Incidence and virulence determinants of verocytotoxin-producing Escherichia coli

JCM Accepts, published online ahead of print on 11 January 2012 J. Clin. Microbiol. doi:10.1128/jcm.05317-11 Copyright 2012, American Society for Microbiology. All Rights Reserved. Surveillance of VTEC infections in Brussels 1 2 Incidence and virulence determinants of verocytotoxin-producing Escherichia coli infections in Brussels-Capital Region, Belgium, 2008-2010 3 4 5 6 7 Glenn Buvens, 1 * Yves De Gheldre, 2 Anne Dediste, 3 Anne-Isabelle de Moreau, 4 Georges Mascart, 5 Anne Simon, 6 Daniël Allemeersch, 7 Flemming Scheutz, 8 Sabine Lauwers, 1 Denis Piérard 1 Belgian Reference Laboratory for VTEC/STEC, Laboratory for Microbiology and Infection 8 Control, Universitair Ziekenhuis Brussel, Vrije Universiteit Brussel; 1 Centre Hospitalier 9 10 11 12 13 14 Interrégional Edith Cavell; 2 Centre Hospitalier Universitaire St-Pierre, Université Libre de Bruxelles; 3 Hôpitaux Iris-Sud site Bracops; 4 Hôpital Universitaire Des Enfants Reine Fabiola, Université Libre de Bruxelles; 5 Cliniques Universitaires St-Luc, Université Catholique de Louvain; 6 Cliniques de l Europe site St-Elisabeth, 7 Brussels, Belgium; and WHO Collaborating Centre for Reference and Research on Escherichia and Klebsiella, Statens Serum Institute, Copenhagen, Denmark 8 15 16 Running title: Surveillance of VTEC infections in Brussels 17 18 19 20 21 * Corresponding author: Glenn Buvens, Belgian Reference Laboratory for VTEC/STEC, Laboratory for Microbiology and Infection Control, Universitair Ziekenhuis Brussel, Laarbeeklaan 101, 1090 Brussels, Belgium, Phone: 0032 2 477 5001, Fax: 0032 2 477 5015, E-mail: gbuvens@vub.ac.be 22 1

23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 Abstract The incidence of verocytotoxin-producing Escherichia coli (VTEC) was investigated by PCR in all human stools from Universitair Ziekenhuis Brussel (UZB) and selected stools from six other hospital laboratories in Brussels-Capital Region (Belgium) collected between April 2008 and October 2010. Selected stools 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 six years of age, and stools containing macroscopic blood. Verocytotoxin genes (vtx) were detected significantly more frequent in stools from patients with selected conditions (2.04%) as compared to unselected stools from UZB (1.20%) (P=0.001). VTEC were most frequently detected in patients with HUS (35.3%), a history of bloody diarrhea (5.15%), and stools containing macroscopic blood (1.85%). Stools of patients up to 17 years of age were significantly more vtx-positive as compared to those from adult patients between 18 and 65 years old (P=0.022). Although stools from patients older than 65 years were also more frequently positive for vtx as compared to those between 18-65 years, this trend was not significant. VTEC were 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/H- (n=34), O26:H11/H- (n=21), O63:H6 (n=8), O111:H8/H- (n=7), and O146:H21/H- (n=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=107), however, accounted for the bulk (75.9%) of isolates. Fifty-nine (55.1%) were positive for vtx1, 36 (33.6%) for vtx2, and 12 (11.2%) carried both vtx1 and vtx2. Pulsed-field gel electrophoresis revealed a wide genetic diversity, however, small clusters of O157, O26 and O63:H6 were identified that could have been part of unidentified outbreaks. Antimicrobial resistance was observed in 63 (44.7%) isolates and 34 (24.1%) showed multi-drug resistance. 2

48 49 50 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 detecting verocytotoxins or vtx genes. 51 52 Keywords: VTEC/STEC, Brussels, prevalence, epidemiology, virulence factors, PFGE 53 3

54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 INTRODUCTION Verocytotoxin-producing Escherichia coli (VTEC), also called Shiga toxin-producing E. coli (STEC), are 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 (28). VTEC are characterized by their 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) have been described with significant differences in biologic activity, serologic reactivity and receptor binding (36). Many pathogenic strains possess intimin (eae) as part of the locus of enterocyte effacement (LEE) that is associated with adhesion to the intestinal epithelium and formation of attachment and effacement lesions. Most also possess the plasmid-borne enterohemolysin (ehxa), that is cytolytic to human microvascular endothelial cells. Additional plasmid-borne virulence factors, such as an extracellular serine protease (espp), a catalase-peroxydase (katp), and a type II secretion system (etpd) were described, but their role in VTEC pathogenesis is unclear. In recent years, new putative virulence factors, such as STEC autoagglutinating adhesin (saa) and subtilase cytotoxin (subab), were described in LEE-negative VTEC strains (47,48). While saa may be of greater importance for attachment in the gut of animals than in humans (9,22), subab is linked to eukaryotic apoptosis following proteolytic cleavage of the endoplasmic reticulum chaperone BiP (44). VTEC of serotypes O157:H7/H- have 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 (1,6,18,24). Global hot spots, in which non-o157 serotypes dominate O157, include France (51), Germany (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 4

79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 0.66 infections per 100,000 persons [ECDC. Annual epidemiological report on communicable diseases in Europe. 2010. (http://ecdc.europa.eu/en/publications/publications/1011_sur_annual_epidemiological_rep ort_on_communicable_diseases_in_europe.pdf)]. In Belgium, this rate was higher with 0.97 per 100,000. Since 1995, surveillance of VTEC infections in Belgium is conducted through a network of sentinel laboratories that refer stools from patients with HUS and 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 foodborne pathogen remains underestimated. At UZB, in addition to stools referred by sentinel laboratories, all collected stool samples 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 prevalence of VTEC and HUS was investigated (49,50), recent detailed data of 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 of six hospitals located in Brussels- Capital Region (BCR) during 2008-2010. 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. 100 5

101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 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 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 get more insight into patient groups at risk for VTEC infection, we expanded the studied population with selected stools from six external hospital laboratories in BCR (n=5,357). Two hospital laboratories in BCR were not included due to the low number of stools submitted to these laboratories. The six external laboratories were asked to submit up to ten selected stools per week. Samples were selected as follows: those from patients with HUS; from patients with a history of bloody diarrhea; from children up to six years old; from epidemiologically linked cases of diarrhea; and stools containing macroscopic (gross) blood. If the number of ten 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 hours 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, number 2008/041. 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 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) and SMAC with cefixime and tellurite (CT-SMAC). A colony sweep was suspended in nutrient broth and used as DNA template in PCR. All stools were 6

126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 investigated for the presence of VTEC by using a multiplex PCR with specific primers aimed at amplifying vtx1, vtx2, and vtx2f genes (45,59). PCR reactions (20 µl) contained 2 µl of bacterial suspension, 200 µm dntps, 10 mm Tris-HCl, 50 mm KCl, 1.5 mm MgCl 2, 1 µm of each primer, and 1.25 U of AmpliTaq Gold polymerase (Applied Biosystems, Belgium). After an initial denaturation of 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/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 assayed colonies 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 serogrouped by agglutination assay using antisera for O157, O26, O103, O111, O121, and O145 (Statens Serum Institute (SSI), Denmark). The H7-antiserum sorbitol-fermentation medium (15) and PCR-RFLP analysis of the flic locus (33) were used for identification of the flagellar (H) antigen. Non-agglutinating 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 (Table 2 and Supplementary Table 1), as proposed by Karmali et al. (29). Seropathotype E, comprising VTEC that do not cause disease in humans, was not taken into account since all VTEC originated from clinical cases. A recently developed PCR-based method was applied for the identification of 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, in preparation), according to the subtyping nomenclature established at the 7 th International Symposium on Shiga Toxin (Verocytotoxin)- 7

151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 Producing Escherichia coli Infections (Buenos Aires, 10 to 13 May 2009). Additional virulence genes eae, ehxa, saa, suba, espp, katp, and etpd were searched for as described previously (7,19,46,47,58). All isolates were screened for the presence of O island 122 (OI- 122)-associated genes pagc, sen, nleb, nlee, efa1, and efa2 (29,61). Isolates with positive PCR results for all six OI-122 genes were defined as isolates carrying a complete OI-122 (COI-122); an incomplete OI-122 was appointed to those isolates with a negative PCR result for at least one OI-122 gene, and isolates with no positive OI-122 PCR result were labeled with 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). PCRgrade water was used as negative control. Pulsed-field gel electrophoresis. PFGE according to the Pulse-Net U.S.A. protocol for E. coli (available at http://www.cdc.gov/pulsenet/) was applied for genomic typing of VTEC isolates. XbaI (BioRad, U.S.A.) macrorestriction patterns were obtained by using a CHEF-DR III (BioRad, U.S.A.) and analyzed with BioNumerics v6.0 (Applied Maths, Belgium) using the Dice coefficient and the UPGMA method (optimization and band tolerance: 1%). Salmonella enterica serovar Braenderup H9812 was used as a size marker in all experiments according to Pulse-Net recommendations. Antimicrobial susceptibility testing. In vitro susceptibility tests were performed by the disk diffusion method for the antimicrobials listed in Table 6 using Neo-Sensitabs tablets (Rosco, 8

176 177 178 179 180 181 Taastrup, Denmark), with interpretation of zones according to CLSI, as described by the manufacturer [Rosco Diagnostica A/S; Neo-Sensitabs users guide, document 3.1.0, 2010 (http: //www.rosco.dk/)]. Statistical analysis. The data were analyzed with Fisher s exact test, Chi square test, or Pearson s corrected Chi square test where appropriate. Probability value of 0.05 was considered significant. 182 9

183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 RESULTS Incidence of VTEC infection and patient groups at risk. A total of 14,705 stools were investigated for the presence of vtx1, vtx2, and vtx2f genes (Table 1). Overall, a positive vtx PCR was 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%). Co-infection of VTEC with Campylobacter spp. (n=10), Salmonella spp. (n=2), and 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 patients with a history of bloody diarrhea (5/97; 5.15%), children up to six years old (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 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 six years old, and stools containing macroscopic blood) was significantly higher as compared to the percentage of vtx-positives among unselected stools from UZB (1.20%) (P=0.001). The rate among stools from UZB (1.20%) did not statistically differ from the rate among stools from patients with uncomplicated diarrhea collected by the external laboratories (35/2,469; 1.41%) (Table 1). 10

208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 The distribution of vtx-positive stools among the selected samples from external laboratories was analyzed in different age groups. The rate of vtx-positive samples among young patients (0-17 years old) (54/2550; 2.12%) was significantly higher as compared to adult patients from 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 as compared to those 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%) as compared to the period 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 such a trend was not observed for non-o157 VTEC infections. No difference was observed between male (102 vtx-positive samples/7,220; 1.41%) and female patients (104 vtx-positive samples/7,485; 1.39%). Phenotypic characteristics of VTEC isolates. A VTEC isolate could be recovered from 140 (67.9%) of 206 vtx-positive stools, including two samples from which two isolates were recovered. Two O111:H8 isolates, one positive for both vtx1 and vtx2 and one only for vtx1, were isolated from a 48-year-old female HUS patient. The PFGE profiles of both isolates differed only in one band. Consequently, both isolates were taken into account as one. Furthermore, two different serotypes O128ab:H- and O176:H- were recovered from a 1-yearold male patient with uncomplicated diarrhea. Thus, a collection of 141 VTEC isolates was established for phenotypic and genotypic characterization. Using slide agglutination, the O-antigen of 133 (94.3%) out of 141 isolates could be identified (Table 2 & Supplementary Table 1). 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), 11

233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 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 less frequently. Twenty (33.3%) O:H serotypes were represented by only one VTEC isolate. Eight isolates were autoagglutinating (Orough), and the O antigen of two isolates could not be typed (Ount:H14). Biochemical properties of all isolates were assessed using classical microbiological techniques. Thirty-three (33/34; 97.1%) O157 isolates did not ferment sorbitol, and were tellurite-resistant. One sorbitol-fermenting O157:H- recovered from a 1-year-old male HUS patient was tellurite-sensitive. Most non-o157 isolates were sorbitol-fermenting (89/107; 83.2%), of which most (75/89; 84.3%) were also tellurite-resistant. Eighteen (18/107; 16.8%) non-o157 isolates were sorbitol negative, of which 15 were tellurite-resistant. Genotypic characteristics of VTEC isolates. PCR was used to investigate the presence of vtx1 and vtx2 subtypes, eae, ehxa, saa, suba, espp, katp, etpd, and 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 a COI-122 (Table 3 and Supplementary Table 1). Fifteen (15/34; 44.1%) also carried vtx1. Most O157 carried the plasmid 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%) for vtx2 (including 18 vtx2f-positive isolates), and 12 (11.2%) carried both vtx1 and vtx2 (Table 3 and Supplementary Table 1). 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 intra-serotype variation. A COI-122 was detected in 12 (11.2%) isolates, comprising O111:H8/H- (n=7), O5:H- (n=2), O80:H- (n=2), and O103:H2 (n=1); 41 (38.3%) carried an incomplete OI-122, and OI-122 was absent in 54 (50.5%). 12

257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 Subtyping of vtx genes. The distribution of vtx subtypes in O157 and eae-positive and negative non-o157 VTEC 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 isolates carried the profile vtx1a + vtx2a (12/34; 35.3%). vtx2c and vtx2a were detected individually in nine (9/34; 26.5%) and seven (7/34; 20.6%) VTEC O157, respectively. Three were positive for both vtx2a and vtx2c, two for both vtx1a + vtx2a and one isolate carried the combination vtx1a + vtx2a + vtx2c. As compared to O157 VTEC, non-o157 isolates showed a wider diversity of vtx genes (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 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 + vtx2c from patients with bloody diarrhea (n=3), non-bloody diarrhea (n=1), and one patient whose diagnosis was not recorded. The second cluster consisted of two VTEC O157:H- isolates positive for vtx1a + 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 were spread in the population. The third cluster contained VTEC O157:H7 13

282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 carrying vtx1a + 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). XbaI patterns of six O63:H6 isolates recovered in the period August 2009 to October 2010 from patients living in different areas showed high genetic similarity (93.1% to 100%), among which four 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 + vtx2a and one only for vtx1a, differed in one band corresponding to the genetic changes following loss of the vtx2a phage. As noted above, these isolates were taken into account as one. Associations of VTEC serotypes, virulence factors, and clinical data. Clinical data were available for 136 (97.1%) of 140 patients for which a VTEC isolate could be recovered (Table 5). Most patients were diagnosed with non-bloody diarrhea (92/136; 67.6%). Nineteen (19) 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%) as compared to patients 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-, O146:H21/H-, and the new serotype OX183:H18. 14

306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 The presence of vtx1 was significantly associated with non-bloody diarrhea and disease other than HUS or diarrhea (P=0.003), but the association of vtx2 with HUS and bloody diarrhea was borderline not significant (P=0.058). Infection with vtx2f-positive VTEC occurred more frequently in young children with uncomplicated diarrhea as compared to diarrhea patients older than six 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, 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 (63/141; 44.7%) isolates showed resistance to at least one antibiotic. In particular, resistance was highest to those 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%), and streptomycin (52/141; 36.9%) which is used for veterinary purposes only. Multi-drug 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 non-bloody diarrhea and abdominal cramps produced a TEM-52 extended-spectrum beta-lactamase (ESBL). There were no significant differences in the occurrence of resistance in O157 and non-o157 VTEC isolates, nor were there any associations with virulence factors (data not shown). 326 15

327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 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 as compared to 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 (27) 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 (factor 10-100) as compared to 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 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. (32), 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 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 (32). 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- 16

352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 effectiveness of routine VTEC screening on public health in our country. In Australia, McPherson et al. estimated the total annual cost of VTEC infections between 2003-2006 of 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 were averted during the 1997 VTEC O157:H7 outbreak, the costs of start-up and five years of operation of the molecular subtyping-based surveillance system PulseNet in the state of Colorado, U.S.A. would have been recovered (12). Infections with non-o157 VTEC were more common in Brussels patients than those with O157 strains. This observation is in line with findings in neighboring countries, such as the Netherlands (60), Germany (2), France (51), Spain (5), and Switzerland (25,26). In contrast to the situation in these continental European countries, the proportion of O157 and non-o157 infections is 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 up to 53% of human VTEC isolates in the United States (18). Analogous with data in other countries (5,6,25), O26:H11/H- was the most frequently isolated non-o157 serogroup in Brussels patients. Although O26 strains have been associated with HUS in Belgium before (10), none were associated with HUS in this study. Most were associated with non-bloody diarrhea and abdominal pain, but two cases of 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 previously not associated with human infection, we compared our results with a database of human VTEC serotypes (http://www.usc.es/ecoli/serotiposhum.htm), and 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, 17

377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 O171:H29, O176:H-, OX182:H34, and OX183:H18) that have either rarely or never been isolated 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 have been frequently associated with HUS and bloody diarrhea, while strains carrying vtx1c or vtx2b were often 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 subtypes 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 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 as compared to previous reports in England, the Netherlands, and Germany (23,52,60). This unusual subtype was the secondmost frequent subtype among non-o157 isolates. All vtx2f VTEC carried intimin, but were negative for other virulence factors investigated. Remarkably, 14/18 (77.7%) of vtx2f VTEC infections occurred in children (age range 0-11 yrs, mean 2 yrs, median 1 yr) suggesting that these VTEC 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 18

402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 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 multi-drug resistance to antimicrobials used in human and veterinary medicine. In concordance with previous data (37), we found no significant difference in the occurrence of resistance among O157 and non-o157 VTEC. There was no association between resistance and virulence genes, which is in contrast to previous studies that showed an enhanced resistance to streptomycin, kanamycin, and tetracycline among non-o157 strains carrying intimin (8,30,37). We identified a new plasmid-borne ESBL of type TEM-52 in one O26:Hisolate (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 (CTX-M-2) (20,31,54). More recently, an 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 of which as much as 908 developed HUS and 50 died [WHO. Outbreaks of E. coli O104:H4 infection: update 30. 2011 (http://www.euro.who.int/en/what-we-do/health-topics/emergencies/international-health- 418 regulations/news/news/2011/07/outbreaks-of-e.-coli-o104h4-infection-update-30)]. The 419 420 421 422 423 424 425 426 O104:H4 outbreak strain showed an unusual combination of pathogenic features typical of VTEC (vtx2a, iha, lpfa O26, lpfa O113, ter, irp2, fyua) and enteroaggregative E. coli (EAggEC) (aggr, agga, siga, sepa, pic, aata, aaic, aap) (4,57), and was more accurately designated as an enteroaggregative VTEC (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 (21,39,40,56), and no natural reservoir has been established. However, two EAggEC-VTEC isolates of serotypes O104:H4 that did not produce ESBL 19

427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 were reported after travel in Egypt and Tunisia, suggesting the presence of a reservoir in North-Aftrica, possibly human (13,56). After this outbreak, all isolates from the present study were tested by PCR for aggr, but 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 it is capable of. It may be possible that the combination of verocytotoxin production with an enteroaggregative instead of the attaching-and-effacing adherence pattern allowed for a more efficient systemic delivery of the toxin, and therefore resulted in a highly virulent strain. The fact that no animal reservoir of EAggEC-VTEC has been established and that recent O104 cases were linked to particular geographical locations indicates that these strains may persist only in specific populations. Nevertheless, because these strains are transmissible through food as well as from person-to-person their dissemination to other parts in the world could occur rapidly. New diagnostic tools should be developed that are able to rapidly recognize the emergence of previously not recognized E. coli clones with new successful combinations of virulence factors. If 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 using molecular tools. These cases represent the top of the iceberg in terms of the total number of VTEC infections, but this approach may help identifying emerging pathogenic serotypes. In conclusion, we showed that VTEC associated with human disease in BCR exhibit broad pheno- and genotypical diversity. The majority of vtx-positive stools in this study were collected from patients with non-bloody diarrhea, and more than 60% of isolates belonged to sorbitol-fermenting non-o157 serotypes that are not recognized when using only culturebased diagnostic techniques. It is recommended that clinical laboratories routinely screen all 20

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