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See detailIdentification of virulotypes and serotypes of enteropathogenic (EPEC) and Shigatoxigenic (STEC) Escherichia coli from healthy cattle at slaughterhouses in Wallonia.
Takaki, Shino; Duprez, Jean-Noël ULg; Fakih, Ibrahim et al

Poster (2016, September)

Escherichia coli producing the attachment-effacement (AE) lesion (EPEC) and/or Shiga toxins (STEC) cause enteritis and (bloody) diarrhoea in young calves and in humans, and are also present in the ... [more ▼]

Escherichia coli producing the attachment-effacement (AE) lesion (EPEC) and/or Shiga toxins (STEC) cause enteritis and (bloody) diarrhoea in young calves and in humans, and are also present in the intestines of healthy cattle. Besides the O157:H7 serotype, which is the main serotype causing STEC outbreaks in the world EPEC and STEC can belong to dozens of O serogroups. Of them, 9 have been frequently identified worldwide: O5, O26, O103, O104, O111, O118, O121, O145 and O165. The aim of this study is to identify the virulotypes and the O serotypes of EPEC and STEC isolated from healthy cattle at slaughterhouses in Wallonia. A total of 245 faeces (216 <1year-old bulls, 25 cows and 4 heifers) were sampled between April and June 2014 in 2 slaughterhouses in Wallonia and grown overnight at 37°C in Lauryl sulfate Enterobacteriaceae selective broth. The enrichment broths were assayed with an stx1, stx2 (Shiga toxin) and eae (AE lesion) triplex PCR and positive broths were inoculated onto 4 agar media: McConkey’s, Chromagar ES, Chromagar ES with tellurite and Chromagar STEC. Up to ten colonies per plate were picked up, sub-cultured and tested by the colony hybridization assay with gene probes targeting the stx1, stx2 and eae genes. The triplex PCR was again performed on all probe-positive isolates. The PCR-positive E. coli were subsequently assayed with two pentaplex PCR targeting the specific genes coding for the ten O serogroups listed above. Of the 2563 sub-cultured isolates, 744 isolates (29%) from 62 animals (25%) tested positive with the colony hybridization assay. Of them, 687 isolates (92%) from 59 animals were positive with the triplex PCR and the results of both tests were in agreement for 617 isolates (83%). One to 29 isolates per animal were probe- and PCR-positive. The positive isolates grew on Chromagar STEC (379; 55%), on Chromagar ES with tellurite (189; 28%), on Chromagar ES (62; 9%) or on McConkey’s agar (57; 8%). The most frequent virulotypes were eae+ (EPEC: 372 isolates; 54%), eae+stx1+ (AE_STEC: 119 isolates; 17%) and stx2+ (STEC: 118 isolates; 17%). In some animals different virulotypes were identified. The serogrouping with the two pentaplex PCR is in progress. AE-STEC, EPEC and STEC are excreted by 25% of the healthy cattle at slaughterhouses in Wallonia and different virulotypes can be excreted by the same animal. Conversely the methodology followed gives no precise idea of the actual level of excretion since the hybridization and PCR were performed after enrichment in selective broth. Therefore multiple isolates belonging to the same virulotype might represent the same clone. Identification of the serogroups and comparison by Pulsed Field Gel Electrophoresis should help to clarify that point. Quantitative (q)PCR is today the best method to quantify bacterial excretion, but is more expensive. The results of the hybridization and PCR correspond to between 80 and 90%. Though the colony hybridization is still useful for large-scale surveillance it needs radioactive probes for highest sensitivity and is more time-consuming than PCR. Therefore the PCR should be the first routine choice if it can be automatized at large scale. Further steps are the confirmation of the PCR results of the 70 isolates with different hybridization and PCR results and the identification of the serogroups with the two pentaplex PCR and later with PCR for the other serogroups, to compare them with isolates from young diarrhoeic calves and from humans. [less ▲]

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See detailIdentification of bovine methicillin resistant staphylococci from Europe, Africa and North America by colony hybridization, PCR and antibiotic sensitivity.
Ngassam Tchamba, Cyrille ULg; Thiry, Damien ULg; Bardiau, Marjorie et al

Conference (2016, September)

Mastitis is the costliest pathology in dairy cattle and staphylococci are the most prevalent bacterial mastitis pathogens worldwide. Antimicrobial treatment of mastitis has led to the selection of ... [more ▼]

Mastitis is the costliest pathology in dairy cattle and staphylococci are the most prevalent bacterial mastitis pathogens worldwide. Antimicrobial treatment of mastitis has led to the selection of resistant staphylococci, of which the Methicillin Resistant S. aureus (MRSA) are the most studied ones. Still, MR has also been described for non-aureus staphylococci (MRS) species. Bovine MRS(A) represent not only a problem in the treatment of mastitis, but also a potential hazard in public health via the inter-Staphylococcus transferability of the mobile “Staphylococcal Cassette Chromosome” (SCC) carrying the mec genes encoding MR and the zoonotic potential of some Staphylococcus species. The aim of this study is the comparison of genetic and phenotypic methods for the identification of MRS(A) isolated from bovine mastitis in European, African and North-American countries. A total of 1168 mastitis-associated staphylococci were isolated between 2005 and 2014 in Belgium, Italy, Switzerland, Senegal, Niger and Canada, and kept at -80°C until further use. Out of them, 867 isolates were identified to S. aureus while 301 isolates were non aureus staphylococci. All 1168 staphylococci were tested genetically by the dot blot hybridization assay on positively charged nylon membranes (Roche) after DNA extraction with 32P-radioactively labelled probes derived from the mecA and mecC genes and phenotypically by growth on “Chrom MRSA ID®” agar plates. Isolates positive at both or either tests were further studied by PCR targeting the same two genes and by the disk diffusion assay to oxacillin and cefoxitin. A total of 265 isolates (23%) were positive at both or either tests. Out of them, 27 S. aureus (10%) but no non-aureus (0%) tested positive both for DNA hybridization with the mecA probe and for growth on “Chrom MRSA ID®” plates. No isolate tested positive with the mecC probe. In addition, 32 S. aureus (12%) and 15 non aureus (6%) were positive with the mecA probe only and 169 S. aureus (64%) and 22 non aureus (8%) grew on “Chrom MRSA ID®” plates only. The S. aureus originate from Belgium (105), Italy (6), Canada (31), Senegal (38) and Niger (48) whereas the non-aureus originate from Belgium (25), Italy (1) and Niger (11). All of them are being tested with the PCR targeting the mecA gene and by the disk diffusion assay to oxacillin and cefoxitin. Most isolates (72%) grew on “Chrom MRSA ID®” plates only while few (18%) were positive to the hybridization with the mecA probe only. This high difference between the results of both tests could be explained by the weak specificity of phenotypic tests comparing to genetic tests. The others 10% of the isolates (S. aureus) which are positive with the two methods (dot blot hybridization and “Chrom MRSA ID®”) can be considered as MRSA mediated by the mecA gene. However, results of PCR and disk diffusion assay will confirm respectively the presence of mec genes and which of the two methods is the most suitable for identifying MRS from mastitis cases in cattle. Comparison of the results of phenotypic and genetic assays will indicate whether other variant(s) than mecA and mecC may be present in MRS. Further genetic and phenotypic studies are needed to (i) identify the non-aureus isolates to the species level; (ii) compare the MRS(A) isolated in the different countries by their biotypes, serotypes, lysotypes, and virulotypes, without forgetting their SCCmec and their clonal complex; and (iii) identify the mec gene variant present in hybridization-positive PCR-negative isolates, if any. [less ▲]

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See detailIdentification of bovine and porcine colistin-resistant mcr1-positive Escherichia coli.
Mainil, Jacques ULg; Muylaert, Adeline ULg; Saulmont, Marc et al

Conference (2016, September)

OBJECTIVE Polymyxins, especially colistin, have been used for years in veterinary medicine and were rediscovered a few years ago as last resort antibiotics in human medicine against multi-resistant Gram ... [more ▼]

OBJECTIVE Polymyxins, especially colistin, have been used for years in veterinary medicine and were rediscovered a few years ago as last resort antibiotics in human medicine against multi-resistant Gram negative bacterial pathogens. For years, only chromosome-mediated resistance to colistin was identified as a consequence of mutation(s) in lipid A-encoding genes. Recently, however, a plasmid-located gene (mcr1) was identified in Gram-negative enterobacteria and has since been found by PCR in several, but not all, bovine, human, porcine and poultry colistin-resistant Escherichia coli (Liu YY et al. Lancet Infect Dis, 2016, 16(2), 161-168; Nordmann P and Poirel L. Clin Microbiol Infect, 2016, 22, 398-400 ; Schwarz S and Johnson AP. J Antimicrob Chemother, 2016, in press, doi: 10.1093/jac/dkw274). The purpose of this study was to compare phenotypic and genetic for the detection of resistance to colistin and of the mcr1 gene in a collection of Escherichia coli isolated from different animal species and from humans. METHODS More than 3000 E. coli isolates from cattle, pigs, dogs, cats, horses, rabbits, chickens ducks and humans were tested for resistance to colistin by growing them on agar plates with 1g/ml of colistin. The Minimal Inhibitory Concentrations (MIC) of and the presence of the mcr1 gene in all growing isolates were determined using the E test® and colony hybridization assay with a mcr1 specific gene probe, respectively. The probe-positive isolates were further tested with the mcr1 gene specific PCR. RESULTS A total of 410 E. coli isolated grew on 1g/ml colistin-containing agar plates. The majority of isolates grew well, but several grew sparsely with only few isolated colonies. As determined by the E test®, MIC of 273 isolates (67%) was 1g/ml of colistin and higher; conversely, MIC of 137 isolates (33%) was lower than 1g/ml of colistin. Of those 410 E. coli isolates, 34 from pigs and bovines (9% of isolates growing on colistin-containing agar plates; 25% of isolates with MIC higher than 1g/ml) hybridized with the mcr1 gene-derived probe: 5 from pigs and 11 from bovines gave black spots (including five from the same calf), while 18 from pigs and one from bovine gave grey spots. All but one pig isolate had a MIC between 1.5 and 16 g/ml of colistin. Fifteen “black spot” probe-positive isolates tested positive with the mcr1 gene specific PCR as did 3 porcine “grey spot” probe-positive isolates, while the remaining 16 isolates repeatedly tested negative even after lowering the annealing temperature. CONCLUSION This study confirms that (i) the results of phenotypic assays for the detection of colistin resistance can not be always trusted; (ii) the mcr1 gene is not the only one mechanism of resistance to colistin; (iii) mcr1 variants may exist that can not be detected by the classical PCR. Phenotypic assays like growth on colistin-containing agar plates can still represent a first base screening assay, although the MIC determination using the E test® confirms a >1g/ml MIC for only 2 out of 3 growing isolates. Presence of mcr1 gene and putative variants (like the most recently described mcr2 gene; Xavier BB et al., Eurosurveillance, 21, 7 July 2016) in all probe-positive isolates will be confirmed after Whole Genome Sequencing that will also allow comparing the mcr1-positive plasmids and isolates from pigs and cattle to similar human E. coli isolates. Further studies should also be performed to identify the colistin resistance mechanism in mec-negative isolates. [less ▲]

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See detailIsolation of bacteriophages against Klebsiella pneumoniae and in vivo activity
Thiry, Damien ULg; Passet, Virginie; Dufour, Nicolas et al

Poster (2016, September)

Klebsiella pneumoniae is a bacterial pathogen able to induce severe healthcare-associated or community-acquired infections in humans and animals. The constant emergence of antibiotic resistant strains ... [more ▼]

Klebsiella pneumoniae is a bacterial pathogen able to induce severe healthcare-associated or community-acquired infections in humans and animals. The constant emergence of antibiotic resistant strains reinforces the need to find alternatives to antibiotic treatments. The use of bacteriophages is a promising approach. The aim of this study was to isolate bacteriophages directed against K. pneumoniae strains and to test their efficacy in a murine model. Bacteriophages against five different K. pneumoniae (2 of capsular type K1 and K2 and 1 undetermined) were isolated and purified from waste water collected in Paris area. The morphology of plaques (zones of bacterial killing) was recorded and several of them were purified three times by successive replating. Phage titers were determined by serial dilutions on their respective hosts as well as on 18 other Klebsiella strains to identify their host range. Kinetics of bacterial lysis were monitored during 15h at 3 multiplicities of infection, in triplicates. For in vivo experiment, a total of 10 mice were inoculated with 200 µl of K. pneumoniae (4.6E+07 CFU) by oral gavage and the level of K. pneumoniae in fecal samples was monitored for 10 days. Five mice did not receive any treatment and 5 other mice received a cocktail of three bacteriophages (8E+07 PFU) at day 4 post-inoculation. A total of 54 bacteriophages were isolated and purified with titers ranging from 2E+5 to 3.6E+10 PFU/ml. The host range study showed that bacteriophages against K. pneumoniae have a specificity related to the capsular type of their bacterial host. Lysis kinetics of bacteria suggested that different phages were isolated. Despite difficulties with the murine intestinal model, evidence was obtained that bacteriophages are able to reduce intestinal carriage. Our results show that bacteriophages isolated against K. pneumoniae are specific for a given capsular type, although further studies are necessary to provide more details on this capsular specificity and its molecular determinants. To fully address the in vivo potential of phages, a reliable mouse model of intestinal carriage of K. pneumoniae strains needs to be established. [less ▲]

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See detailMediation analysis to estimate direct and indirect milk losses associated with bacterial load in bovine subclinical mammary infections
Detilleux, Johann ULg; Theron, Léonard ULg; Duprez, Jean-Noël ULg et al

in Animal (2016)

Milk losses associated with mastitis can be attributed to either effects of pathogens per se (i.e. direct losses) or to effects of the immune response triggered by the presence of mammary pathogens (i.e ... [more ▼]

Milk losses associated with mastitis can be attributed to either effects of pathogens per se (i.e. direct losses) or to effects of the immune response triggered by the presence of mammary pathogens (i.e. indirect losses). Test-day milk somatic cell counts (SCC) and number of bacterial colony forming units (CFU) found in milk samples are putative measures of the level of immune response and of the bacterial load, respectively. Mediation models, in which one independent variable affects a second variable which, in turn, affects a third one, are conceivable models to estimate direct and indirect losses. Here, we evaluated the feasibility of a mediation model in which test-day SCC and milk were regressed toward bacterial CFU measured at three selected sampling dates, 1 week apart. We applied this method on cows free of clinical signs and with records on up to 3 test-days before and after the date of the first bacteriological samples. Most bacteriological cultures were negative (52.38%), others contained either staphylococci (23.08%), streptococci (9.16%), mixed bacteria (8.79%) or were contaminated (6.59%). Only losses mediated by an increase in SCC were significantly different from null. In cows with three consecutive bacteriological positive results, we estimated a decreased milk yield of 0.28 kg per day for each unit increase in log2-transformed CFU that elicited one unit increase in log2-transformed SCC. In cows with one or two bacteriological positive results, indirect milk loss was not significantly different from null although test-day milk decreased by 0.74 kg per day for each unit increase of log2-transformed SCC. These results highlight the importance of milk losses that are mediated by an increase in SCC during mammary infection and the feasibility of decomposing total milk loss into its direct and indirect components. © The Animal Consortium 2016 [less ▲]

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See detailIdentification of Shiga toxin-producing (STEC) and enteropathogenic (EPEC) Escherichia coli in diarrhoeic calves and comparative genomics of O5 bovine and human STEC
Fakih, Ibrahim; Thiry, Damien ULg; Duprez, Jean-Noël ULg et al

in Veterinary Microbiology (2016)

Escherichia coli producing Shiga toxins (Stx) and the attaching-effacing (AE) lesion (AE-STEC) are responsible for (bloody) diarrhoea in humans and calves while the enteropathogenic E. coli (EPEC ... [more ▼]

Escherichia coli producing Shiga toxins (Stx) and the attaching-effacing (AE) lesion (AE-STEC) are responsible for (bloody) diarrhoea in humans and calves while the enteropathogenic E. coli (EPEC) producing the AE lesion only cause non-bloody diarrhoea in all mammals. The purpose of this study was (i) to identify the pathotypes of enterohaemolysin-producing E. coli isolated between 2009 and 2013 on EHLY agar from less than 2 month-old diarrhoeic calves with a triplex PCR targeting the stx1, stx2, eae virulence genes; (ii) to serotype the positive isolates with PCR targeting the genes coding for ten most frequent and pathogenic human and calf STEC O serogroups; and (iii) to compare the MLSTypes and virulotypes of calf and human O5 AE-STEC after Whole Genome Sequencing using two server databases (www.genomicepidemiology.org). Of 233 isolates, 206 were triplex PCR-positive: 119 AE-STEC (58%), 78 EPEC (38%) and 9 STEC (4%); and the stx1+eae+ AE-STEC (49.5%) were the most frequent. Of them, 120 isolates (84% of AE-STEC, 23% of EPEC, 22% of STEC) tested positive with one O serogroup PCR: 57 for O26 (47.5%), 36 for O111 (30%), 10 for O103 (8%) and 8 for O5 (7%) serogroups. The analysis of the draft sequences of 15 O5 AE-STEC could not identify any difference correlated to the host. As a conclusion, (i) the AE-STEC associated with diarrhoea in young calves still belong to the same serogroups as previously (O5, O26, O111) but the O103 serogroup may be emerging, (ii) the O5 AE-STEC from calves and humans are genetically similar [less ▲]

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See detailA 3-year long study of Staphylococcus aureus isolates from subclinical mastitis in three Azawak zebu herds at the Sahelian experimental farm of Toukounous, Niger
Issa Ibrahim, Abdoulkarim; Duprez, Jean-Noël ULg; Bada-alambdeji, Rianatou et al

in Tropical Animal Health and Production (2016), 48(2), 321-329

bovine mastitis. The aim of the present work was to follow in three herds and during the 3 years the clonality of S. aureus isolated from California Mastitis Test (CMT)-positive cows at the experimental ... [more ▼]

bovine mastitis. The aim of the present work was to follow in three herds and during the 3 years the clonality of S. aureus isolated from California Mastitis Test (CMT)-positive cows at the experimental station of Toukounous (Niger) by (i) comparing their pulsed field gel electrophoresis (PFGE) fingerprints, (ii) identifying their virulotypes by PCR amplification and (iii) assessing the production of capsule and the formation of biofilm. The 88 S. aureus isolates belonged to 14 different pulsotypes, 3 of them being predominant: A (30 %), D (27 %), B (15 %). A and B pulsotypes had the highest profile similarity coefficient (94 %), while others had similarity coefficients under 60 %. Seventy-five S. aureus isolates were further studied for their virulotypes, capsular antigens and biofilm production. Most surface factor-, leukocidin- and haemolysin-, but not the enterotoxin-encoding genes were detected in the majority (>75 %) of the isolates and were evenly distributed between the A, B and D pulsotype isolates. The majority of the 72 S. aureus positive with the cap5H or cap8H PCR produced the CP5 (82 %) or the CP8 (88 %) capsular antigen, respectively. Biofilm production by the 57 icaA-positive isolates was strong for 8 isolates, moderate for 31 isolates but weak for 18 isolates, implying that the icaA gene may not be expressed in vitro by one third of the positive isolates. Similar to other studies, those results confirm that a restricted number of S. aureus clones circulate within the three herds at Toukounous and that their specific virulence-associated properties must still be further studied [less ▲]

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See detailExistence of two groups of Staphylococcus aureus strains isolated from bovine mastitis based on biofilm formation, intracellular survival, capsular profile and agr-typing.
Bardiau, Marjorie ULg; Caplin, Jonathan; Detilleux, Johann ULg et al

in Veterinary microbiology (2016), 185

Staphylococcus (S.) aureus is recognised worldwide as an important pathogen causing contagious acute and chronic bovine mastitis. Chronic mastitis account for a significant part of all bovine cases and ... [more ▼]

Staphylococcus (S.) aureus is recognised worldwide as an important pathogen causing contagious acute and chronic bovine mastitis. Chronic mastitis account for a significant part of all bovine cases and represent an important economic problem for dairy producers. Several properties (biofilm formation, intracellular survival, capsular expression and group agr) are thought to be associated with this chronic status. In a previous study, we found the existence of two groups of strains based on the association of these features. The aim of the present work was to confirm on a large international and non-related collection of strains the existence of these clusters and to associate them with case history records. In addition, the genomes of eight strains were sequenced to study the genomic differences between strains of each cluster. The results confirmed the existence of both groups based on capsular typing, intracellular survival and agr-typing: strains cap8-positive, belonging to agr group II, showing a low invasion rate and strains cap5-positive, belonging to agr group I, showing a high invasion rate. None of the two clusters were associated with the chronic status of the cow. When comparing the genomes of strains belonging to both clusters, the genes specific to the group "cap5-agrI" would suggest that these strains are better adapted to live in hostile environment. The existence of these two groups is highly important as they may represent two clusters that are adapted differently to the host and/or the surrounding environment. [less ▲]

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See detailPoint d'actualité : Chlamydia ou Chlamydophila ?
Mainil, Jacques ULg

Learning material (2016)

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See detailAntibiothérpaie chez le chien : choix raisonné et raisonnable
Mainil, Jacques ULg

Scientific conference (2015, December)

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See detailEpidemiology and Diagnosis of Q Fever in Animals and Humans in the 21st Century
Mainil, Jacques ULg; Monseur, Christine ULg; Saegerman, Claude ULg et al

Scientific conference (2015, November 13)

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See detailUpdate on Shigatoxigenic (STEC) and Attaching/Effacing (AEEC) Escherichia coli in Europe
Mainil, Jacques ULg

Scientific conference (2015, November)

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See detailAllelic variation contributes to bacterial host specificity
Yue, Min; Han, Xiangan; De Masi, Leon et al

in Nature Communications (2015)

Understanding the molecular parameters that regulate cross-species transmission and host adaptation of potential pathogens is crucial to control emerging infectious disease. Although microbial pathotype ... [more ▼]

Understanding the molecular parameters that regulate cross-species transmission and host adaptation of potential pathogens is crucial to control emerging infectious disease. Although microbial pathotype diversity is conventionally associated with gene gain or loss, the role of pathoadaptive nonsynonymous single-nucleotide polymorphisms (nsSNPs) has not been systematically evaluated. Here, our genome-wide analysis of core genes within Salmonella enterica serovar Typhimurium genomes reveals a high degree of allelic variation in surface-exposed molecules, including adhesins that promote host colonization. Subsequent multinomial logistic regression, MultiPhen and Random Forest analyses of known/suspected adhesins from 580 independent Typhimurium isolates identifies distinct host-specific nsSNP signatures. Moreover, population and functional analyses of host-associated nsSNPs for FimH, the type 1 fimbrial adhesin, highlights the role of key allelic residues in host-specific adherence in vitro. Together, our data provide the first concrete evidence that functional differences between allelic variants of bacterial proteins likely contribute to pathoadaption to diverse hosts. [less ▲]

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See detailVeterinary Medicine education in Europe, Belgium and Wallonia : history and evolution
Mainil, Jacques ULg

Scientific conference (2015, July)

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