Isabelle Ote

Genotypic characterization by polymerase chain reaction of Staphylococcus aureus isolates associated with bovine mastitis.

Staphylococcus aureus is recognized worldwide as a pathogen causing many serious diseases in humans and animals, and is the most common aetiological agent of clinical and subclinical bovine mastitis. The importance of evaluating the combination of S. aureus virulence factors has been emphasized both in human and veterinary medicine, and knowledge about the genetic variability within different S. aureus populations would help in the design of efficient treatments. The aim of the present study was to determine the genetic profiles of S. aureus strains isolated from milk of cows suffering from clinical and subclinical mastitis in Belgium. The presence of about forty virulence-associated genes was investigated by specific polymerase chain reaction (PCR) amplification. A high number of genotypic subtypes were observed, demonstrating further the large variation in the presence of virulence genes in S. aureus isolates and the considerable diversity of strains populations that are able to cause mastitis in cows. In accordance with other studies, we showed that some genes are associated with mastitis-causing S. aureus isolates, whereas others are absent or rarely present. We also further highlighted the presence of conserved gene combinations, namely the enterotoxigenic egc-cluster and the bovine pathogenicity island SaPIbov. Importantly, the presence of isolates carrying genes coding for toxins involved in important human infections makes the milk of cows with mastitis a potential reservoir for these toxins, and therefore a potential danger in human health, which strengthens the importance to consider raw milk consumption and its processing very carefully.

The varicella-zoster virus ORF47 kinase interferes with host innate immune response by inhibiting the activation of IRF3.

The innate immune response constitutes the first line of host defence that limits viral spread and plays an important role in the activation of adaptive immune response. Viral components are recognized by specific host pathogen recognition receptors triggering the activation of IRF3. IRF3, along with NF-κB, is a key regulator of IFN-β expression. Until now, the role of IRF3 in the activation of the innate immune response during Varicella-Zoster Virus (VZV) infection has been poorly studied. In this work, we demonstrated for the first time that VZV rapidly induces an atypical phosphorylation of IRF3 that is inhibitory since it prevents subsequent IRF3 homodimerization and induction of target genes. Using a mutant virus unable to express the viral kinase ORF47p, we demonstrated that (i) IRF3 slower-migrating form disappears; (ii) IRF3 is phosphorylated on serine 396 again and recovers the ability to form homodimers; (iii) amounts of IRF3 target genes such as IFN-β and ISG15 mRNA are greater than in cells infected with the wild-type virus; and (iv) IRF3 physically interacts with ORF47p. These data led us to hypothesize that the viral kinase ORF47p is involved in the atypical phosphorylation of IRF3 during VZV infection, which prevents its homodimerization and subsequent induction of target genes such as IFN-β and ISG15.

Associations between properties linked with persistence in a collection of Staphylococcus aureus isolates from bovine mastitis

Staphylococcus aureus is recognized worldwide as a pathogen causing many serious diseases in humans and animals, and is one of the most important etiological agents of clinical and subclinical bovine mastitis. The aim of the present study was to investigate and correlate properties, that may be associated with persistent mastitis, of S. aureus strains isolated from milk of cows suffering from mastitis: (i) expression of capsular antigens (CP5 or CP8) by specific ELISA; (ii) intracellular survival by invasion of MAC-T cells; and (iii) biofilm production by spectrophotometry analysis after growth in TSBglc. The results showed that (i) the proportion of strains expressing capsular antigen was higher in cap8- than in cap5-positive isolates; (ii) a correlation was observed between the capsular profile and the intracellular survival as well as the biofilm production; and (iii) the capsular profile, biofilm production and intracellular survival were associated with only two agr-groups. Statistical and clustering analysis allowed us to establish different profiles that could be associated with in vivo persistence. Indeed, isolates belonging to agr group II, expressing the capsular antigen CP8 and showing low intracellular survival are probably better adapted to an extracellular niche. Conversely, isolates belonging to agr group I that do not express any capsular antigen (CP5 or CP8) but show high intracellular survival are probably better adapted to an intracellular niche.

Genotypic and phenotypic characterization of methicillin-resistant Staphylococcus aureus (MRSA) isolated from milk of bovine mastitis.

The aim of this study was to evaluate the presence of methicillin‐resistant Staphylococcus aureus (MRSA) among a (S. aureus) collection (n = 430) isolated from milk of cows suffering from mastitis in Belgium and to compare their genotypic as well as phenotypic characteristics. Pulsed field gel electrophoresis (PFGE) and PCR‐based typing techniques (MLST, spa, SCCmec, and agr typing) have been applied and supplemented by capsule serotyping, biofilm production quantification and antimicrobial susceptibility testing. Nineteen MRSA were isolated. Seven distinct ApaI PFGE patterns were observed. All isolates, except one, were identified as ST398 strains. Three spa types (t011, t567 and t108) and two SCCmec types (IV and V) were identified. All isolates belonged to agr type I and capsule type 5 and were Panton‐Valentine leukocidin (PVL) negative. All isolates produced biofilm in TSBglc, whereas the majority did not in milk serum. Twelve resistance patterns were observed, with almost two‐thirds of the isolates being resistant to at least six antibiotics, including penicillin and tetracycline. Our study confirms that the emerging ST398 LA‐MRSA clone has attained Belgian cattle. With regard to genotypic and phenotypic typing, the 19 MRSA isolated in this study form a homogenous group and do not differ much from one another, neither from what has been previously described.

Varicella-Zoster Virus IE4 Protein Interacts with SR Proteins and Exports mRNAs through the TAP/NXF1 Pathway

Available data suggest that the Varicella-Zoster virus (VZV) IE4 protein acts as an important regulator on VZV and cellular genes expression and could exert its functions at post-transcriptional level. However, the molecular mechanisms supported by this protein are not yet fully characterized. In the present study, we have attempted to clarify this IE4-mediated gene regulation and identify some cellular partners of IE4. By yeast two-hybrid and immunoprecipitation analysis, we showed that IE4 interacts with three shuttling SR proteins, namely ASF/SF2, 9G8 and SRp20. We positioned the binding domain in the IE4 RbRc region and we showed that these interactions are not bridged by RNA. We demonstrated also that IE4 strongly interacts with the main SR protein kinase, SRPK1, and is phosphorylated in in vitro kinase assay on residue Ser-136 contained in the Rb domain. By Northwestern analysis, we showed that IE4 is able to bind RNA through its arginine-rich region and in immunoprecipitation experiments the presence of RNA stabilizes complexes containing IE4 and the cellular export factors TAP/NXF1 and Aly/REF since the interactions are RNase-sensitive. Finally, we determined that IE4 influences the export of reporter mRNAs and clearly showed, by TAP/NXF1 knockdown, that VZV infection requires the TAP/NXF1 export pathway to express some viral transcripts. We thus highlighted a new example of viral mRNA export factor and proposed a model of IE4-mediated viral mRNAs export.

Characterization of methicillin-resistant Staphylococcus pseudintermedius isolated from dogs and cats

The aim of this study was to explore the presence of methicillin‐resistant Staphylococcus pseudintermedius (MRSP) in a collection of S. pseudintermedius strains isolated from dogs and cats with dermatitis in Japan and to compare their genotypic and phenotypic characteristics. Clonal relationships were determined by pulse field gel electrophoresis (PFGE), staphylococcal chromosomal cassette mec (SCCmec) typing, and multilocus sequence typing (MLST). Biofilm formation assay was performed using safranin staining in microplates. Three virulence genes coding for S. intermedius exfoliative toxin and Panton‐Valentine leukocidin (siet, lukS‐PV and lukF‐PV) were searched for in a collection of strains. Antimicrobial resistance against 15 antibiotics was studied by a disc diffusion method. Twenty‐seven MRSP were isolated. According to PFGE results the isolates were not closely related except for a few strains. MLST showed that the strains belonged to five groups, ST71 and ST26 being the two most prevalent. Three types of SCCmec (II, II–III and V) were identified. All isolates were siet‐positive but PVL‐negative. Most strains (except for two) produced strong biofilm in tryptic soy broth with glucose. Seventy‐eight percent of the isolates were resistant or intermediate to twelve or more antibiotics. Our study demonstrates that the ST71 lineage is widespread in Japan and that ST26 could represent an emerging lineage. Moreover, most of our strains are capable of forming strong biofilm and possess siet gene, two virulence characteristics that probably help the bacteria to persist and spread. Finally, our MRSP strains show a strong resistance profile to antibiotics commonly used in veterinary medicine.

Varicella-zoster virus induces the formation of dynamic nuclear capsid aggregates.

The first step of herpesviruses virion assembly occurs in the nucleus. However, the exact site where nucleocapsids are assembled, where the genome and the inner tegument are acquired, remains controversial. We created a recombinant VZV expressing ORF23 (homologous to HSV-1 VP26) fused to the eGFP and dually fluorescent viruses with a tegument protein additionally fused to a red tag (ORF9, ORF21 and ORF22 corresponding to HSV-1 UL49, UL37 and UL36). We identified nuclear dense structures containing the major capsid protein, the scaffold protein and maturing protease, as well as ORF21 and ORF22. Correlative microscopy demonstrated that the structures correspond to capsid aggregates and time-lapse video imaging showed that they appear prior to the accumulation of cytoplasmic capsids, presumably undergoing the secondary egress, and are highly dynamic. Our observations suggest that these structures might represent a nuclear area important for capsid assembly and/or maturation before the budding at the inner nuclear membrane.

The Varicella-Zoster virus IE4 protein: A conserved member of the herpesviral mRNA export factors family and a potential alternative target in antiherpetic therapies

During a viral infection, in addition to cellular mRNAs, amounts of viral mRNAs have to be efficiently transported to the cytoplasm for translation. It is now established that herpesviruses encode a conserved gene family whose proteins act as viral mRNA export factors that mediate nucleocytoplasmic transport of viral transcripts and eventually modulate through this mechanism the antiviral response. This conserved family of proteins contains the IE4 protein of the Varicella-Zoster virus (VZV). Here, we compared the functional characteristics of IE4 with those of its herpesviral homologues and proposed a model by which IE4 would be able to recruit the essential TAP/NXF1 receptor to viral transcripts. Moreover, on the basis of their crucial roles in the infectious cycle, these conserved viral factors should be considered as alternative targets in therapeutic approaches. Here, we discussed the possibility of developing antiherpetic agents targeting IE4 or its herpesviral homologues.