Identification des gènes de Escherichia coli entérohémorragique exprimés pendant l'infection de macrophages humains

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Interaction d'Escherichia coli entérohémorragique (EHEC) avec Acanthamoeba castellanii et rôle du régulon Pho chez les EHEC

Les EHEC de sérotype O157:H7 sont des agents zoonotiques d’origine alimentaire ou hydrique. Ce sont des pathogènes émergeants qui causent chez l’humain des épidémies de gastro-entérite aiguë et parfois un syndrome hémolytique-urémique. Les EHEC réussissent leur transmission à l’humain à partir de leur portage commensal chez l’animal en passant par l’étape de survie dans l’environnement. L’endosymbiose microbienne est une des stratégies utilisées par les bactéries pathogènes pour survivre dans les environnements aquatiques. Les amibes sont des protozoaires vivants dans divers écosystèmes et connus pour abriter plusieurs agents pathogènes. Ainsi, les amibes contribueraient à transmettre les EHEC à l'humain. La première partie de mon projet de thèse est centrée sur l'interaction de l’amibe Acanthamoeba castellanii avec les EHEC. Les résultats montrent que la présence de cette amibe prolonge la persistance des EHEC, et ces dernières survivent à leur phagocytose par les amibes. Ces résultats démontrent le potentiel réel des amibes à héberger les EHEC et à contribuer à leur transmission. Cependant, l’absence de Shiga toxines améliore leur taux de survie intra-amibe. Par ailleurs, les Shiga toxines sont partiellement responsables de l’intoxication des amibes par les EHEC. Cette implication des Shiga toxines dans le taux de survie intracellulaire et dans la mortalité des amibes démontre l’intérêt d’utiliser les amibes comme modèle d'interaction hôte/pathogène pour étudier la pathogénicité des EHEC. Durant leur cycle de transmission, les EHEC rencontrent des carences en phosphate inorganique (Pi) dans l’environnement. En utilisant conjointement le système à deux composantes (TCS) PhoB-R et le système Pst (transport spécifique de Pi), les EHEC détectent et répondent à cette variation en Pi en activant le régulon Pho. La relation entre la virulence des EHEC, le PhoB-R-Pst et/ou le Pi environnemental demeure inconnue. La seconde partie de mon projet explore le rôle du régulon Pho (répondant à un stress nutritif de limitation en Pi) dans la virulence des EHEC. L’analyse transcriptomique montre que les EHEC répondent à la carence de Pi par une réaction complexe impliquant non seulement un remodelage du métabolisme général, qui est critique pour sa survie, mais aussi en coordonnant sa réponse de virulence. Dans ces conditions le régulateur PhoB contrôle directement l’expression des gènes du LEE et de l’opéron stx2AB. Ceci est confirmé par l’augmentation de la sécrétion de l’effecteur EspB et de la production et sécrétion de Stx2 en carence en Pi. Par ailleurs, l’activation du régulon Pho augmente la formation de biofilm et réduit la motilité chez les EHEC. Ceci corrèle avec l’induction des gènes régulant la production de curli et la répression de la voie de production d’indole et de biosynthèse du flagelle et du PGA (Polymère β-1,6-N-acétyle-D-glucosamine).

Occurrence of non-O157 Shiga toxin-producing Escherichia coli in dogs with diarrhea

Shiga toxigenic Escherichia coli (STEC) and Attaching and effacing E. coli (AEEC) have been associated with diarrhea illness in dogs. From January to December 2006, 92 E. coli isolates from 25 diarrheic dogs were analyzed, by screening for the presence of Shiga toxin-producing (stx 1 and stx 2) and intimin (eae) genes. Twelve isolates were detected by PCR to harbor the Shiga toxin genes (7 the stx 1 (7.6%); 5 the stx 2 (5.4%); and none both of them). Nine (9.8%) of the E. coli isolates studied were eae positive non Shiga toxin-producing. Thirteen (62.0%) isolates, carrying stx or eae gene, also showed a hemolysin production. The strains with virulence genes were also examined for resistance to 12 antimicrobial agents. Resistances to cephalothin (85.7%), streptomycin (81.0%), amoxicillin (71.4%) and gentamicin (71.4%) were predominantly observed.

Beef Carcass Contamination by Shiga Toxin-Producing Escherichia coli Strains in an Abattoir in Brazil: Characterization and Resistance to Antimicrobial Drugs

A survey was performed to estimate the frequency of Escherichia coli and Shiga toxin-producing E. coli (STEC) in carcasses obtained from an abattoir in Brazil between February 2006 and June 2007. A total of 216 beef carcasses were sampled at three stages of the slaughter process-preevisceration, postevisceration, and postprocessing-during the rain and dry seasons, respectively. Of the carcasses sampled, 58%, were preevisceration E. coli positive, 38% were postevisceration positive, and 32% postprocessing positive. At the postprocessing stage, the isolation of E. coli was twice as high in the rain season. E. coli was isolated from 85 carcasses of which only 3 (1.4%) were positive for stx-encoding genes. No E. coli O157 serogroup isolates were detected. No antimicrobial resistance was found in nine of the isolates (10% of the total). The most frequent resistances were seen against cephalothin (78%), streptomycin (38%), nalidixic acid (36%), and tetracycline (30%). Multidrug resistance (MDR) to three or more antimicrobial agents was determined in 28 (33%) E. coli isolates. The presence of STEC and MDR strains among the isolates in the beef carcasses emphasizes the importance of proper handling to prevent carcass contamination.

The effect of volatile fatty acids in acidified fermented piggery effluent on shiga-toxigenic and non-toxic resident strains of Escherichia coli

Aims: To examine the effects of acidified acidogenically fermented piggery effluent containing Volatile Fatty Acids (VFA) on shiga-toxigenic and resident strains of Escherichia coli (E. coli) as part of the development of a waste treatment process. Methods and Results: Four shiga-toxigenic E. coli strains (O157:H7, 091.H-, 0111.H-, and 0123.H-) and four non-toxic resident enzootic strains were all killed by 3 h treatment with fermented piggery effluent liquor (153 mmol l(-1) total VFA) at pH 4.3. The shiga-toxigenic strains showed greater sensitivity after 1 h of treatment. The fermented liquor at pH 6.8 was not inhibitory. Conclusions: The shiga-toxigenic strains were no more resistant to the toxic effects of VFA than the non-toxic strains tested. Significance and Impact of the Study: Shiga-toxigenic strains and resident enzootic non-toxigenic strains are equally susceptible to inactivation by this waste treatment process and by acidified VFA in general.

Comparison between human and animal isolates of Shiga toxin-producing Escherichia coli O157 from Australia

There is very little human disease associated with enterohaemorrhagic Escherichia coli O157 in Australia even though these organisms are present in the animal population. A group of Australian isolates of E. coli O157:H7 and O157:H- from human and animal sources were tested for the presence of virulence markers and compared by XbaI DNA macrorestriction analysis using pulsed-field gel electrophoresis (PFGE). Each of 102 isolates tested contained the gene eae which encodes the E. coli attaching and effacing factor and all but one carried the enterohaemolysin gene, ehxA, found on the EHEC plasmid. The most common Shiga toxin gene carried was stx(2c), either alone (16%) or in combination with stx(1) (74%) or stx(2) (3%) PFGE grouped the isolates based on H serotype and some clusters were source specific. Australian E. coli O157:H7 and H- isolates from human, animal and meat sources carry all the virulence markers associated with EHEC disease in humans therefore other factors must be responsible for the low rates of human infection in Australia.

Horizontal transmission of Shiga toxin-producing Escherichia coli within groups of dairy calves

To examine the dissemination of Shiga-toxigenic Escherichia coli (STEC) within cattle groups, dairy calves on two farms utilizing different calf-rearing practices were exposed to a traceable STEC strain. Test strain dissemination differed significantly between farms, with a higher prevalence being associated with group penning. Pen floors and calf hides may be the main environmental mechanisms of transmission. Dairy calf husbandry represents a control point for reducing on-farm STEC prevalence.

Phenotypic and genotypic characteristics of shiga toxin-producing Escherichia coli strains isolated from children in São Paulo, Brazil

The biochemical and serological characteristics, virulence properties, and genetic relatedness of Shiga toxin-producing Escherichia coli (STEC) strains isolated in São Paulo, from April 1989 through March 1990, were determined. This is also the first report on clinic findings of human STEC infections in Brazil. The only three STEC strains identified in that period were lysine decarboxylase negative, belonged to serotype O111ac: non-motile, were Stx1 producers, carried the eae and astA genes, and 2 of them also presented the EHEC-hly sequence. The children carrying STEC were all boys, with less than two years old, and had no previous history of hospitalization. None of them presented blood in stools. Vomiting, cough and coryza were the most common clinical manifestations observed. Although the STEC strains were isolated during summer months, and presented similar phenotypic and genotypic characteristics, carbohydrate fermentation patterns and PFGE analysis suggested that these diarrheal episodes were not caused by a single clone.

Biochemical properties, enterohaemolysin production and plasmid carriage of Shiga toxin-producing Escherichia coli strains

Thirty-eight strains of Shiga toxin-producing Escherichia coli (STEC) were characterised in terms of biochemical properties, enterohaemolysin production and plasmid carriage. A wide variation in the biochemical properties was observed among the STEC, with 14 distinct biotypes identified. Biotype 1 was the most common, found in 29% of the strains. Enterohaemolysin production was detected in 29% of the strains. Most of the bacterial strains (95%) carried one or more plasmids and considerable heterogeneity in size and combinations was observed. Seven distinct plasmid profiles were identified. The most common profile, characterised by the presence of a single plasmid of ~90 kb, was found in 50% of these strains. These data indicate extensive diversity among STEC strains. No correlation was found among biotype, serotype, enterohaemolysin production and plasmid profile.

Le facteur D du complément: toxine urémique [Factor D of complement: Uremic toxin?].

Factor D is an essential enzyme of the alternative pathway of complement. Its catabolism is mainly renal. The concentration of factor D increases with renal failure, and is approximately 10-fold higher in patients with end-stage renal disease. The accumulation of factor D is responsible for an enhancement of alternative pathway activation. Whether this excess of factor D has pathophysiological consequences remains to be determined, however, complement activation might participate in adverse effects during hemodialysis and in the progression of renal injury.

Implications of free Shiga toxin-converting bacteriophages occurring outside bacteria for the evolution and the detection of Shiga toxin-producing Escherichia coli

In this review we highlight recent work that has increased our understanding of the distribution of Shiga toxin-converting phages that can be detected as free phage particles, independently of Shiga toxin-producing bacteria (STEC). Stx phages are a quite diverse group of temperate phages that can be found in their prophage state inserted within the STEC chromosome, but can also be found as phages released from the cell after activation of their lytic cycle. They have been detected in extraintestinal environments such as water polluted with feces from humans or animals, food samples or even in stool samples of healthy individuals. The high persistence of phages to several inactivation conditions makes them suitable candidates for the successful mobilization of stx genes, possibly resulting in the genes reaching a new bacterial genomic background by means of transduction, where ultimately they may be expressed, leading to Stx production. Besides the obvious fact that Stx phages circulating between bacteria can be, and probably are, involved in the emergence of new STEC strains, we review here other possible ways in which free Stx phages could interfere with the detection of STEC in a given sample by current laboratory methods and how to avoid such interference.

Characterizing RecA-Independent Induction of Shiga toxin2-encoding Phages by EDTA Treatment

The bacteriophage life cycle has an important role in Shiga toxin (Stx) expression. The induction of Shiga toxin-encoding phages (Stx phages) increases toxin production as a result of replication of the phage genome, and phage lysis of the host cell also provides a means of Stx toxin to exit the cell. Previous studies suggested that prophage induction might also occur in the absence of SOS response, independently of RecA.

Définition et prise en charge de l'échec d'une première injection de toxine botulique Botox(®) 200 U pour hyperactivité détrusorienne neurogène : résultats de l'enquête DETOX [Definition of botulinum toxin failure in neurogenic detrusor overactivity: Preliminary results of the DETOX survey].

OBJECTIVE: There is currently no guideline regarding the management of neurogenic detrusor overactivity (NDO) refractory to intra-detrusor botulinum toxin injections. The primary objective of the present study was to find a consensus definition of failure of botulinum toxin intra-detrusor injections for NDO. The secondary objective was to report current trends in the managment of NDO refractory to botulinum toxin. METHODS: A survey was created, based on data drawn from current literature, and sent via e-mail to all the experts form the Group for research in neurourology in french language (GENULF) and from the comittee of neurourology of the French urological association (AFU). The experts who did not answer to the first e-mail were contacted again twice. Main results from the survey are presented and expressed as numbers and proportions. RESULTS: Out of the 42 experts contacted, 21 responded to the survey. Nineteen participants considered that the definition of failure should be a combination of clinical and urodynamics criteria. Among the urodynamics criteria, the persistence of a maximum detrusor pressure>40cm H2O was the most supported by the experts (18/21, 85%). According to the vast majority of participants (19/21, 90.5%), the impact of injections on urinary incontinence should be included in the definition of failure. Regarding the management, most experts considered that the first line treatment in case of failure of a first intra-detrusor injection of Botox(®) 200 U should be a repeat injection of Botox(®) at a higher dosage (300 U) (15/20, 75%), regardless of the presence or not of urodynamics risk factors of upper tract damage (16/20, 80%). CONCLUSION: This work has provided a first overview of the definition of failure of intra-detrusor injections of botulinum toxin in the management of NDO. For 90.5% of the experts involved, the definition of failure should be clinical and urodynamic and most participants (75%) considered that, in case of failure of a first injection of Botox(®) 200 U, repeat injection of Botox(®) 300 U should be the first line treatment. Level of proof 4.