Community-wide outbreak of Escherichia coli O157:H7 associated with consumption of frozen beef burgers.

On 24-25 October 2005 a cluster of five haemolytic uraemic syndrome (HUS) cases was reported in southwest France. An investigation was undertaken to identify the outbreak source and implement control measures. Cases were defined as individuals with HUS or diarrhoea with isolation of Escherichia coli O157:H7 in stools or a positive antibody response to E. coli O157 lipopolysaccharide, resident in southwest France with symptom onset after 19 September 2005. Sixty-nine identified patients had symptom onset between 5 October and 3 November 2005, including 17 cases of HUS. One brand of frozen beef burgers produced on 22 August 2005 was consumed by all patients in the week before symptom onset. E. coli O157:H7 strains from patients, patients' burgers and the manufacturing plant were genetically related. This is the largest community-wide outbreak of E. coli O157:H7 in France to date and the first associated with consumption of contaminated frozen beef burgers.

Clonal relationship among Vibrio cholerae O1 El Tor strains causing the largest cholera epidemic in Kenya in the late 1990s.

Eighty Vibrio cholerae O1 strains selected to represent the 1998-to-1999 history of the largest cholera epidemic in Kenya were characterized by ribotyping, antimicrobial susceptibility, and random amplified polymorphic DNA patterns. Except for 19 strains from 4 local outbreaks in North Eastern Province along the Somalia border, the other 61 strains from 25 outbreaks occurring in districts scattered around the country were all ribotype B27 and resistant to chloramphenicol, spectinomycin, streptomycin, sulfamethoxazole, and trimethoprim. The 61 strains showed similar and specific amplified DNA patterns. These findings indicate that the predominant strains that caused the Kenyan epidemic had a clonal origin and suggest that ribotype B27 strains, which first appeared in West Africa in 1994, have had a rapid spread to eastern Africa.

O148 Shiga toxin-producing Escherichia coli outbreak: microbiological investigation as a useful complement to epidemiological investigation.

An outbreak of Shiga toxin-producing Escherichia coli (STEC) O148 infection occurred among wedding attendees in France in June 2002. A retrospective cohort study was performed and ten cases were identified, including two adults with haemolytic uraemic syndrome (HUS). The analytical study revealed that > 80% of affected individuals had eaten lightly roasted mutton and poultry pâté, but only the consumption of pâté tended to be associated with illness (relative risk 3.4; 95% CI 0.8-14.4). Left-overs (cooked mutton and raw offal) and processed foods (pâté) from the same batches as served at the party were sampled. Human, food and environmental samples were examined for the Shiga toxin (stx) gene and virulence traits by PCR. Stx-positive samples were cultured for STEC. HUS cases were tested for serum antibodies against 26 major STEC serogroups. An STEC O26 strain (stx1, eae, ehxA) was isolated from one case with diarrhoea, and an STEC O148 strain (stx2c) from one case of HUS. Serum antibodies against O26 were not detected in either of these patients; antibodies against O148 were not tested. Three STEC strains were isolated from the mutton and the offal (stx2c, O148), and two from the pâté (stx2c, O-X and O-Y). The isolates from the mutton were indistinguishable from the human stx2c isolate, whereas the pâté isolates differed. Although four different STEC strains were identified in patients and foods, the results of molecular subtyping, in conjunction with analysis of food consumption patterns, strongly suggested that this outbreak was caused by mutton contaminated with STEC O148.

Escherichia coli O157 outbreak associated with fresh unpasteurized goats' cheese.

A family cluster of three cases of Escherichia coli O157 infection was identified in France. Two cases developed haemolytic-uraemic syndrome. The source was fresh unpasteurized goats' cheese, produced by an independent producer. Three E. coli O157 strains, isolated from one HUS case and faeces of one cow and one goat, were indistinguishable by toxin type and PFGE pattern.

Case management of a multidrug-resistant Shigella dysenteriae serotype 1 outbreak in a crisis context in Sierra Leone, 1999-2000.

From December 1999 to the end of February 2000, 4218 cases of dysentery were reported in Kenema district, southeastern Sierra Leone, by a Médecins Sans Frontières team operating in this region. Shigella dysenteriae serotype 1 was isolated from the early cases. The overall attack rate was 7.5% but higher among children under 5 years (11.2%) compared to the rest of the population (6.8%) (RR = 1.6; 95% CI 1.5-1.8). The case fatality ratio was 3.1%, and higher for children under 5 years (6.1% vs. 2.1%) (RR = 2.9; 95% CI 2.1-4.1). A case management strategy based on stratification of affected cases was chosen in this resource-poor setting. Patients considered at higher risk of death were treated with a 5 day ciprofloxacin regimen in isolation centres. Five hundred and eighty-three cases were treated with a case fatality ratio of 0.9%. Patients who did not have signs of severity when seen by health workers were given hygiene advice and oral rehydration salts. This strategy was effective in this complex emergency.

Outbreak of Pseudomonas putida bacteraemia in a neonatal intensive care unit.

During the period of 9-27 March 2001, Pseudomonas putida strains were recovered from 10 neonates hospitalized in the neonatal intensive care unit of Farhat Hached Hospital, Sousse (Tunisia). Seven neonates developed bacteraemia, and three had an umbilical catheter-related infection (without bacteraemia). A total of 18 isolates were cultured from blood (N = 11) and catheters (N = 7). These isolates were identified as P. putida by routine biochemical methods (API 20 NE, bioMérieux, Lyon, France). Restriction endonuclease DNA profiles were determined by pulsed-field gel electrophoresis using two endonucleases XbaI and SpeI. They yielded the same patterns showing that the outbreak was caused by a single clone of P. putida. Although the antiseptic solutions used to clean the umbilicus were implicated circumstantially as probable sources, they were not sampled and so this could not be confirmed.

Two outbreaks of Salmonella enteritidis phage type 8 linked to the consumption of Cantal cheese made with raw milk, France, 2001.

Salmonelloses are one are the main causes of foodborne infections in industrialised countries. In France, the incidence of human salmonellosis recorded by the National Reference Centre for Salmonella and Shigella (CNRSS) in 2001 was 21 cases per 100,000 inhabitants, and Salmonella serotype Enteritidis represented 39% of cases (1). This article reports the investigation results of two community outbreaks of salmonellosis that occurred simultaneously in the south west of France, and which were linked to the consumption of cheese made from raw milk.

Corynebacterium macginleyi isolation from conjunctival swab in Italy.

Corynebacterium macginleyi was isolated from conjunctival swabs of a farmer suffering from purulent conjunctivitis. This species has only recently been reported in Switzerland and Germany to be exclusively isolated from ocular surfaces. This represents the first isolation of C. macginleyi in Italy indicating that its circulation is not geographically limited.

A molecular and phenotypic study of Vibrio cholerae in Iran.

Vibrio cholerae is again the subject of attention on account of the current increase in the world-wide incidence of cholera. In this study, 200 clinical isolates of V. cholerae serotypes O1 and non-O1, non-O139, were collected from different provinces in Iran. The isolates were subjected to biochemical analysis, antibiogram, PCR of toxin genes, plasmid profile, ribotyping and pulsed-field gel electrophoresis (PFGE). The analysis of plasmid content showed that 33-96% of V. cholerae isolated from different provinces carry a large plasmid. PCR analysis of V. cholerae O1 showed that the genes encoding cholera toxin (ctx), toxin co-regulated pilus (tcp), accessory cholera enterotoxin (ace) and zonula occludens toxin (zot) were present in 55-97% of isolates in different provinces. Restriction fragment length polymorphism (RFLP) of BglI-digested DNA probed with five oligonucleotides revealed three different ribotype patterns in isolates of V. cholerae O1. The ribotype pattern B21 of V. cholerae O1 El Tor was found to be the predominant pattern in the isolates studied. V. cholerae non-O1, non-O139 isolates showed a single ribotype pattern. PFGE analysis also showed 10 different patterns amongst the isolates, 9 of which were in V. cholerae O1. Overall, the analysis of polymorphism of ribotypes and PFGE patterns of the isolates showed that the provinces in Iran were affected by a limited number of clones of V. cholerae O1 and non-O1, non-O139 strains.

Minced beef and human salmonellosis: review of the investigation of three outbreaks in France.

The synthesis of investigations of three salmonellosis outbreaks which occured in France between 1998 and 2000 confirms the role of the consumption of minced beef and highlights the importance of prevention measures.

Computer identification of Shigella species by rRNA gene restriction patterns.

We describe a MluI ribotyping scheme for Shigella which approaches correlation with serotyping. One hundred and seventeen reference strains and previously serotyped clinical isolates representing the 57 Shigella serotypes and biotypes were included in this study. A total of 51 distinct ribotypes were obtained and a database was built with them. The number of bands composing each ribotype varied from 9 to 15. The fragments ranged in size from 1.6 to 18.8 kbp. One hundred and eleven clinical isolates were successfully identified in a double blind study with standard biochemical/serologic methods, by automatic comparison of their ribotypes with our database using the software Taxotron.

Molecular and phenotypic characterization of potentially new Shigella dysenteriae serotype.

From September 1997 to November 1998, the French National Center for Salmonella and Shigella received 22 Shigella isolates recovered from 22 different patients suffering from dysentery. None of these isolates reacted with any of the antisera used to identify established Shigella serotypes, but all of them agglutinated in the presence of antisera to a previously described potentially new Shigella dysenteriae serotype (represented by strain 96-204) primarily isolated from stool cultures of imported diarrheal cases in Japan. All French isolates, as well as strain 96-204, showed biochemical reactions typical of S. dysenteriae and gave positive results in a PCR assay for detection of the plasmid ipaH gene coding for invasiveness. No Shiga toxin gene was detected by PCR. These isolates were indistinguishable by molecular analysis of ribosomal DNA (ribotyping) and seemed to be related to S. dysenteriae serotypes 3 and 12. However, further characterization by restriction of the amplified O-antigen gene cluster clearly distinguished this new serotype from all other Shigella or Escherichia coli serotypes.

Clonal relationships among Shigella serotypes suggested by cryptic flagellin gene polymorphism.

The presence of cryptic fliC alleles in the genomes of 120 strains representative of the four Shigella species was investigated. One fragment was obtained by PCR amplification of fliC, with a size varying from 1.2 to 3.2 kbp, depending on the species or serotype. After digestion with endonuclease HhaI, the number of fragments in patterns varied from three to nine, with sizes of between 115 and 1,020 bp. Patterns sharing most of their bands were grouped to constitute an F type. A total of 17 different F types were obtained from all strains included in this study. A unique pattern was observed for each the following serotypes: Shigella dysenteriae 1, 2, 8, and 10 and S. boydii 7, 13, 15, 16, and 17. On the contrary, S. dysenteriae serotype 13 and S. sonnei biotype e were each subdivided into two different F types. S. flexneri serotypes 3a and X could be distinguished from the cluster containing S. flexneri serotypes 1 to 5 and Y. S. flexneri serotype 6 clustered with S. boydii serotypes 1, 2, 3, 4, 6, 8, 10, 11, 14, and 18 and S. dysenteriae serotypes 4, 5, 6, 7, 9, 11, and 12. Two other clusters were outlined: one comprising S. dysenteriae serotypes 3, 12, 13 (strain CDC598-77), 14, and 15 and the other one joining S. boydii serotypes 5 and 9. None of the 17 fliC patterns was found in the fliC HhaI pattern database previously described for Escherichia coli. Overall, this work supports the hypothesis that Shigella evolved from different ancestral strains of E. coli. Moreover, the method outlined here is a promising tool for the identification of some clinically important Shigella strains as well as for confirmation of atypical isolates as Shigella spp.

Legionella gresilensis sp. nov. and Legionella beliardensis sp. nov., isolated from water in France.

Novel Legionella-like isolates, strains Montbéliard A1T and Gréoux 11 D13T, isolated from two different French water sources, were studied taxonomically and phylogenetically. Morphological and biochemical characterization revealed that they were Gram-negative, aerobic, non-spore-forming bacilli with a cut-glass appearance that grew only on L-cysteine-supplemented buffered charcoal yeast extract agar. Phenotypic characterization using fatty acid and ubiquinone profiles and SDS-PAGE analysis confirmed that they were closely related, but distinct from, other species of the genus Legionella, since serotyping could not relate them to any existing serogroup. Genotypic profiles generated by randomly amplified polymorphic DNA and 16S-23S rDNA spacer region PCR analyses were unique for each of these isolates. DNA-DNA relatedness values of strains Montbéliard A1T and Gréoux 11 D13T to each other and to other Legionella type strains were less than 25%. Phylogenetic affiliation of these organisms obtained by 16S rDNA sequence comparisons confirmed that they were distinct from any other known Legionella species. All the above results confirm that these strains constitute two novel species for which the names Legionella gresilensis sp. nov. (type strain Gréoux 11 D13T = ATCC 700509T = CIP 106631T) and Legionella beliardensis sp. nov. (type strain Montbéliard A1T = ATCC 700512T = CIP 106632T) are proposed.

Superficial contamination of bovine carcasses by Escherichia coli O157:H7 in a slaughterhouse in Normandy (France).

In 1997, analyses were carried out on 255 bovine carcasses to determine the extent of superficial contamination by E. coli O157. A 50-cm(2) meat sample taken from all carcasses was collected and tested using immunomagnetic separation method to detect E. coli O157. One strain of E. coli O157 bacterium was isolated and sent to the reference national center (Institut Pasteur, Paris). The strain was confirmed as E. coli O157:H7 and found to contain two out of the three pathogenicity genes (eae and EHEC-hlyA) necessary for enteropathogenicity. Shiga toxin genes were not detected. Superficial contamination of E. coli O157:H7 was established, but at low level (0.4%).

Investigation of concurrent outbreaks of gastroenteritis and typhoid fever following a party on a floating restaurant, France, March 1998.

A retrospective cohort study was carried out to investigate concurrent outbreaks of gastroenteritis and typhoid fever that occurred among guests of a supper on a floating restaurant in France in March 1998. A total of 133 guests (attack rate = 90%) reported gastroenteritis within 12 days of the supper. Twenty-seven guests developed typhoid fever (attack rate = 18%) of whom 15 were confirmed by stool or blood culture. All patients with typhoid fever had had an initial gastroenteritis. The results suggest that the same food items served during the supper, chicken and rice, were the vehicles of both gastroenteritis and typhoid fever, but the authors could not determine the specific source of infection. Initial gastroenteritis has been described as a clinical manifestation of typhoid fever but whether or not these two syndromes (gastroenteritis and typhoid fever) were due to the same etiology remains unclear in this outbreak.

Identification of Escherichia coli O-serogroups by restriction of the amplified O-antigen gene cluster (rfb-RFLP).

The precise serotyping of clinical Escherichia coli isolates is a crucial step for diagnostic and epidemiological purposes. Epidemiological knowledge associated with serotyping is so important that no alternative method may be considered if it does not correlate with serotyping. Unfortunately, E. coli are difficult to serotype. Genes specifically involved in O-antigen synthesis are clustered in E. coli, Shigella and Salmonella. Published oligonucleotide sequences complementary to JUMPstart and the gnd gene (the conserved flanking sequences upstream and downstream of O-antigen gene clusters, respectively) were used to amplify the O-antigen gene cluster of representative strains of 148 E. coli O-serogroups. A unique amplified fragment was observed for each serogroup (size ranging from 1.7 to 20 kbp). Clearly identifiable and reproducible O-patterns were obtained for the great majority of O-serogroups after MboII digestion of amplified products. The number of bands composing each pattern varied from five to 25. A database was built with the patterns obtained. A total of 147 O-patterns were obtained. Thirteen O-serogroups were subdivided into different O-patterns. However, each of 13 other O-patterns was shared by two or more O-serogroups. 0-serogroups of clinical isolates were deduced accurately from O-patterns in all cases, even for some rough or nonagglutinating isolates. The restriction method (rfb-RFLP) may prove to be better than serotyping since 100% of strains are typable, which is not the case with serotyping.

Identification of Escherichia coli flagellar types by restriction of the amplified fliC gene.

A total of 182 strains of Escherichia coli (133 reference strains, 22 clinical strains, nine nonmotile strains and 18 strains derived from K-12) were characterized by HhaI restriction of the amplified flagellin gene (fliC). The amplified fliC product was a single band between 0.9 and 2.6 kbp. With the collection of reference strains which represented 48 flagellar types (H-types), a total of 62 patterns (F-types) were observed after HhaI restriction. A single F-type was associated with each of 39 H-types and more than one F-type was associated with the other nine H-types. Antigenically related H-types 12 and 45 gave a single F-type. The determination of HhaI-fliC F-types could allow deduction of all H-types and subdivision of some of these. Application of this identification system to 22 E. coli clinical isolates yielded nine F-patterns and the deduced H-types were confirmed by serotyping in all cases. Nine nonmotile strains were studied and their F-types were also identified. The proposed determination of fliC restriction patterns should be helpful for epidemiological studies.

Outbreak of typhoid fever on the French Riviera.

A case-control study was conducted to establish the source of a community outbreak of typhoid fever in Utelle, France, a village located in the Alpes-Maritimes district of the French Riviera. Thirteen confirmed cases of typhoid fever and 41 confirmed community controls were included in the study. Cases and controls did not differ regarding ingestion of water. Multivariate logistic regression analysis identified consumption of pork meats during a village festival as the only statistically significant risk factor for typhoid fever after adjusting for age and sex (odds ratio, 76.0; 95% confidence interval, 3.5-1660). Assessment of food-handling procedures at the inn where the food had been prepared showed that the refrigeration and cooking facilities were inadequate to maintain a proper sanitary environment during the preparation of a meal for 350 people. Although the exact cause of the epidemic could not be confirmed, food contaminated by a chronic typhi carrier is the most plausible hypothesis.

Streptococcus infantarius sp. nov., Streptococcus infantarius subsp. infantarius subsp. nov. and Streptococcus infantarius subsp. coli subsp. nov., isolated from humans and food.

Eighteen strains isolated from human specimens or from food products were characterized as atypical variants of mannitol-negative Streptococcus bovis. They were tested for extended biochemical criteria, ribotyping and DNA-DNA hybridization in order to define their taxonomic status. These strains were demonstrated to constitute a DNA relatedness group that includes strains of DNA group 4 of Farrow et al. (1984). Comparative analysis of 16S rRNA sequences demonstrated that these strains represent a new species which belongs to the Streptococcus bovis/Streptococcus equinus complex and which has been provisionally named S. infantarius by Bouvet et al. (1997). Biotyping and ribotyping allowed differentiation of these strains from the aesculin-positive strains of S. bovis belonging to the previously described biotypes I, II.1 and II.2. The results of the ribotyping and hybridization assays demonstrated the presence of two different DNA subgroups within the 18 strains. On the basis of these data, the names S. infantarius subsp. infantarius (aesculin-negative for five strains out of seven, including the type strain HDP 90056T = NCDO 599T) and S. infantarius subsp. coli (aesculin-positive, reference strain HDP 90248 = NCDO 2620) are proposed as the names for these two subspecies within the S. infantarius species.