Publications of Jacques Mainil
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See detailGenetic characterization of Shigatoxigenic and enteropathogenic Escherichia coli O80:H2 from diarrheic and septicemic calves and relatedness to human Shigatoxigenic E. coli O80:H2
Habets, Audrey ULiege; Crombé, F.; Nakamura, K. et al

in European Journal of Applied Microbiology (2020)

Aim: The purpose of this work was to identify and genetically characterize enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) O80:H2 from diarrhoeic and septicaemic calves in ... [more ▼]

Aim: The purpose of this work was to identify and genetically characterize enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) O80:H2 from diarrhoeic and septicaemic calves in Belgium and to comparing them with human EHEC after whole genome sequencing. Methods and Results: Ten EHEC and 21 EPEC O80 identified by PCR between 2009 and 2018 from faeces, intestinal content and a kidney of diarrhoeic or septicaemic calves were genome sequenced and compared to 19 human EHEC identified between 2008 and 2019. They all belonged to the O80: H2 serotype and ST301, harboured the eaeξ gene, and 23 of the 29 EHEC contained the stx2d gene. Phylogenetically, they were distributed in two major sub-lineages: one comprised a majority of bovine EPEC whereas the second one comprised a majority of stx2d bovine and human EHEC. Conclusions: Not only EPEC but also EHEC O80:H2 are present in diarrhoeic and septicaemic calves in Belgium and are genetically related to human EHEC. Significance and Impact of the Study: These findings support the need to assess cattle as potential source of contamination of humans by EHEC O80:H2 and to understand the evolution of bovine and human EHEC and EPEC O80: H2. [less ▲]

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See detailHarley William Moon (1936-2018)
Bertschinger, Hans; Duchesnes, Christiane ULiege; Mainil, Jacques ULiege et al

in FEMS Microbiology Letters (2020), 367(fnz012),

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See detailIn vitro and in vivo assessment of phage therapy against Staphylococcus aureus causing bovine mastitis
Ngassam Tchamba, Cyrille ULiege; Duprez, Jean-Noël ULiege; Fergestad, M. et al

in Journal of Global Antimicrobial Resistance (2020)

Objectives The aim of this study was to assess the efficacy of lytic bacteriophages onStaphylococcus aureus causing bovine mastitis, by in vitro and in vivo assays using Galleria mellonella and murine ... [more ▼]

Objectives The aim of this study was to assess the efficacy of lytic bacteriophages onStaphylococcus aureus causing bovine mastitis, by in vitro and in vivo assays using Galleria mellonella and murine mastitis models. Methods Between May and December 2016, tenS. aureus (five methicillin-resistant and five methicillin-sensitive) isolates were isolated from milk samples of cattle with mastitis in Belgium and Norway. The isolates were assessed in vitro for their susceptibility to four lytic bacteriophages (Romulus, Remus, ISP and DSM105264) and subsequently in vivo in G. mellonella larvae and in murine mastitis model. Results Romulus, Remus and ISP showed a lytic activity against theS. aureus isolates in vitro. A larvae survival rate below 50% was observed at four days post inoculation in the groups infected with a methicillin-sensitive S. aureus isolate and treated with these three phages in vivo. An incomplete recovery of the mice mastitis was observed at 48 hours post inoculation in the groups infected and treated with the ISP phage in vivo. Conclusions The observations are much more pronounced statistically between the infected-PBS treated and infected-phage treated groups inG. mellonella and murine mastitis model demonstrating an effect of the phages against S. aureus associated with bovine mastitis. [less ▲]

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See detailDifferential dynamics and impacts of prophages and plasmids on the pangenome and virulence factor repertoires of Shiga toxin-producing Escherichia coli O145:H28
Nakamura, K.; Murase, K.; Sato, M. P. et al

in Microbial Genomics (2020), 6(1),

Phages and plasmids play important roles in bacterial evolution and diversification. Although many draft genomes have been generated, phage and plasmid genomes are usually fragmented, limiting our ... [more ▼]

Phages and plasmids play important roles in bacterial evolution and diversification. Although many draft genomes have been generated, phage and plasmid genomes are usually fragmented, limiting our understanding of their dynamics. Here, we performed a systematic analysis of 239 draft genomes and 7 complete genomes of Shiga toxin (Stx)-producing Escherichia coli O145:H28, the major virulence factors of which are encoded by prophages (PPs) or plasmids. The results indicated that PPs are more stably maintained than plasmids. A set of ancestrally acquired PPs was well conserved, while various PPs, including Stx phages, were acquired by multiple sublineages. In contrast, gains and losses of a wide range of plasmids have frequently occurred across the O145:H28 lineage, and only the virulence plasmid was well conserved. The different dynamics of PPs and plasmids have differentially impacted the pangenome of O145:H28, with high proportions of PP- and plasmid-associated genes in the variably present and rare gene fractions, respectively. The dynamics of PPs and plasmids have also strongly impacted virulence gene repertoires, such as the highly variable distribution of stx genes and the high conservation of a set of type III secretion effectors, which probably represents the core effectors of O145:H28 and the genes on the virulence plasmid in the entire O145:H28 population. These results provide detailed insights into the dynamics of PPs and plasmids, and show the application of genomic analyses using a large set of draft genomes and appropriately selected complete genomes. [less ▲]

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See detailIdentification of non-conventional serotypes of enteropathogenic Escherichia coli (EPEC) isolated from diarrheic calves
Habets, Audrey ULiege; Duprez, Jean-Noël ULiege; Saulmont, Marc et al

Poster (2019, June)

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 ... [more ▼]

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. [less ▲]

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See detailVirulotyping of Shiga toxin-producing (STEC) and enteropathogenic (EPEC) Escherichia coli isolated from recto-anal mucosal swabs of young diarrheic and non-diarrheic calves
Habets, Audrey ULiege; Duprez, Jean-Noël ULiege; Engelen, Frédéric et al

Poster (2019, June)

Shiga toxin-producing Escherichia coli (STEC) are food-borne pathogens causing severe disease in humans worldwide, whose most important virulence factor is the production of Shiga toxins (Stx1, Stx2 ... [more ▼]

Shiga toxin-producing Escherichia coli (STEC) are food-borne pathogens causing severe disease in humans worldwide, whose most important virulence factor is the production of Shiga toxins (Stx1, Stx2). Additionally, most STEC also possess the eae gene encoding for the intimin protein, which is responsible for the formation of the attaching and effacing (A/E) lesions (AE-STEC) and is also typical of enteropathogenic E. coli (EPEC). AE-STEC colonize the mucosa of the recto-anal junction of healthy adult cattle, making recto-anal mucosal swabs (RAMS) more sensitive than fecal culture to isolate them. In addition, AE-STEC also colonize the small and large intestines of young calves causing diarrhea. Conversely, the carrier state of non-diarrheic young calves is unknown. Finally, most studies address the most pathogenic O157:H7 AE-STEC serotypes, although several other non-O157:H7 AE-STEC serotypes are becoming increasingly important with regard to human outbreaks. The objective of the present study was to evaluate the colonization by and shedding of AE-STEC in young diarrheic and non-diarrheic dairy calves. RAMS from 233 young calves in 3 farms with non-O157:H7 AE-STEC in older animals were overnight enriched in Lauryl-Sulfate broth and tested with a triplex PCR targeting the eae, stx1 and stx2 genes. Positive broths were subsequently streaked on four selective agar plates (Figure 1). Up to 5 colonies per plate will be picked-up, tested by the colony hybridization assay with probes for the same genes and confirmed by the same triplex PCR.A total of 152 broths (65%) tested positive with the triplex PCR after overnight enrichment: 23 AE-STEC, 122 STEC and 7 EPEC. In farm #1, 2 RAMS tested positive for AE-STEC, 45 for STEC and 3 for EPEC. In farm #2, 3 RAMS tested positive for AE-STEC, 42 for STEC and 4 for EPEC. In farm #3, 18 RAMS tested positive for AE-STEC and 35 for STEC. All positive broths are being streaked on the selective agar plates and the 5 picked-up colonies are being prepared for the colony hybridization assay. Of the positive RAM’s the majority (80%) harbored STEC and a minority AE-STEC (15%) or EPEC (5%). This low number of EPEC-positive samples is different from previous results obtained in adult cattle in two slaughterhouses following the same methodology. In the same survey, 80% of the PCR-positive broths gave colony hybridization-positive and triplex PCR-positive colonies. Confirmed positive colonies will be O:H serotyped by PCR and all results will be associated with presence/absence of diarrhea in the calves. [less ▲]

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See detailNew Bacteriophages against Emerging Lineages ST23 and ST258 of Klebsiella pneumoniae and Efficacy Assessment in Galleria mellonella Larvae
Thiry, Damien ULiege; Passet, Virginier; Danis-Wlodarczyk, Katarzyna et al

in Viruses (2019), 11(5),

Klebsiella pneumoniae is a bacterial pathogen of high public health importance. Its polysaccharide capsule is highly variable but only a few capsular types are associated with emerging pathogenic ... [more ▼]

Klebsiella pneumoniae is a bacterial pathogen of high public health importance. Its polysaccharide capsule is highly variable but only a few capsular types are associated with emerging pathogenic sublineages. The aim of this work is to isolate and characterize new lytic bacteriophages and assess their potential to control infections by the ST23 and ST258 K. pneumoniae sublineages using a Galleria mellonella larvae model. Three selected bacteriophages, targeting lineages ST258 (bacteriophages vB_KpnP_KL106-ULIP47 and vB_KpnP_KL106-ULIP54) and ST23 (bacteriophage vB_KpnP_K1-ULIP33), display specificity for capsular types KL106 and K1, respectively. These podoviruses belong to the Autographivirinae subfamily and their genomes are devoid of lysogeny or toxin-associated genes. In a G. mellonella larvae model, a mortality rate of 70% was observed upon infection by K. pneumoniae ST258 and ST23. This number was reduced to 20% upon treatment with bacteriophages at a multiplicity of infection of 10. This work increases the number of characterized bacteriophages infecting K. pneumoniae and provides information regarding genome sequence and efficacy during preclinical phage therapy against two prominent sublineages of this bacterial species. [less ▲]

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See detailAssessment of two selective agar media to isolate colistin-resistant bovine Escherichia coli: Correlation with minimal inhibitory concentration and presence of mcr genes
Thiry, Damien ULiege; Berrah, Anis ULiege; Evrard, Julien et al

in Journal of microbiological methods (2019), 159

The identification of colistin-resistant enterobacteria in veterinary medicine is impaired by the absence of first-line reliable phenotypic assay. The purpose of this study was to assess two selective ... [more ▼]

The identification of colistin-resistant enterobacteria in veterinary medicine is impaired by the absence of first-line reliable phenotypic assay. The purpose of this study was to assess two selective agar media for the detection of colistin-resistant bovine pathogenic Escherichia coli. A total of 158 E. coli (46 R <resistant>, 96 I <intermediate> and 16 S <sensitive> at the disk diffusion assay) isolated between 2013 and 2018 from <3 month-old calves suffering enteritis or septicaemia, were (i) tested by the broth dilution assay to determine colistin Minimal Inhibitory Concentrations (MIC); (ii) streaked on CHROMID® Colistin_R and CHROMagar™ COL-APSE agar plates; (iii) submitted to a pentaplex PCR to identify the presence of mcr-1 to mcr-5 genes. Of the 92 E. coli growing on both agar media, 90 had a MIC > 2.0 μg/ml as had the 3 E. coli that grew only on the CHROMID® Colistin_R agar medium and one E. coli that grew on neither agar media. Therefore, the positive predictive values of the CHROMID® Colistin_R and CHROMagar™ COL-APSE agar media were both 0.98 whereas their negative predictive values were 0.98 and 0.94, respectively. Also noteworthy 43 of the 46 R isolates had a MIC > 2.0 μg/ml and grew on both selective media as did half of the 96 I isolates and only 1 of the S isolates. Conversely, only 30 of the 90 isolates that grew on both agar media and with a MIC > 2.0 μg/ml tested positive for the mcr-1 or mcr-2 genes with the pentaplex PCR. These two selective agar media can be used to reliably detect colistin-resistant E. coli. Positive growth was highly correlated with R results at the disk diffusion assay, but not with the presence of mcr genes. Copyright © 2019. Published by Elsevier B.V. [less ▲]

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See detailCharacteristics of Shiga toxin producing- and enteropathogenic Escherichia coli of the emerging serotype O80:H2 isolated from humans and diarrhoeic calves in Belgium
De Rauw, K.; Thiry, Damien ULiege; Caljon, B. et al

in Clinical Microbiology and Infection (2019), 25

Objectives: Recently a highly virulent Escherichia coli O80:H2 pathotype carrying Shiga toxin genes, the intimin subtype eaeξ, and genes associated with the extraintestinal pathogenic E. coli (ExPEC) pS88 ... [more ▼]

Objectives: Recently a highly virulent Escherichia coli O80:H2 pathotype carrying Shiga toxin genes, the intimin subtype eaeξ, and genes associated with the extraintestinal pathogenic E. coli (ExPEC) pS88 plasmid was described in France. In this study we examine the relatedness of Belgian E. coli O80:H2 isolated from humans and diarrhoeic calves as well their similarities with the French pathotype. Methods: Eighteen Belgian E. coli O80:H2 strains (nine human Shiga toxin-producing E. coli (STEC) (2008–2016), two bovine STEC (1987) and seven bovine atypical enteropathogenic E. coli (aEPEC) (2009–2015)) were characterized with conventional PCR, disc diffusion susceptibility testing and whole genome sequencing. Results: Only nine sporadic human STEC O80:H2 cases have been detected in Belgium. All patients were female, just two of them suffered from haemolytic uremic syndrome. All studied strains had the eaeξ subtype, belonged to the multi-locus sequence type ST-301, and carried virulence genes associated with the type III secretion system and effectors not encoded by the locus of enterocyte effacement (LEE). Multiple genes of the pS88 plasmid were detected in all but two strains (one human and one calf STEC). The Shiga toxin subtypes stx1a (n = 3; one human, two calf), stx2a (n = 2) and stx2d (n = 6) were detected. All strains were multidrug resistant, two were extended-spectrum β-lactamase positive. Core genome MLST revealed that some human and calf E. coli differed by only 22 loci. Conclusions: The STEC/ExPEC O80:H2 pathotype was present in calves in Belgium as early as 1987, but human infections have been rare and mostly mild. The human STEC and bovine aEPEC cluster together and have the potential to be as virulent as the French isolates, as shown by their similar gene content. © 2018 European Society of Clinical Microbiology and Infectious Diseases [less ▲]

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See detailComparison of quantitative PCR and MALDI-TOF mass spectrometry assays for identification of bacteria in milk samples from cows with subclinical mastitis
Ngassam Tchamba, C.; Rao, Anne-Sophie ULiege; Boyen, F. et al

in Journal of Applied Microbiology (2019), 127

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See detailCHROMAgar Mastitis medium: assessment of the new formulation and comparison with the previous one
DIDESSE, Elsa; Duprez, Jean-Noël ULiege; Mainil, Jacques ULiege et al

Poster (2018, October 26)

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See detailIsolation of sero-pathotype specific bacteriophages against unconventional Shiga toxin-producing (STEC) and enteropathogenic (EPEC) Escherichia coli from diarrheic calves.
Habets, Audrey ULiege; Duprez, Jean-Noël ULiege; Iguchi, Atsushi et al

Conference (2018, October 26)

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 ... [more ▼]

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. [less ▲]

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