Identification of Shigatoxigenic and enteropathogenic Escherichia coli serotypes in healthy young dairy calves in Belgium by recto-anal mucosal swabbing

Enterohemorrhagic Escherichia coli (EHEC), enteropathogenic E. coli (EPEC), and Shigatoxigenic E. coli (STEC) are carried by healthy adult cattle and even more frequently by young calves in their intestinal tract, especially at the height of the recto-anal junction. The purpose of the present study was to assess the presence of ten EHEC, EPEC, and/or STEC O serotypes (O5, O26, O80, O103, O111, O118, O121, O145, O157, and O165) in calves sampled via recto-anal mucosal swabs (RAMS) at three dairy farms in Belgium. A total of 233 RAMS were collected on three consecutive occasions from healthy <6-month-old Holstein-Friesian calves and submitted to a PCR targeting the eae, stx1, and stx2 genes after non-selective overnight enrichment growth. The 148 RAMS testing positive were streaked on four (semi-)selective agar media; of the 2146 colonies tested, 294 from 69 RAMS were PCR-confirmed as EHEC, EPEC, or STEC. The most frequent virulotype was eae+ EPEC and the second one was stx1+ stx2+ STEC, while the eae+ stx1+ and eae+ stx1+ stx2+ virulotypes were the most frequent among EHEC. The majority of EHEC (73%) tested positive for one of the five O serotypes detected (O26, O103, O111, O145, or O157) vs. 23% of EPEC and 45% of STEC. Similarly, more RAMS (73%) harbored EHEC isolates positive for those five serotypes compared to EPEC (53%) or STEC (52%). This survey confirms that (i) healthy young dairy calves are asymptomatic carriers of EHEC and EPEC in Belgium; (ii) the carrier state rates, the virulotypes, and the identified O serotypes differ between farms and in time; and (iii) a majority of EPEC belong to so far unidentified O serotypes

Virulence of Shigatoxigenic and Enteropathogenic Escherichia coli O80:H2 in Galleria mellonella Larvae: Comparison of the Roles of the pS88 Plasmids and STX2d Phage

peer reviewedThe invasiveness properties of Shigatoxigenic and enteropathogenic Escherichia coli (STEC and EPEC) O80:H2 in humans and calves are encoded by genes located on a pS88-like ColV conjugative plasmid. The main objectives of this study in larvae of the Galleria mellonella moth were therefore to compare the virulence of eight bovine STEC and EPEC O80:H2, of two E. coli pS88 plasmid transconjugant and STX2d phage transductant K12 DH10B, of four E. coli O80:non-H2, and of the laboratory E. coli K12 DH10B strains. Thirty larvae per strain were inoculated in the last proleg with 10 μL of tenfold dilutions of each bacterial culture corresponding to 10 to 106 colony-forming units (CFUs). The larvae were kept at 37 °C and their mortality rate was followed daily for four days. The main results were that: (i) not only the STEC and EPEC O80:H2, but also different E. coli O80:non-H2 were lethal for the larvae at high concentrations (from 104 to 106 CFU) with some variation according to the strain; (ii) the Stx2d toxin and partially the pS88 plasmid were responsible for the lethality caused by the E. coli O80:H2; (iii) the virulence factors of E. coli O80:non-H2 were not identified. The general conclusions are that, although the Galleria mellonella larvae represent a useful first-line model to study the virulence of bacterial pathogens, they are more limited in identifying their actual virulence properties

Consensus on exercise reporting template (Cert): Modified delphi study

© 2016 American Physical Therapy Association. Background. Exercise interventions are often incompletely described in reports of clinical trials, hampering evaluation of results and replication and implementation into practice. Objective. The aim of this study was to develop a standardized method for reporting exercise programs in clinical trials: the Consensus on Exercise Reporting Template (CERT). Design and Methods. Using the EQUATOR Network’s methodological framework, 137 exercise experts were invited to participate in a Delphi consensus study. A list of 41 items was identified from a meta-epidemiologic study of 73 systematic reviews of exercise. For each item, participants indicated agreement on an 11-point rating scale. Consensus for item inclusion was defined a priori as greater than 70% agreement of respondents rating an item 7 or above. Three sequential rounds of anonymous online questionnaires and a Delphi workshop were used. Results. There were 57 (response rate=42%), 54 (response rate=95%), and 49 (response rate=91%) respondents to rounds 1 through 3, respectively, from 11 countries and a range of disciplines. In round 1, 2 items were excluded; 24 items reached consensus for inclusion (8 items accepted in original format), and 16 items were revised in response to participant suggestions. Of 14 items in round 2, 3 were excluded, 11 reached consensus for inclusion (4 items accepted in original format), and 7 were reworded. Sixteen items were included in round 3, and all items reached greater than 70% consensus for inclusion. Limitations. The views of included Delphi panelists may differ from those of experts who declined participation and may not fully represent the views of all exercise experts. Conclusions. The CERT, a 16-item checklist developed by an international panel of exercise experts, is designed to improve the reporting of exercise programs in all evaluative study designs and contains 7 categories: materials, provider, delivery, location, dosage, tailoring, and compliance. The CERT will encourage transparency, improve trial interpretation and replication, and facilitate implementation of effective exercise interventions into practice

Virulotypes and phylogenetic relationships study between O80:H2 Shigatoxinogenic and Enteropathogenic strains of Escherichia coli and Stx bacteriophage mediated stx2d gene transduction

Récemment, un pathotype d'Escherichia coli O80:H2 très virulent et peu commun, portant les gènes de la toxine de Shiga (stx), le sous-type rare de l’intimine eaeξ, et des gènes associés au plasmide pS88 des E. coli pathogènes extraintestinaux (ExPEC) a été décrit en Europe. Celui-ci est impliqué dans le syndrome urémique et hémolytique (SHU) avec bactériémie chez l’homme et il est devenu le deuxième sérotype d’E. coli shigatoxinogènes (STEC) le plus fréquemment isolé en 2016 en France (Bruyand et al., 2019). Il est également le troisième sérotype d’E. coli entérohémorragiques (AE-STEC) le plus fréquemment isolé parmi les cas de SHU en Europe en 2019 chez l’homme (EFSA 2021). Des E. coli entéropathogènes (EPEC) O80:H2 ont de plus été détectées chez des jeunes veaux diarrhéiques (Thiry et al., 2017) ; des AE-STEC O80:H2 ont également été détectées, bien que plus rarement chez des bovins adultes (Blanco et al., 2004 ; Cointe et al., 2018) ainsi que chez un veau diarrhéique en 1987 en Belgique (Thiry et al., 2017 ; De Rauw et al., 2019). L’objectif général de cette thèse a été de poursuivre l’identification du sérotype O80:H2 parmi des souches EPEC et AE-STEC isolées de veaux malades et sains en Belgique, d’étudier leur virulence et de mieux comprendre le rôle du phage Stx2d dans la pathogénicité et dans l’évolution de ces souches. L’objectif de la première étude a été d'identifier le sérotype O80:H2 parmi des EPEC et AE-STEC isolées de veaux diarrhéiques et septicémiques et de comparer génétiquement ces AE-STEC bovines avec les AE-STEC O80:H2 humaines. Dans cette étude, dix AE-STEC et 21 EPEC O80 ont été identifiées à partir de fèces, de contenus intestinaux et d'un rein de veaux diarrhéiques ou septicémiques. Leurs génomes ont été séquencés et comparés à 19 AE-STEC humaines. Elles appartenaient toutes au sérotype O80:H2 et au ST301, hébergeaient le gène eaeξ, et 23 des 29 AE-STEC contenaient le gène stx2d. D'un point de vue phylogénétique, elles étaient réparties en deux sous-lignées principales : l'une comprenait une majorité d'EPEC bovines tandis que la seconde comprenait une majorité d’AE-STEC bovines et humaines comprenant le gène stx2d. Les AE-STEC, les EPEC et les STEC sont portées par les bovins adultes sains dans leur tractus intestinal, plus particulièrement dans le colon à la hauteur de la jonction recto-anale (Beutin et Fach, 2015). L'objectif de la seconde étude a été d'évaluer la présence du sérotype O80:H2 dans des souches AE- STEC et EPEC isolées de veaux laitiers sains âgés de moins de 6 mois prélevés par écouvillonnage au niveau la muqueuse recto-anale (RAMS) dans trois exploitations. Dans cette étude, 233 RAMS ont été prélevés à trois occasions consécutives sur des veaux Holstein et ont été soumis à une PCR ciblant les gènes eae, stx1 et stx2. Au total, 148 RAMS positifs ont été ensemencés sur quatre milieux gélosés (semi-)sélectifs ; sur les 2146 colonies testées, 294 provenant de 2 69 RAMS ont été confirmées par PCR comme étant des AE-STEC, EPEC ou STEC avec une majorité d’AE-STEC (73 %). Aucun isolat n’était positif pour le sérogroupe O80. Les gènes de la toxine de Shiga (stx) ont été transférés à de nombreuses bactéries, dont l'une est E. coli O157:H7 (Wick et al., 2005). Le point crucial de la virulence des STEC réside dans le fait que les toxines de Shiga ne sont pas codées par un gène chromosomique d’E. coli mais que leurs gènes stx sont situés dans les génomes de bactériophages qui se trouvent dans leurs hôtes en tant que prophages (Los et al., 2011). Les études d'acquisition et de stabilité du phage Stx sont donc cruciales en termes de santé publique et animale. Les objectifs de cette troisième étude ont été d'isoler et de caractériser le phage Stx2d d’une souche STEC O80:H2, d'étudier la transduction du gène médiée par ce phage vers des souches non-STEC et ensuite, d’évaluer la survie de larves de Galleria mellonella inoculées avec ces souches transduites. Trois phages tempérés ont été induits et isolés de souches STEC bovine O80:H2 par rayonnement UV. Le gène stx2d d’un de ces phages a été transduit dans cinq souches non-STEC et son génome a été analysé. Trois souches : K12-MG1655, K12-DH5a et O80:H26 ont été transduites avec succès. Ces transferts ont été confirmés par une PCR ciblant le gène stx2d. L’ADN génomique du phage a été extrait puis analysé. Le phage fait partie de la classe des Caudoviricetes, il est stable dans les nouvelles souches STEC après trois repiquages successifs et présente des résistances à température et à pH modérés. Son génome présente de nombreuses protéines impliquées dans le cycle lysogénique. Les expériences en modèle Galleria mellonella ont montré que les souches transduites provoquaient des taux de mortalité significativement plus élevés que les souches non transduites respectives. En conclusion, non seulement les EPEC mais aussi les AE-STEC O80:H2 sont présentes chez les veaux diarrhéiques et septicémiques en Belgique et sont génétiquement apparentées aux AE-STEC humaines. Les jeunes veaux laitiers en bonne santé sont des porteurs sains d'AE-STEC et d'EPEC en Belgique ; le sérotype O80:H2 restant non identifié à ce jour dans cette catégorie d’animaux. Ces résultats confirment la nécessité de comprendre l'évolution des AE-STEC bovines et humaines et des EPEC O80:H2. Pour cela, l’étude sur le transfert de gène de la toxine de Shiga médiée par le phage Stx2d a montré que celui-ci peut être transféré à des souches non-STEC et contribuer à leur virulence dans le modèle des larves de Galleria mellonella. La surveillance des souches (AE)-STEC O80:H2 est essentielle au vu de leur présence dans certaines catégories d’animaux et du risque potentiel représenté par la transduction du gène stx

Rasa3 controls turnover of endothelial cell adhesion and vascular lumen integrity by a Rap1-dependent mechanism

Rasa3 is a GTPase activating protein of the GAP1 family which targets R-Ras and Rap1. Although catalytic inactivation or deletion of Rasa3 in mice leads to severe hemorrhages and embryonic lethality, the biological function and cellular location of Rasa3 underlying these defects remains unknown. Here, using a combination of loss of function studies in mouse and zebrafish as well as in vitro cell biology approaches, we identify a key role for Rasa3 in endothelial cells and vascular lumen integrity. Specific ablation of Rasa3 in the mouse endothelium, but not in megakaryocytes and platelets, lead to embryonic bleeding and death at mid-gestation, recapitulating the phenotype observed in full Rasa3 knock-out mice. Reduced plexus/sprouts formation and vascular lumenization defects were observed when Rasa3 was specifically inactivated in mouse endothelial cells at the postnatal or adult stages. Similar results were obtained in zebrafish after decreasing Rasa3 expression. In vitro, depletion of Rasa3 in cultured endothelial cells increased β1 integrin activation and cell adhesion to extracellular matrix components, decreased cell migration and blocked tubulogenesis. During migration, these Rasa3-depleted cells exhibited larger and more mature adhesions resulting from a perturbed dynamics of adhesion assembly and disassembly which significantly increased their life time. These defects were due to a hyperactivation of the Rap1 GTPase and blockade of FAK/Src signaling. Finally, Rasa3-depleted cells showed reduced turnover of VE-cadherin-based adhesions resulting in more stable endothelial cell-cell adhesion and decreased endothelial permeability. Altogether, our results indicate that Rasa3 is a critical regulator of Rap1 in endothelial cells which controls adhesions properties and vascular lumen integrity; its specific endothelial cell inactivation results in occluded blood vessels, hemorrhages and early embryonic death in mouse, mimicking thus the Rasa3-/- mouse phenotype

Identification of non-conventional serotypes of enteropathogenic Escherichia coli (EPEC) isolated from diarrheic calves

Enteropathogenic Escherichia coli (EPEC) are subdivided into “typical (t) EPEC” producing the “Bundle Forming Pili” (BFP) type 4 fimbriae and isolated from humans, and “atypical (a) EPEC” not producing the BFP and isolated from animals and humans. aEPEC are indeed quite frequently associated with diarrhoea in young calves. Although calf aEPEC can belong to the O26:H11 and O80:H2 serotypes, most serotypes remain unidentified (Mainil and Fairbrother, 2014). The general purpose of this project is to identify the serotypes of aEPEC isolated from diarrheic calves and to compare them with calf and human Shiga toxin-producing E. coli (STEC) belonging to the same serotypes. The specific purpose of the study reported here was therefore to test 41 and 35 un-typed aEPEC and STEC for five non-conventional O serogroups (O123/186, O156, O177, O182, O183) recently identified in 5 calf aEPEC and STEC that previously tested negative, using two multiplex PCRs and to confirm the positive results with the uniplex PCRs. Twenty-three aEPEC (56%) tested positive with the multiplex and uniplex PCRs: 9 for the O123/186 serogroups (these two serogroups cannot be distinguished by PCR), 12 for the O177 and 2 for the O182 serogroups. In addition, the PCRs also detected 8 STEC (23%): 1 for the O123/186, 2 for the O156, 1 for the O177, 2 for the O182 and 2 for the O183 serogroups.  Besides the classical O26:H11 serotype, calf aEPEC belong to several non-conventional serogroups/-types, like O80:H2 and those identified in this study, though still other serogroups/-types remain to identify. The further steps are: (i) comparison of these calf aEPEC with calf and human STEC belonging to the same serogroups/-types; (ii) investigation to answer the following question: are these calf aEPEC true aEPEC, or STEC derivatives that have lost stx genes, or STEC precursors that could acquire stx genes in the future? (iii) identification of still other serogroups/-types amongst the remaining un-typed calf aEPEC

Isolation of sero-pathotype specific bacteriophages against unconventional Shiga toxin-producing (STEC) and enteropathogenic (EPEC) Escherichia coli from diarrheic calves.

Escherichia coli producing the Shiga toxins (STEC) and/or the attaching-effacing (AE) lesion (EPEC) cause enteritis and (bloody) diarrhoea in young calves and in humans. STEC and EPEC can belong 7 serogroups frequently identified worldwide: O26, O103, O111, O121, O145, O157 and O165. Beside these classical “gang of 7” serogroups, unconventional serogroups can be identified as previously demonstrated with the O80 EPEC. The use of active and specific bacteriophages as biocontrol agents seems to be a promising alternative against unconventional STEC/EPEC and have been employed for diagnostic. The first aim of this project was to identify those 6 unconventional serogroups among 76 STEC and EPEC isolated between 2008 and 2015 from diarrheic calves at ARSIA. Two triplex PCRs have been applied either for the O146_O182_O183 serogroups or for the O123/186_O156_O177 serogroups. The second objective of this project was to isolate specific bacteriophages against these unconventional serogroups from wastewater or farm slurry. So far, the first triplex PCR identified 4 O182-positive and 2 O183-positive STEC and EPEC. The second triplex PCR identified 10 O123-186-positive, 2 O156-positive and 13 O177-positive STEC and EPEC. A total of 4 potentially specific bacteriophages respectively active against O5, O123, O146, O186 were obtained. The further steps of this study will be to perform: (i) the PFGE profile comparison of the calf and healthy cattle PCR-positive STEC and EPEC between themselves and with human STEC and EPEC; (ii) the identification of still other unconventional serogroups among STEC and EPEC from diarrheic calves; (iii) the host range spectrum of the 4 potentially specific bacteriophages

Identification of unconventional Shiga toxin-producing (STEC) and enteropathogenic (EPEC) Escherichia coli from diarrheic calves.

Introduction Escherichia coli producing the Shiga toxins (STEC) and/or the attaching-effacing (AE) lesion (AE-STEC) cause enteritis and (bloody) diarrhoea in young calves and in humans, and are also present in the intestines of healthy cattle. AE-STEC in humans can belong to scores of O serogroups with 7 most frequent and pathogenic ones (O157, O26, O103, O111, O121, O145 and O165). Conversely the majority of AE-STEC in young calves belong to a narrower range of serogroups (O26, O111, O118, O5). Besides AE-STEC, E. coli producing only the AE lesion (EPEC) are also frequently associated with diarrhoea in young calves. Half of them belong to the O26 and O80 serogroups,, but the serotypes of the second half of isolates remain unidentified (Fakih et al. 2017; Thiry et al., 2018). Materials and Methods Different unconventional serogroups (O123/186, O156, O177, O182, O183) were identified by PCR in 5 STEC or EPEC that tested negative for the pre-cited 12 serogroups (Iguchi et al., 2015). The aim of this study was therefore to identify those 5 serogroups and the O146 serogroup amongst 76 AE-STEC, STEC and EPEC isolated between 2008 and 2015 from diarrheic calves at ARSIA (Fakih et al., 2017). Two triplex PCRs were applied either for the O146_O182_O183 serogroups or for the O123/186_O156_O177 serogroups. Discussion So far the first triplex PCR identified 4 O182-positive and 2 O183-positive STEC and EPEC. These 6 PCR-positive isolates were confirmed by either uniplex PCR. Two O182-positive isolates were AE-STEC and 2 were EPEC whereas the two O183-positive isolate were STEC. The second triplex PCR identified 10 O123-186-positive, 2 O156-positive and 13 O177-positive STEC and EPEC. These 25 PCR-positive isolates were confirmed by the three uniplex PCR. One O123/186-positive isolate was AE-STEC and 9 were EPEC. One O156-positive isolate was AE-STEC and one was STEC and finally, 12 O177-positive isolates were EPEC and 1 was STEC. The further steps of the work are: (i) the identification of the same 6 serogroups amongst a collection of (AE-)STEC and EPEC isolated from healthy cattle at slaughterhouse; (ii) the identification of still other serogroups amongst the remaining untyped calf (AE-)STEC and EPEC; (iii) the comparison of the calf-positive STEC and EPEC with human (AE-)STEC and EPEC belonging to the same serogroups. References Fakih, I., Thiry, D., Duprez, J. N., Saulmont, M., Iguchi, A., Piérard, D., … Mainil, J. G. (2017). Identification of Shiga toxin-producing (STEC) and enteropathogenic (EPEC) Escherichia coli in diarrhoeic calves and comparative genomics of O5 bovine and human STEC. Veterinary Microbiology, 202, 16–22. Iguchi, A., Iyoda, S., Seto, K., Morita-Ishihara, T., Scheutz, F., & Ohnishi, M. (2015). Escherichia coli O-genotyping PCR: A comprehensive and practical platform for molecular O serogrouping. Journal of Clinical Microbiology, 53(8), 2427–2432. https://doi.org/10.1128/JCM.00321-15 Thiry, D., De Rauw, K., Takaki, S., Duprez, J. N., Iguchi, A., Piérard, D., … Mainil, J. G. (2018). Low prevalence of the “gang of seven” and absence of the O80:H2 serotypes among Shigatoxigenic and enteropathogenic Escherichia coli (STEC and EPEC) in intestinal contents of healthy cattle at two slaughterhouses in Belgium in 2014. Journal of Applied Microbiology, 124(3), 867–873