Incidence and virulence determinants of verocytotoxin-producing Escherichia coli infections in the Brussels-Capital Region, Belgium, in 2008-2010.

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 = 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 = 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/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%) 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.

Virulence profiling and quantification of verocytotoxin-producing Escherichia coli O145:H28 and O26:H11 isolated during an ice cream-related hemolytic uremic syndrome outbreak.

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 identified. Laboratory confirmation of O145:H28 infection was obtained for three hemolytic uremic syndrome patients, 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.

Antimicrobial resistance testing of verocytotoxin-producing Escherichia coli and first description of TEM-52 extended-spectrum ß-lactamase in serogroup O26.

We have investigated the antimicrobial resistance of verocytotoxin-producing Escherichia coli (VTEC) strains isolated from humans, animals, food, and the environment in Belgium. Resistance was more frequent in non-O157 strains from humans than in O157 strains from humans or other sources, and among non-O157 VTEC strains, intimin-positive strains were more resistant than intimin-negative strains. We also report the first VTEC strain producing an IncI1 extended-spectrum ß-lactamase encoded by plasmid-borne bla(TEM-52); this ß-lactamase was previously associated with Salmonella enterica and E. coli isolates from different origins.

Characterization of Bordetella pertussis clinical isolates that do not express the tracheal colonization factor.

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.

Third Belgian multicentre survey of antibiotic susceptibility of anaerobic bacteria.

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.

Pneumococcal serogroups and serotypes in severe pneumococcal pneumonia in Belgian children: theoretical coverage of the 7-valent and 9-valent pneumococcal conjugate vaccines.

BACKGROUND: Although the causative pneumococcal serotypes of invasive diseases are already extensively studied, few data are available about the pneumococcal serotypes additionally isolated from broncho-alveolar lavage samples in childhood pneumonia. STUDY AIM: To identify the causative pneumococcal serotypes in culture proven childhood community acquired pneumonia (CAP) and to calculate the effectiveness of the heptavalent and nonavalent pneumococcal vaccine (7- and 9-valent PnV) in severe pneumococcal pneumonia. METHODS: All pneumococcal isolates stored from broncho-alveolar lavage, blood culture and pleural fluid in healthy children with CAP were characterized. RESULTS: Seventy children (median age 2 years 3.5 months) could be included. The most prevalent serotypes were: SGT1 (21.4%), SGT6 (20.0%), SGT19 (12.8%), SGT23 (10.0%), and SGT14 (7.1%). SGT1 was especially prevalent in complicated cases and children >5 years. This first ranking of SGT1 is not reported in invasive pneumococcal disease studies. The overall theoretical coverage of the 7-valent PnV and the 9-valent PnV for pneumococcal pneumonia was 45.7% and 72.8%. The theoretical coverage of both vaccines was equal for non-invasive pneumonia (64%) but the theoretical coverage of the 9-valent PnV for invasive pneumonia was much higher (79% vs. 37.2%). Antibiotic susceptibility to penicillin was 84%, 70% to tetracycline and 61% to erythromycin; however only one strain (MIC = 4 mg/L) was highly resistant to penicillin. CONCLUSIONS: Based on this serotyping, the theoretical coverage of the 7-valent PnV for proven pneumococcal pneumonia is good but decreases with age. A 9-valent PnV containing SGT1 could significantly increase the coverage, especially for invasive pneumonia. According to these data, penicillin remains the first choice antibiotic treatment for childhood CAP in Belgium.

Simple Algorithm for Identification of Bordetella pertussis Pertactin Gene Variants.

Studies performed in several countries have demonstrated the recent emergence and subsequent dominance of circulating Bordetella pertussis strains harboring pertactin and pertussis toxin variants not included in pertussis vaccines. Determination of the pertactin gene variants is commonly performed using a time-consuming and expensive sequence analysis. We developed a simple and reliable pertactin typing algorithm suitable for large-scale screening. The assay correctly identified all pertactin alleles in representative strains. The typing of 231 clinical strains of B. pertussis routinely isolated in Belgium showed that this algorithm was adequate to identify less-frequent prn types like prn9 and prn11.

Biliary tract infection in patients with acute biliary pancreatitis.

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 < 0.001), serum concentrations of glucose (132 +/- 4 vs. 149 +/- 6 mg/dL, p < 0.02) and alanine aminotransferase (ALT) (304 +/- 28 vs. 226 +/- 25 IU/L, p < 0.05) and hematocrit (43.4 +/- 0.4% vs. 41.7 +/- 0.5%, p < 0.05), Glasgow pancreatitis score (1.58 +/- 0.11 vs. 1.97 +/- 0.10, p < 0.01) and APACHE II score (6.20 +/- 0.38 vs. 7.82 +/- 0.35, p < 0.005). The prediction of the presence of bacteria in bile by each of these individual parameters, however, was of variable accuracy. From 82 patients with positive bile cultures, a total of 150 microorganisms were isolated, including 66 gram-positive aerobes, 66 gram-negative facultative anaerobes, 15 obligate anaerobes, and three fungi. The most common organisms were Escherichia coli (20.6%), followed by enterococci (18%) and streptococci (15.3%). CONCLUSION: Patients with acute biliary pancreatitis who manifest abnormalities of one or more of the above-mentioned risk factors are more likely to have positive bile cultures. Whether such patients might benefit from early antibiotic therapy directed against both gram-negative bacilli and gram-positive cocci needs to be determined.

In vitro activity of ertapenem against anaerobes isolated from the respiratory tract.

Ertapenem is a novel parenteral carbapenem with a long serum half-life. Its spectrum of activity is similar to that of imipenem and meropenem against Gram-positive bacteria, Enterobacteriaceae and fastidious Gram-negative bacteria but it is less active against Pseudomonas aeruginosa and Acinetobacter spp. Several studies were performed in the United States but only one European study has shown that ertapenem has an excellent activity against anaerobes. The objectives of the present study were to test the activity of ertapenem against anaerobes isolated prospectively from the lower and upper respiratory tracts, and to compare their susceptibility with that of anaerobic isolates from other body sites. Fifty-three isolates from the respiratory tract, as well as 50 isolates from various other body sites were tested with E-tests against six antibiotics. For respiratory isolates and for isolates from other sites, MIC 90 values (mg/l) were, respectively, >32 and >32 for penicillin, 0.38 and 0.75 for amoxicillin/clavulanate, 48 and >256 for ceftriaxone, 0.12 and 0.75 for ertapenem, 12 and >256 for clindamycin and 2 and 12 for moxifloxacin. The higher susceptibility of respiratory tract isolates was mainly due to the different distribution of isolated species: only three respiratory isolates but 22 other isolates belonged to the Bacteroides fragilis group. This study confirms the excellent anti-anaerobic activity of ertapenem against anaerobic isolates from the respiratory tract.

Prevalence of fragilysin gene in Bacteroides fragilis isolates from blood and other extraintestinal samples.

Of 166 Bacteroides fragilis isolates, 26.2% of 103 isolates from blood and 20.6% of 63 extraintestinal isolates harbored the fragilysin gene (difference not statistically significant). Clinical characteristics and evolution were comparable in patients with B. fragilis bacteremia with or without this enterotoxin. Fragilysin seems not to be an important virulence factor in B. fragilis disease.

Molecular typing of Bordetella pertussis isolates recovered from Belgian children and their household members.

Recently, a moderate increase in the prevalence of pertussis, possibly contracted from adults, has been observed among unvaccinated children. During a 3-year period, we prospectively enrolled 93 index patients with a polymerase chain reaction (PCR) and/or culture result positive for Bordetella pertussis. Among 63 household contacts of 28 index patients, PCR and culture for B. pertussis identified 25 B. pertussis-positive persons. Nineteen of 25 B. pertussis-positive household contacts were asymptomatic. Isolates were available from 10 families of both index patients and household contacts for molecular typing by pulsed-field gel electrophoresis (PFGE) and for genotyping of pertactin and pertussis toxin by sequence-specific PCR and sequencing. PFGE demonstrated homogeneity among the isolates recovered from within each family but heterogeneity among the isolates recovered from different families. B. pertussis isolates recovered from index patients and their household contacts were indistinguishable by molecular typing, demonstrating that identical strains can cause full pertussis disease in children and asymptomatic infection in adults and adolescents.

Comparison of two methods for serotyping Ureaplasma urealyticum clinical isolates.

A newly developed enzyme linked immunosorbent assay (ELISA) method using monoclonal antibodies (MAbs) to the 14 serotypes of Ureaplasma urealyticum was compared to immunofluorescence assay (IFA) for serotyping U. urealyticum clinical isolates. Of the 102 vaginal isolates of U. urealyticum, five strains were lost and were excluded from analysis. Of the 97 strains analysed, a total of 86 (89%) strains were typeable by ELISA and a total of 89 (92%) strains were typeable by IFA. Eighty-six strains were typeable by both methods, three by IFA only and eight strains were not typeable neither by ELISA nor by IFA. Of the 86 strains typeable by both methods, complete concordance in serotyping results was found. The three strains not typeable by ELISA were typeable as serotype 4 by IFA. These three strains were reanalysed by ELISA after major modifications of the antigen preparation and were typeable as serotype 4. In conclusion, the ELISA was found suitable for serotyping clinical isolates. However, since the ELISA had a somewhat lower performance than IFA, strains not typeable by ELISA, should be retested by another technique such as IFA.

Hemolytic uremic syndrome in Belgium: incidence and association with verocytotoxin-producing Escherichia coli infection.

OBJECTIVE: To evaluate the incidence of hemolytic uremic syndrome (HUS) in Belgium and to determine the role of verocytotoxin-producing Escherichia coli O157:H7 and other serotypes (non-O157 VTEC). METHODS: Twenty-two centers, including the seven university hospitals, registered prospectively all cases of HUS; they collected clinical samples for isolation of VTEC strains and serum for detection of specific O-lipopolysaccharide antibodies. RESULTS: Forty-seven cases of HUS (including five incomplete cases) were recorded. Three cases were seen in non-residents. The incidence of complete HUS in Belgian residents was 4.3 cases/100 000 in children <5 years old, 1.8 cases/100 000 when all children <15 years were considered, and 0.42/100 000 when patients of all ages were taken into account. By combining bacteriologic and serologic results, evidence of VTEC infection was obtained in 64% of the patients, mainly but not exclusively in children with prodromal diarrhea. The 13 VTEC isolates belonged to serotypes O157:H7 (nine isolates), O26:H11, O121:H---, O145:H--- and O172:H--- (one each) and all produced VT2 (+VT2vh-a in three O157 strains) and were positive for the eaeA gene. CONCLUSIONS: The incidence rate found in this study and the high mortality and morbidity linked with this syndrome warrant further registration of pediatric and post-diarrheic adult HUS cases and also examination of stools for both O157 and non-O157 VTEC strains. For effective prevention of this disease, further study of the serotypes and accessory virulence factors associated with HUS is needed.

Comparison of Auxacolor with API 20 C Aux in yeast identification.

OBJECTIVE: To compare Auxacolor with API 20 C Aux for identification of yeasts. METHODS: A total of 206 isolates belonging to 25 species was used in this study. Conventional yeast identification methods were used as a reference. RESULTS: With API 20 C Aux, the correct identification rate was 89.3% after 2 days, while 94.7% of the strains were correctly identified after 3 days. One of 14 strains of Candida tropicalis and 10 of 16 strains of Trichosporon cutaneum were not correctly identified. With Auxacolor, the percentages of correct identification after 1 and 2 days were 60.1% and 69.4%, respectively. Most strains of 11 of the 20 species considered in the system were correctly identified, including several of the most frequent yeast species. Several less commonly encountered yeast species were not correctly identified. Suggestions for improvement of the Auxacolor system are given. CONCLUSIONS: For the most frequent yeast species, Auxacolor, after adaptation and correction of the identification table, provides a useful alternative to API 20 C Aux. For less frequently encountered yeast species, the use of API 20 C Aux is preferable.

Isolation and virulence factors of verocytotoxin-producing Escherichia coli in human stool samples.

OBJECTIVE: To evaluate the isolation rate of O157 and non-O157 verocytotoxin-producing Escherichia coli (VTEC) strains, to study the occurrence of additional virulence factors and to correlate these with clinical symptoms. METHODS: Over more than 5 years, 17 296 unduplicated fecal samples submitted for routine culture were screened for VTEC by a single PCR detecting VT1, VT2 and its variants. Verocytotoxin B subunit genotypes of the isolates obtained by testing individual colonies in positive samples were determined by a polymerase chain reaction---restriction fragment length polymorphism (PCR---RFLP) technique, the eaeA gene and the 60-MDa virulence plasmid by PCR, and the hemolytic phenotype by using CaCl2-washed blood agar. RESULTS: Verocytotoxin genes were found in 1.02% of the samples. Non-O157 VTEC strains were isolated in 0.66% and O157 in 0.17%. Overall, VTEC was less frequently isolated than Campylobacter and Salmonella but more frequently than Yersinia and Shigella. All cases except two siblings were epidemiologically unrelated. Cases of hemolytic uremic syndrome (HUS) were only observed in association with serogroup O157, which seems to be more pathogenic than the non-O157 strains. Among non-O157 VTEC strains, eaeA-positive strains are more frequently associated with clinical symptoms than are eaeA-negative strains. Other virulence factors correlate less closely with the presence of symptoms. CONCLUSIONS: VTEC is the third bacterial intestinal pathogen in our study population. All stool samples from patients with diarrhea should be screened for the most frequent serogroup, O157, or, if this is not possible, at least those from patients with bloody diarrhea. Non-O157 VTEC strains, especially if they are eaeA positive, are also associated with diarrhea, more often non-bloody. PCR or the new commercially available immunoassays could be used in selected cases, e.g. in patients suffering from HUS and in cases of outbreaks.