O104:H4 that did not produce ESBL were reported after travel in Egypt and Tunisia, suggesting the presence of a reservoir in North Africa, possibly human (13, 56). After this outbreak, all isolates from the present study were tested by PCR for aggR but were found negative (data not shown), confirming that EAggEC-VTEC strains are extremely rare in Europe. The aggR gene is the master regulator gene of EAggEC virulence and is a primary diagnostic target (41). The 2011 German O104:H4 outbreak demonstrated the genomic plasticity of E. coli and the adaptive evolution of which it is capable. It may be possible that the combination of verocytotoxin production with an enteroaggregative pattern instead of the attaching-and-effacing adherence pattern allowed for more-efficient systemic delivery of the toxin and therefore resulted in a highly virulent strain. The facts that no animal reservoir of EAggEC-VTEC has been established and that recent O104 cases have been linked to particular geographical locations indicate that these strains may persist only in specific populations. Nevertheless, because these strains are transmissible through food as well as from person to person, they could be disseminated rapidly to other parts in the world. New diagnostic tools that can rapidly recognize the emergence of previously unrecognized E. coli clones with new, successful combinations of virulence factors should be developed. If screening is not systematically done on all cultured stools, at least those from HUS patients should be routinely screened for the presence of O157 and non-O157 VTEC serotypes by using molecular tools. These cases represent the “tip of the iceberg” in terms of the total number of VTEC infections, but this approach may help identify emerging pathogenic serotypes. In conclusion, we showed that VTEC associated with human disease in BCR exhibits broad phenotypic and genotypic diversity. The majority of vtx-positive stools in this study were collected from patients with nonbloody diarrhea, and more than 60% of isolates belonged to sorbitol-fermenting non-O157 serotypes that are not recognized when only culture-based diagnostic techniques are used. It is recommended that clinical laboratories routinely screen all stools for both O157 and non-O157 VTEC using selective culture media and a method of detecting verocytotoxins or vtx
1344
jcm.asm.org
genes, as recently indicated by the Centers for Disease Control and Prevention (CDC) (11). However, keeping in mind the cost, workload, and local differences in VTEC incidence (especially in low-prevalence areas), some clinical laboratories could choose to determine the incidence of VTEC in their test population prior to adopting routine VTEC screening. ACKNOWLEDGMENTS This research was supported by grant 2007-29 of the “Prospective Research for Brussels” program of Innoviris (Brussels-Capital Region) to G.B. All authors declare no conflicts of interest.
REFERENCES 1. Andreoli SP, Trachtman H, Acheson DW, Siegler RL, Obrig TG. 2002. Hemolytic uremic syndrome: epidemiology, pathophysiology, and therapy. Pediatr. Nephrol. 17:293–298. 2. Beutin L, Krause G, Zimmermann S, Kaulfuss S, Gleier K. 2004. Characterization of Shiga toxin-producing Escherichia coli strains isolated from human patients in Germany over a 3-year period. J. Clin. Microbiol. 42:1099 –1108. 3. Bielaszewska M, Friedrich AW, Aldick T, Schurk-Bulgrin R, Karch H. 2006. Shiga toxin activatable by intestinal mucus in Escherichia coli isolated from humans: predictor for a severe clinical outcome. Clin. Infect. Dis. 43:1160 –1167. 4. Bielaszewska M, et al. 22 June 2011. Characterisation of the Escherichia coli strain associated with an outbreak of haemolytic uraemic syndrome in Germany, 2011: a microbiological study. Lancet Infect. Dis. [Epub ahead of print.] doi:10.1016/S1473-3099(11)70165-7. 5. Blanco JE, et al. 2004. Serotypes, virulence genes, and intimin types of Shiga toxin (verotoxin)-producing Escherichia coli isolates from human patients: prevalence in Lugo, Spain, from 1992 through 1999. J. Clin. Microbiol. 42:311–319. 6. Brooks JT, et al. 2005. Non-O157 Shiga toxin-producing Escherichia coli infections in the United States, 1983–2002. J. Infect. Dis. 192:1422–1429. 7. Brunder W, Schmidt H, Frosch M, Karch H. 1999. The large plasmids of Shiga-toxin-producing Escherichia coli (STEC) are highly variable genetic elements. Microbiology 145(Pt 5):1005–1014. 8. Buvens G, Bogaerts P, Glupczynski Y, Lauwers S, Piérard D. 2010. Antimicrobial resistance testing of verocytotoxin-producing Escherichia coli and first description of TEM-52 extended-spectrum beta-lactamase in serogroup O26. Antimicrob. Agents Chemother. 54:4907– 4909. 9. Buvens G, Lauwers S, Piérard D. 2010. Prevalence of subtilase cytotoxin in verocytotoxin-producing Escherichia coli isolated from humans and raw meats in Belgium. Eur. J. Clin. Microbiol. Infect. Dis. 29:1395–1399. 10. Buvens G, et al. 2011. Virulence profiling and quantification of verocytotoxin-producing Escherichia coli O145:H28 and O26:H11 isolated during an ice cream-related hemolytic uremic syndrome outbreak. Foodborne Pathog. Dis. 8:421– 426. 11. Centers for Disease Control and Prevention. 2006. Importance of culture confirmation of Shiga toxin-producing Escherichia coli infection as illustrated by outbreaks of gastroenteritis—New York and North Carolina, 2005. MMWR Morb. Mortal. Wkly. Rep. 55:1042–1045. 12. Elbasha EH, Fitzsimmons TD, Meltzer MI. 2000. Costs and benefits of a subtype-specific surveillance system for identifying Escherichia coli O157:H7 outbreaks. Emerg. Infect. Dis. 6:293–297. 13. European Centre for Disease Prevention and Control. 2011. Shiga toxin/verotoxin-producing Escherichia coli in humans, food and animals in the EU/EEA, with special reference to the German outbreak strain STEC O104. ECDC, Stockholm, Sweden. http://ecdc.europa.eu/en/publications /Publications/1106_TER_EColi_joint_EFSA.pdf. 13a.European Centre for Disease Prevention and Control. 2010. Surveillance report. Annual epidemiological report on communicable diseases in Europe, 2010. ECDC, Stockholm, Sweden. http://ecdc.europa.eu/en /publications/Publications/1011_SUR_Annual_Epidemiological_Report _on_Communicable_Diseases_in_Europe.pdf. 14. European Food Safety Authority. 5 July2011. Tracing seeds, in particular fenugreek (Trigonella foenum-graecum) seeds, in relation to the Shiga toxin-producing E. coli (STEC) O104:H4 2011 outbreaks in Germany and France. EFSA-Q-2011-00817. European Food Safety Authority, Parma, Italy. http://www.efsa.europa.eu/en/supporting/pub/176e.htm.
Journal of Clinical Microbiology
Surveillance of VTEC Infections in Brussels
15. Farmer JJ III, Davis BR. 1985. H7 antiserum-sorbitol fermentation medium: a single tube screening medium for detecting Escherichia coli O157:H7 associated with hemorrhagic colitis. J. Clin. Microbiol. 22:620 – 625. 16. Frank C, et al. 2011. Epidemic profile of Shiga-toxin-producing Escherichia coli O104:H4 outbreak in Germany—preliminary report. N. Engl. J. Med. 365:1771–1780. 17. Friedrich AW, et al. 2002. Escherichia coli harboring Shiga toxin 2 gene variants: frequency and association with clinical symptoms. J. Infect. Dis. 185:74 – 84. 18. Reference deleted. 19. Hedican EB, et al. 2009. Characteristics of O157 versus non-O157 Shiga toxin-producing Escherichia coli infections in Minnesota, 2000 –2006. Clin. Infect. Dis. 49:358 –364. 20. Herold S, Paton JC, Paton AW. 2009. Sab, a novel autotransporter of locus of enterocyte effacement-negative Shiga-toxigenic Escherichia coli O113:H21, contributes to adherence and biofilm formation. Infect. Immun. 77:3234 –3243. 21. Ishii Y, et al. 2005. Extended-spectrum beta-lactamase-producing Shiga toxin gene (Stx1)-positive Escherichia coli O26:H11: a new concern. J. Clin. Microbiol. 43:1072–1075. 22. Iyoda S, et al. 2000. Inducible stx2 phages are lysogenized in the enteroaggregative and other phenotypic Escherichia coli O86:HNM isolated from patients. FEMS Microbiol. Lett. 191:7–10. 23. Jenkins C, et al. 2003. Distribution of the saa gene in strains of Shiga toxin-producing Escherichia coli of human and bovine origins. J. Clin. Microbiol. 41:1775–1778. 24. Jenkins C, et al. 2003. Subtyping of virulence genes in verocytotoxinproducing Escherichia coli (VTEC) other than serogroup O157 associated with disease in the United Kingdom. J. Med. Microbiol. 52:941–947. 25. Johnson KE, Thorpe CM, Sears CL. 2006. The emerging clinical importance of non-O157 Shiga toxin-producing Escherichia coli. Clin. Infect. Dis. 43:1587–1595. 26. Kappeli U, Hachler H, Giezendanner N, Beutin L, Stephan R. 2011. Human infections with non-O157 Shiga toxin-producing Escherichia coli, Switzerland, 2000 –2009. Emerg. Infect. Dis. 17:180 –185. 27. Kappeli U, Hachler H, Giezendanner N, Cheasty T, Stephan R. 2011. Shiga toxin-producing Escherichia coli O157 associated with human infections in Switzerland, 2000 –2009. Epidemiol. Infect. 139:1097–1104. 28. Karch H, Meyer T. 1989. Single primer pair for amplifying segments of distinct Shiga-like-toxin genes by polymerase chain reaction. J. Clin. Microbiol. 27:2751–2757. 29. Karch H, Tarr PI, Bielaszewska M. 2005. Enterohaemorrhagic Escherichia coli in human medicine. Int. J. Med. Microbiol. 295:405– 418. 30. Karmali MA, et al. 2003. Association of genomic O island 122 of Escherichia coli EDL 933 with verocytotoxin-producing Escherichia coli seropathotypes that are linked to epidemic and/or serious disease. J. Clin. Microbiol. 41:4930 – 4940. 31. Kobayashi H, et al. 2001. Prevalence and characteristics of Shiga toxinproducing Escherichia coli from healthy cattle in Japan. Appl. Environ. Microbiol. 67:484 – 489. 32. Kon M, et al. 2005. Cefotaxime-resistant Shiga toxin-producing Escherichia coli O26:H11 isolated from a patient with diarrhea. Kansenshogaku Zasshi 79:161–168. 33. Machado J, Grimont F, Grimont PA. 2000. Identification of Escherichia coli flagellar types by restriction of the amplified fliC gene. Res. Microbiol. 151:535–546. 34. Marcon MJ, Kiska DL, Riddell SW, Gilligan P. 2011. Should all stools be screened for Shiga toxin-producing Escherichia coli? J. Clin. Microbiol. 49:2390 –2397. 35. McPherson M, Kirk MD, Raupach J, Combs B, Butler JR. 2011. Economic costs of Shiga toxin-producing Escherichia coli infection in Australia. Foodborne Pathog. Dis. 8:55– 62. 36. Melton-Celsa AR, O’Brien AD. 1998. Structure, biology, and relative toxicity of Shiga toxin family members for cells and animals, p 121–128. In Kaper JB, O’Brien AD (ed), Escherichia coli O157:H7 and other Shiga toxin-producing E. coli strains. ASM Press, Washington, DC. 37. Mora A, et al. 2005. Antimicrobial resistance of Shiga toxin (verotoxin)producing Escherichia coli O157:H7 and non-O157 strains isolated from humans, cattle, sheep and food in Spain. Res. Microbiol. 156:793– 806. 38. Morabito S, et al. 2001. Detection and characterization of Shiga toxinproducing Escherichia coli in feral pigeons. Vet. Microbiol. 82:275–283. 39. Morabito S, et al. 1998. Enteroaggregative, Shiga toxin-producing Esch-
April 2012 Volume 50 Number 4
40.
41. 42. 43.
44. 45. 46. 47. 48.
49. 50. 51. 52. 53. 54. 55. 56. 57.
58.
59. 60. 61. 62.
erichia coli O111:H2 associated with an outbreak of hemolytic-uremic syndrome. J. Clin. Microbiol. 36:840 – 842. Mossoro C, et al. 2002. Chronic diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome associated with HEp-2 adherent Escherichia coli in adults infected with human immunodeficiency virus in Bangui, Central African Republic. J. Clin. Microbiol. 40:3086 –3088. Nataro JP, Bopp CA, Fields PI, Kaper JB, Strockbine NA. 2011. Escherichia, Shigella, and Salmonella, p 603– 626. In Versalovic J, et al (ed), Manual of clinical microbiology, 10th ed. ASM Press, Washington, DC. Nataro JP, Kaper JB. 1998. Diarrheagenic Escherichia coli. Clin. Microbiol. Rev. 11:142–201. Orth D, et al. 2007. The Shiga toxin genotype rather than the amount of Shiga toxin or the cytotoxicity of Shiga toxin in vitro correlates with the appearance of the hemolytic uremic syndrome. Diagn. Microbiol. Infect. Dis. 59:235–242. Paton AW, et al. 2006. AB5 subtilase cytotoxin inactivates the endoplasmic reticulum chaperone BiP. Nature 443:548 –552. Paton AW, Paton JC. 1998. Detection and characterization of Shiga toxigenic Escherichia coli by using multiplex PCR assays for stx1, stx2, eaeA, enterohemorrhagic E. coli hlyA, rfbO111, and rfbO157. J. Clin. Microbiol. 36:598 – 602. Paton AW, Paton JC. 2002. Direct detection and characterization of Shiga toxigenic Escherichia coli by multiplex PCR for stx1, stx2, eae, ehxA, and saa. J. Clin. Microbiol. 40:271–274. Paton AW, Srimanote P, Talbot UM, Wang H, Paton JC. 2004. A new family of potent AB(5) cytotoxins produced by Shiga toxigenic Escherichia coli. J. Exp. Med. 200:35– 46. Paton AW, Srimanote P, Woodrow MC, Paton JC. 2001. Characterization of Saa, a novel autoagglutinating adhesin produced by locus of enterocyte effacement-negative Shiga-toxigenic Escherichia coli strains that are virulent for humans. Infect. Immun. 69:6999 –7009. Piérard D, et al. 1999. A case-control study of sporadic infection with O157 and non-O157 verocytotoxin-producing Escherichia coli. Epidemiol. Infect. 122:359 –365. Piérard D, Stevens D, Moriau L, Lior H, Lauwers S. 1997. Isolation and virulence factors of verocytotoxin-producing Escherichia coli in human stool samples. Clin. Microbiol. Infect. 3:531–540. Pradel N, et al. 2000. Prevalence and characterization of Shiga toxinproducing Escherichia coli isolated from cattle, food, and children during a one-year prospective study in France. J. Clin. Microbiol. 38:1023–1031. Prager R, Fruth A, Siewert U, Strutz U, Tschape H. 2009. Escherichia coli encoding Shiga toxin 2f as an emerging human pathogen. Int. J. Med. Microbiol. 299:343–353. Rivero MA, Passucci JA, Rodriguez EM, Parma AE. 2010. Role and clinical course of verotoxigenic Escherichia coli infections in childhood acute diarrhoea in Argentina. J. Med. Microbiol. 59:345–352. Roest HI, et al. 2007. Antibiotic resistance in Escherichia coli O157 isolated between 1998 and 2003 in The Netherlands. Tijdschr. Diergeneeskd. 132:954 –958. (In Dutch.) Scallan E, et al. 2011. Foodborne illness acquired in the United States— major pathogens. Emerg. Infect. Dis. 17:7–15. Scavia G, et al. 2011. Similarity of Shiga toxin-producing Escherichia coli O104:H4 strains from Italy and Germany. Emerg. Infect. Dis. 17:1957–1958. Scheutz F, et al. 2011. Characteristics of the enteroaggregative Shiga toxin/verotoxin-producing Escherichia coli O104:H4 strain causing the outbreak of haemolytic uraemic syndrome in Germany, May to June 2011. Euro Surveill. 16(24):pii⫽19889. Schmidt H, Scheef J, Huppertz HI, Frosch M, Karch H. 1999. Escherichia coli O157:H7 and O157:H⫺ strains that do not produce Shiga toxin: phenotypic and genetic characterization of isolates associated with diarrhea and hemolytic-uremic syndrome. J. Clin. Microbiol. 37:3491–3496. Schmidt H, et al. 2000. A new Shiga toxin 2 variant (Stx2f) from Escherichia coli isolated from pigeons. Appl. Environ. Microbiol. 66:1205–1208. van Duynhoven YT, et al. 2008. Prevalence, characterisation and clinical profiles of Shiga toxin-producing Escherichia coli in The Netherlands. Clin. Microbiol. Infect. 14:437– 445. Wickham ME, et al. 2006. Bacterial genetic determinants of non-O157 STEC outbreaks and hemolytic-uremic syndrome after infection. J. Infect. Dis. 194:819 – 827. York MK, Rodrigues-Wong P, Church DL. 2010. Fecal culture for aerobic pathogens of gastroenteritis, p. 3.8.1.1–3.8.5.1. In Garcia LS (ed), Clinical microbiology procedures handbook, 3rd ed. ASM Press, Washington, DC.
jcm.asm.org 1345
