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A hybrid strain of enteroaggregative and Shiga toxin 2-producing Escherichia coli (EAEC-STEC) serotype O104:H4 strain caused a large outbreak of haemolytic uraemic syndrome and bloody diarrhoea in 2011 in Europe. Two surveys were performed in the European Union (EU) and European Economic Area (EEA) countries to assess their laboratory capabilities to detect and characterise this previously uncommon STEC strain. Prior to the outbreak, 11 of the 32 countries in this survey had capacity at national reference laboratory (NRL) level for epidemic case confirmation according to the EU definition. During the outbreak, at primary diagnostic level, nine countries reported that clinical microbiology laboratories routinely used Shiga toxin detection assays suitable for diagnosis of infections with EAEC-STEC O104:H4, while 14 countries had NRL capacity to confirm epidemic cases. Six months after the outbreak, 22 countries reported NRL capacity to confirm such cases following initiatives taken by NRLs and the European Centre for Disease Prevention and Control (ECDC) Food- and Waterborne Disease and Zoonoses laboratory network. These data highlight the challenge of detection and confirmation of epidemic infections caused by atypical STEC strains and the benefits of coordinated EU laboratory networks to strengthen capabilities in response to a major outbreak.

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An enteroaggregative Verotoxin (Vtx)-producing Escherichia coli strain of serotype O104:H4 has recently been associated with an outbreak of haemolytic-uremic syndrome and bloody diarrhoea in humans mainly in Germany, but also in 14 other European countries, USA and Canada. This O104:H4 E. coli strain has often been described as an enterohaemorrhagic E. coli (EHEC), i.e. a Vtx-producing E. coli with attaching and effacing properties. Although both EHEC and the German O104:H4 E. coli strains indeed produce Vtx, they nevertheless differ in several other virulence traits, as well as in epidemiological characteristics. For instance, the primary sources and vehicles of typical EHEC infections in humans are ruminants, whereas no animal reservoir has been identified for enteroaggregative E. coli (EAggEC). The present article is introduced by a brief overview of the main characteristics of Vtx-producing E. coli and EAggEC. Thereafter, the O104:H4 E. coli outbreak is compared to typical EHEC outbreaks and the virulence factors and host specificity of EHEC and EAggEC are discussed. Finally, a renewed nomenclature of Vtxproducing E. coli is proposed to avoid more confusion in communication during future outbreaks and to replace the acronym EHEC that only refers to a clinical condition.

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In September-October 2007, a mixed-serotype outbreak of verocytotoxin-producing Escherichia coli (VTEC) O145:H28 and O26:H11 occurred in the province of Antwerp, Belgium. Five girls aged between 2 and 11 years developed hemolytic uremic syndrome, and seven other coexposed persons with bloody diarrhea were iden- tified. Laboratory confirmation of O145:H28 infection was obtained for three hemolytic uremic syndrome pa- tients, one of whom was coinfected with O26:H11. The epidemiological and laboratory investigations revealed ice cream as the most likely source of the outbreak. The ice cream was produced at a local dairy farm using pasteurized milk. VTEC of both serotypes with indistinguishable pulsed-field gel electrophoresis patterns were isolated from patients, ice cream, and environmental samples. Quantitative analysis of the ice cream indicated concentrations of 2.4 and 0.03 CFU=g for VTEC O145 and O26, respectively. Virulence typing revealed that the repertoire of virulence genes carried by the O145:H28 outbreak strain was comparable to that of O157 VTEC and more exhaustive as compared to the O26:H11 outbreak strain and nonrelated clinical strains belonging to these serotypes. Taken together, these data suggest that O145:H28 played the most important role in this outbreak.

AIMS: This study evaluated a typing method of O26:H11 enterohaemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC) based on the variation in genomic location and copy numbers of IS621. METHODS AND RESULTS: Two multiplex PCRs, targeting either the left (5') or right (3') IS/chromosome junction of 12 IS621 insertion sites and one PCR specific of another truncated copy, were developed. Thirty-eight amplification profiles were observed amongst a collection of 69 human and bovine O26:H11 EHEC and EPEC. Seventy-one per cent of the 45 EHEC and EPEC with identical IS621 fingerprints within groups of two, three or four isolates had >85% pulsed field gel electrophoresis (PFGE) profile similarity, including four groups of epidemiologically related EHEC or EPEC, while most of the groups had CONCLUSIONS: The IS621 fingerprinting and the PFGE are complementary typing assays of EHEC and EPEC; though, the former is less discriminatory. SIGNIFICANCE AND IMPACT OF THE STUDY: The IS621 printing method represents a rapid (24 h) first-line surveillance and typing assay, to compare and trace back O26:H11 EHEC and EPEC during surveys in farms, multiple human cases and outbreaks.

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The susceptibilities of 824 Bacteroides fragilis group isolates against nine antibiotics were evaluated in a Europe-wide study involving 13 countries. Species determination, by different methods, was carried out on all but one isolate. Resistance rates were evaluated according to species and geographical areas via CLSI and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints. The present data were compared with those obtained 10 and 20 years ago at a European level. High-level resistance (MIC ≥ 64 mg/L) to ampicillin was observed in 44.5% of the strains, which is a significant increase relative to 20 years ago (16%). Piperacillin/tazobactam was more active than amoxicillin/clavulanic acid (3.1% and 10.4% resistance, respectively), again with a resistance increase relative to earlier studies. Dramatic increases in resistance were observed for cefoxitin, clindamycin and moxifloxacin, with rates of 17.2%, 32.4% and 13.6%, respectively. The lowest resistances were found for imipenem, metronidazole and tigecycline (1.2%

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Preliminary evaluation of multiplex PCR-based O157 IS-printing method for subtyping of verocytotoxin-producing Escherichia coli O157 G. Buvens, O. Soetens, S. Lauwers, D. Piérard Belgian Reference Laboratory for VTEC/EHEC, Department of Microbiology and Infection Control, Universitair Ziekenhuis Brussel, Brussels, Belgium Background: Verocytotoxin-producing Escherichia coli (VTEC) are frequent causes of bloody diarrhea (BD) and the hemolytic-uremic syndrome (HUS). Whereas more than 200 serotypes have been described, O157:H7/non-motile are the most commonly associated with BD and HUS. Strong phylogenetic similarity between O157 isolates has been shown by genomic cluster analysis. Only highly discriminative but time-consuming techniques like pulsed-field gel electrophoresis (PFGE) and multilocus variable number of tandem repeats analysis (MLVA) are useful for O157 subtyping. For efficient infection control rapid techniques are needed. Ooka et al. recently described a multiplex PCR-based subtypingmethod based on the variable location of Insertion Sequence 629 (IS629) among VTEC O157 strains (O157 IS-printing) (1). Objectives: To evaluate O157 IS-printing as compared to PFGE. Materials and methods: Thirty-seven VTEC O157 isolates were included. O157 IS-printing was applied as described (1). PFGE was performed according to CDC PulseNet protocol for E. coli. Banding patterns were analyzed using GelCompar II. Results: Using PFGE, indistinguishable band patterns were obtained for 12 O157:H7 isolates associated with an outbreak during the summer of 2008, for 2 isolates from 2 siblings and for 2 O157:H7 recovered from 1 patient, respectively. Twenty-one other epidemiologically unrelated isolates showed distinct band patterns. O157 IS-printing yielded similar clustering as PFGE with the exception of 3 pairs of PFGE distinct isolates that clustered together. Conclusions: O157 IS-printing can be performed within 5 hours while several days are needed for PFGE, reducing the workload dramatically. No specialized equipment is needed, and a large number of strains can be analyzed simultaneously. Although less discriminative, it is a suitable tool for rapid subtyping of VTEC O157 isolates. Acknowledgements: Research funded by grant 2007-29 of "Prospective Research for Brussels" program of the Brussels-Capital Region to G.B. 1. Ooka T, Terajima J, Kusumoto M et al. J Clin Microbiol 2009;47:2888-94

Initial evaluations of the COBAS(R) AmpliPrep/COBAS(R) TaqMan(R) HIV-1 Test (CAP/CTM) showed good performance but afterwards reports about under-quantification became available. The aim of this study was to investigate if the problem is solved with the second version of this assay, the COBAS(R) AmpliPrep/COBAS(R) TaqMan(R) HIV-1 Test, version 2.0 (CAP/CTM v2.0). Remaining plasma of 375 consecutive HIV-1 positive samples with viral load >/=4000 copies/ml was collected in 3 labs. Samples were diluted and retested with our routine method COBAS(R) AmpliPrep/COBAS(R) AMPLICOR(R) HIV-1 MONITOR Test v1.5 in the ultrasensitive mode (CAP/CA PHS) as well as with CAP/CTM and CAP/CTM v2.0 tests. An absolute difference between the results of two methods of >/=0.71 log10 copies/ml (cp/ml) was defined as moderately discrepant and an absolute difference of >/=0.93 log10 cp/ml as severely discrepant. In addition, criteria for considering the new methods equivalent to the routine method were formulated. CAP/CTM compared to CAP/CA PHS: 36 (9.5%) and 20 (5.3%) samples were respectively considered as moderately and severely under-quantified by CAP/CTM. The mean difference between CAP/CTM and CAP/CA PHS was -0.32 log10 cp/ml. Eight out of 19 of the severely under-quantified samples were from patients infected with HIV-1 subtype B strain. CAP/CTM v2.0 compared to CAP/CA PHS: No sample was moderately or severely under-quantified by CAP/CTM v2.0. A mean difference of +0.08 log10 cp/ml was found with CAP/CTM v2.0 compared to CAP/CA PHS. The under-quantification problem of the CAP/CTM kit was clearly demonstrated. Criteria for equivalence of CAP/CTM v2.0 to the routine test CAP/CA PHS were fulfilled.

Accumulating evidence suggests that differences in pathogenic potential of Verocytotoxin-producing Escherichia coli (VTEC) are due to specific virulence characteristics encoded on horizontally acquired pathogenicity islands such as the locus of enterocyte effacement (LEE), and O Island 122 (OI 122). Whereas intimin and enterohemolysin are used in VTEC diagnostics for many years, the prevalence of OI 122 genes in VTEC associated with human disease in Belgium is unknown. In addition to the characterization of a limited number of VTEC O157, we compared the distribution of OI 122 and other virulence genes in non-O157 VTEC isolated from patients with and without the hemolytic uremic syndrome (HUS). One hundred and twenty-nine non-O157 VTEC consecutively isolated from routinely submitted stool samples (2 from HUS patients), seventeen non-O157 VTEC isolates from HUS patients, and twenty-one O157 VTEC (of which 10 from HUS patients) were included in this study. PCR was used for the detection of VT1, VT2, eae, multiple plasmid genes (ehx, saa, espP, katP, and etpD), and 9 ORFs of OI 122. All O157 VTEC isolates (16 O157:H7 and 5 O157:H-) carried VT2, eae, and all plasmid genes, except one sorbitol-fermenting O157:H- isolate which lacked espP and katP. Four isolates were carrying both VT1 and VT2. All O157 VTEC possessed a complete OI 122 (COI 122) with all nine ORFs present. The virulence factors present in 19 non-O157 VTEC isolates recovered from HUS patients were compared to those present in the 127 isolates from patients without HUS. While VT1 was significantly more present in VTEC not associated with HUS (pTwenty-seven out of 146 (18.5%) non-O157 isolates carried a COI 122, 81 (55.5%) had an incomplete OI 122, and in 38 (26%) isolates OI 122 was not detected. Isolates associated with HUS (7/19; 36.8%) carried significantly more often a COI 122 than non-HUS isolates (20/127; 15.7%) (pOur study confirms the association of OI 122 with O157 and with severe disease induced by non-O157. This genomic island could be considered in addition to the toxin type and other virulence factors for determining the pathogenicity of non-O157 clinical isolates. Our data show that the prevalence of COI 122 in non-O157 VTEC in Belgium is lower compared to other reports. * research supported by Brussels-Capital Region grant PRFB 2007-29 allowed to G Buvens

In order to evaluate the prevalence of infections with Verocytotoxin-producing Escherichia coli (VTEC) in the Brussels-Capital Region, clinical stool samples collected by 6 of the 9 hospital laboratories for microbiology were investigated. In addition to routinely submitted stool samples from UZ Brussel, all other participating laboratories were asked to submit ten stools per week for VTEC screening. Samples to include in this study were selected as follows: those from patients with HUS; from patients with a history of bloody diarrhea; from children Stool samples were cultured on sorbitol MacConkey (SMAC) agar and SMAC with cefixime and tellurite. A colony sweep and single colonies were tested for VTEC using a multiplex PCR targeting VT1, VT2, and their subtypes. Isolated VTEC were tested for the presence of the eae and ehx genes, and latex agglutination was used to identify serogroups O157, O26, O103, O111, O121, and O145. A total of 4240 stool samples were screened for VTEC between January and October 2008, with 67 (1.58%) positive PCR results. VTEC were isolated from 46 (68.6%) PCR positive stools. Twenty-four (52.2%) isolates were positive for VT1, 14 (30.4%) for VT2 (including 3 VT2f), and 8 for both VT1 and VT2. The eae and ehx genes were present in 30 (65.2%) out of 46 isolates. Eighteen (39.1%) isolates belonged to the six previously mentioned O serogroups, comprising 8 O157, 5 O26, 3 O111, and 2 O103. The highest prevalence of VTEC was detected in stools from patients with a history of bloody diarrhea (8.8%) followed by stools containing macroscopic blood (2.4%). Prevalence among young children (2.06%) was significantly higher than in random diarrhea samples (0.67%) (pThis surveillance shows an increased prevalence of VTEC compared to previous data collected in our lab. The majority of isolates (82.6%) belonged to non-O157 serogroups. Comparable proportions were recently reported in the Netherlands. Our data show that clinical laboratories should consider testing samples from patients with HUS, bloody diarrhea or a history of bloody diarrhea for the presence of VTEC. Screening of pediatric stool samples can also be considered. * research supported by Brussels-Capital Region grant PRFB 2007-29 to G Buvens

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Bordetella pertussis, the causative agent of whooping cough, has remained endemic and there is a resurgence in some countries despite vaccination. Bordetella pertussis produces a wide range of virulence factors which are assumed to play an important role in infection and transmission, including tracheal colonization factor (TcfA). Here we show that clinical isolates belonging to distinct lineages may lose their ability to produce TcfA. Irreversible and reversible loss occurred, respectively, by recombination between repeats leading to deletion of the tcfA gene and by mutations in a polymorphic G-track. These phenomena may reflect adaptation to distinct niches.

Objectives: To collect recent data on the susceptibility of anaerobes and to compare them with results from previous studies. Methods: Four hundred and forty-three anaerobic clinical isolates from various body sites were prospectively collected from October 2003 to February 2005 in nine Belgian hospitals. MICs were determined for nine anti-anaerobic and three recently developed antibiotics. Results: Most Gram-negative bacilli except Fusobacterium spp. were resistant to penicillin. Piperacillin/tazobactam, metronidazole, chloramphenicol, meropenem and amoxicillin/clavulanic acid were very active against all groups, but only 86% of Bacteroides fragilis group strains were susceptible to the latter. Cefoxitin, cefotetan and clindamycin were less active. In particular, only 62%, 52% and 48% of B. fragilis group strains were susceptible, respectively. Clindamycin shows a continuing decrease in activity, as 83% were still susceptible in 1987 and 66% in 1993-94. Anti-anaerobic activity of the new antibiotics is interesting, with MIC50 and MIC90 of 1 and > 32 mg/L for moxifloxacin, 2 and 4 mg/L for linezolid and 0.5 and 8 mg/L for tigecycline. Conclusions: The susceptibility of anaerobic bacteria remains stable in Belgium, except for clindamycin, which shows a continuous decrease in activity. However, for each of the tested antibiotics, at least a few resistant organisms were detected. Consequently, for severe infections involving anaerobic bacteria, it could be advisable to perform microbiological testing instead of relying on known susceptibility profiles. Periodically monitoring background susceptibility remains necessary to guide empirical therapy.

Temocillin is a narrow spectrum penicillin with high stability to most beta-lactamases including AmpC types and extended-spectrum types (ESBLs). We have analysed its in vitro activity against 652 clinical isolates of Enterobacteriaceae prospectively collected from patients hospitalised in intensive care units at seven different university hospitals in Belgium in 2005. Strains were screened for ESBL production using cefotaxime and ceftazidime screen agar plates and by double ESBL E-tests. The MIC of temocillin and of five comparators was determined using the E-test method. ESBLs were characterized at one central laboratory by isoelectric focusing, PCR for bla genes of the SHV, TEM, and CTX-M families, and by DNA sequencing. The prevalence of ESBL-producing Enterobacteriaceae averaged 11.8% and ranged between 3.0 and 29% in the different hospitals. Meropenem exhibited the highest in vitro activity overall (mode MIC 0.064 mug; MIC(90); 0.19 mug/ml), whereas ceftazidime (MIC(90) > 256 mug/ml) and ciprofloxacin (MIC(90) > 32 mug/ml) scored the worst. Temocillin was active against more than 90% of the isolates including most AmpC- and ESBL-producing isolates. These data indicate the well preserved activity of temocillin over the years against Enterobacteriaceae and show the wide variation in prevalence of ESBL-producing Enterobacteriaceae isolates in Belgian intensive care units. Prospective clinical studies are, however, needed to validate the usefulness of temocillin in the treatment of microbiologically documented infections caused by ESBL- and/or AmpC- overproducing nosocomial Enterobacteriaceae pathogens.

Twee meisjes van 11 en 13 jaar die in juni 2006 samen met hun ouders en twee vriendinnen op een vakantieboerderij logeerden, maakten kort daarop een hemorragische colitis door. Ze werden besmet door een enterohemorragische Escherichia coli O157: H7 (EHEC). In de daaropvolgende week ontwikkelde één van de patiënten een hemolytisch uremisch syndroom waarvoor ze gedialyseerd werd. Tijdens het verblijf op de boerderij waren ze betrokken bij het verzorgen van de dieren. Ook speelden ze in de stallen en dronken ze van verse ongepasteuriseerde koemelk. Het melkstaal dat nadien genomen werd uit de melkopslagtank was negatief voor EHEC. Uit de stof-, bodem- en meststalen van de runderen werden drie stammen geïsoleerd die genetisch identiek waren aan de humane E. coli O157: H7-stam.

EHEC O157 were isolated from faeces of Belgian and Polish beef slaughter cattle. In Belgium, 1281 faecal samples were analysed by immunomagnetic separation [IMS] after enrichment in buffered peptone water from June 1998 till July 1999. Eighty-one samples (6.3%) were positive for E. coli O157. Phage type 8 was most frequently found. Bulls between 1 and 2 years old, slaughtered in September and October were most frequently found positive. Atypical biochemical features were observed in some isolates: 22 (27%) isolates were urease positive and 1 (1.2%) isolate was unable to ferment lactose. In Poland, 551 faecal samples, taken from January 1999 till December 1999, were examined using exactly the same techniques. Four faecal samples (0.7%) were positive for O157 EHEC, yielding seven phage type 8 isolates. All positive samples were from cattle younger than 2 years. Positive samples occurred in August, September and October.

BACKGROUND: The presence of infective microorganisms in the bilio-pancreatic tract is believed to be important in both the onset and outcome of acute biliary pancreatitis. In this study, the characteristics of bile colonization or infection in human pancreatitis were investigated in order to optimize prophylactic antibiotic therapy. METHODS: In 174 patients, 22 clinical and biological factors were recorded prospectively on admission and compared with the bacteriological findings at the time of surgery. RESULTS: There was a significant difference between patients with negative or positive bile cultures in six parameters: Age (57.7 +/- 1.7 vs. 68.5 +/- 1.5 years, p

Verocytotoxine-producerende Escherichia coli (VTEC) veroorzaakt bloederige of vloeibare diarree die naar een hemorragische colitis of een hemolytisch uremisch syndroom (HUS) kan evolueren. VTEC van het serotype O157:H7 kan in het routine diagnostisch laboratorium opgespoord worden op het sorbitol-McConkey-agarmedium, maar men raadt meestal aan om dit enkel op geselecteerde stalen toe te passen. Het is dus nodig om gerichte klinische inlichtingen aan het laboratorium mee te delen. Andere serotypes zijn moeilijk op te sporen en in de meest ernstige gevallen, bijvoorbeeld HUS of uitbraken, zal men beroep doen op een gespecialiseerd laboratorium. Deze organismen worden in circa 1% van de fecesmonsters afgezonderd in het AZ-VUB. Het meest pathogene serotype, O157:H7, dat slechts een vijfde van de isolaten vertegenwoordigt, is nochtans verantwoordelijk voor drie vierde van de HUS-gevallen. In tegenstelling met andere landen zijn er nog geen epidemieën gerapporteerd in België, maar waakzaamheid blijft nodig voor dit organisme dat massale voedselinfectie-uitbraken heeft veroorzaakt in de Verenigde Staten, Groot-Brittannië en Japan.