Evaluation of the presence of the bap gene in Staphylococcus aureus isolates recovered from human and animals species.

International audienceThe implication of biofilm in chronic bacterial infection in many species has triggered an increasing interest in the characterization of genes involved in biofilm formation. The bap gene is a newly identified gene that encodes the biofilm-associated protein, BAP, which is involved in biofilm formation in Staphylococcus aureus. So far the bap gene has only been found in a small proportion of S. aureus strains from bovine mastitis in Spain. In order to study the presence of the bap gene in S. aureus isolates obtained from other species and various locations, a collection of 262 isolates was tested by PCR, using published primers and dot-blot. The results indicated that none of the isolates carried the bap gene suggesting that the prevalence of this gene among S. aureus isolates should be very low

Difference in virulence between Staphylococcus aureus isolates causing gangrenous mastitis versus subclinical mastitis in a dairy sheep flock

Staphylococcus aureus mastitis in dairy sheep ranges from subclinical mastitis to lethal gangrenous mastitis. Neither the S. aureus virulence factors nor the host-factors or the epidemiological events contributing to the different outcomes are known. In a field study in a dairy sheep farm over 21 months, 16 natural isolates of S. aureus were collected from six subclinical mastitis cases, one lethal gangrenous mastitis case, nasal carriage from eight ewes and one isolate from ambient air in the milking room. A genomic comparison of two strains, one responsible for subclinical mastitis and one for lethal gangrenous mastitis, was performed using multi-strain DNA microarrays. Multiple typing techniques (pulsed-field-gel-electrophoresis, multiple-locus variable-number, single-nucleotide polymorphisms, randomly amplified polymorphic DNA, spa typing and sas typing) were used to characterise the remaining isolates and to follow the persistence of the gangrenous isolate in ewes’ nares. Our results showed that the two strains were genetically closely related and they shared 3 615 identical predicted open reading frames. However, the gangrenous mastitis isolate carried variant versions of several genes (sdrD, clfA-B, sasA, sasB, sasD, sasI and splE) and was missing fibrinogen binding protein B (fnbB) and a prophage. The typing results showed that this gangrenous strain emerged after the initial subclinical mastitis screening, but then persisted in the flock in the nares of four ewes. Although we cannot dismiss the role of host susceptibility in the clinical events in this flock, our data support the hypothesis that S. aureus populations had evolved in the sheep flock and that S. aureus genetic variations could have contributed to enhanced virulence

Staphylococcus aureus seroproteomes discriminate ruminant isolates causing mild or severe mastitis

Staphylococcus aureus is a major cause of mastitis in ruminants. In ewe mastitis, symptoms range from subclinical to gangrenous mastitis. S. aureus factors or host-factors contributing to the different outcomes are not completely elucidated. In this study, experimental mastitis was induced on primiparous ewes using two S. aureus strains, isolated from gangrenous (strain O11) or subclinical (strain O46) mastitis. Strains induced drastically distinct clinical symptoms when tested in ewe and mice experimental mastitis. Notably, they reproduced mild (O46) or severe (O11) mastitis in ewes. Ewe sera were used to identify staphylococcal immunoreactive proteins commonly or differentially produced during infections of variable severity and to define core and accessory seroproteomes. Such SERological Proteome Analysis (SERPA) allowed the identification of 89 immunoreactive proteins, of which only 52 (58.4%) were previously identified as immunogenic proteins in other staphylococcal infections. Among the 89 proteins identified, 74 appear to constitute the core seroproteome. Among the 15 remaining proteins defining the accessory seroproteome, 12 were specific for strain O11, 3 were specific for O46. Distribution of one protein specific for each mastitis severity was investigated in ten other strains isolated from subclinical or clinical mastitis. We report here for the first time the identification of staphylococcal immunogenic proteins common or specific to S. aureus strains responsible for mild or severe mastitis. These findings open avenues in S. aureus mastitis studies as some of these proteins, expressed in vivo, are likely to account for the success of S. aureus as a pathogen of the ruminant mammary gland

Molecular Basis of Virulence in Staphylococcus aureus Mastitis

S. aureus is one of the main pathogens involved in ruminant mastitis worldwide. The severity of staphylococcal infection is highly variable, ranging from subclinical to gangrenous mastitis. This work represents an in-depth characterization of S. aureus mastitis isolates to identify bacterial factors involved in severity of mastitis infection

Caractérisation moléculaire d'isolats de Staphylococcus aureus responsables de mammites chez des ovins laitiers dans le Sud-Est de la France

C est l étude de quelques caractères phénotypiques et du génotype de souches de Staphylococcus aureus isolées de cas de mammites chez des brebis laitières dans la région Provence Alpes Côte d Azur (PACA). Le génotypage (PFGE, MLST, RAPD-PCR, RFLP) a montré que ces souches de S. aureus étaient génétiquement proches. Les souches étudiées ont exprimé une forte capacité relative d adhérence et de formation de slime, malgré l absence du gène bap (codant la biofilm-associated protein) mais avec la présence des gènes icaA-D chez la majorité des souches. Quarante six souches ont été testées vis à vis de 10 antibiotiques et ont montré une faible résistance en relation avec l absence de plasmides. La production d entérotoxines A-D in vitro à partir de 15 souches a souligné que la relative homogénéité génétique des souches cachait une variabilité phénotypique. Cent treize isolats provenant des trois espèces laitières ont étés comparés en utilisant la technique des puces à ADN avec le marqueur Cy5 (187 gènes de virulence). Les résultats ont mis en évidence certaines signatures génétiques en relation avec la clonalité des souches régionales ainsi que des gènes spécifiques aux souches ovins-caprins versus bovins . L adaptation au portage nasal ne semble pas le fait de particularités génétiques propres. La partie II a permis par la comparaison de deux souches, l une responsable de mammite subclinique et l autre de mammite gangreneuse, de montrer que sur un fond de souches endémiques (étudiées dans la partie I) certaines souches avaient, avec un potentiel génétique différent, le pouvoir de provoquer des pathologies mammaires plus sévères.This study characterized phenotypic and genotypic traits of Staphylococcus aureus isolates recovered from dairy sheep farm in Provence-Alpes-Côte d Azur (PACA). The typing by PFGE, MLST, RAPD-PCR, RFLP shown that the strains were genetically related. Among forty six strains tested for adherence and slime production, 39% and 26% were positive respectively despite the absence of bap gene (coding the biofilm-associated protein) but with the presence of the intercellular adhesion locus (icaA-D). The strains were mainly susceptible to ten antibiotics (absence of plasmids). The in vitro production of enterotoxins A-D in fifteen isolates illustrated the phenotypic variability for this parameter in these genetically related strains. A genomic comparative study was carried out on 113 S. aureus isolates recovered from sheep, goats, cows, from nostrils recovered from regional sheep and from other dairy sheep farms in France. For this purpose, a single-dye DNA microarray had been developed for simultaneous characterization of 187 genes implicated in S. aureus virulence. The screening of the 113 isolates allowed us to identify a few genes or alleles which were specifically associated with the regional dairy sheep in correlation to their genetic clonality. Some genes had been found to be specific to small ruminants versus cows. The nasal carriage isolates had identical genetic specificity in comparison with mastitis isolates. The S. aureus strains compared in the part two shown that gangrenous mastitis had genetic particularities (presence of genes of virulence) in comparison with the strains responsible of subclinical mastitis.NICE-BU Sciences (060882101) / SudocSudocFranceF

Characterization of 26 isolates of Staphylococcus aureus, predominantly from dairy sheep, using four different techniques of molecular epidemiology.

Little information is available regarding the molecular epidemiology of Staphylococcus aureus-induced mastitis in dairy sheep. In this study, 4 different typing techniques were compared in typing 26 S. aureus isolates, predominantly from cases of subclinical mastitis in dairy ewes. The 4 techniques were pulsed-field gel electrophoresis (PFGE), restriction fragment length polymorphism (RFLP) on 2 genes (coagulase and clumping factor B), randomly amplified polymorphic DNA-polymerase chain reaction (PCR) (RAPD-PCR), and multilocus sequence typing (MLST). On the basis of discriminatory power as the key parameter of typing systems, MLST and PFGE were found to be the most powerful techniques. The MLST and PFGE could contribute to epidemiological surveillance and evaluation of mastitis control programs, by documenting prevalence and dissemination of endemic clones in infected populations. The results of this study show that a single clone of S. aureus is widely distributed in infected ewe mammary glands

Characterization of Staphylococcus aureus isolates recovered from dairy sheep farms (agr group, adherence, slime, resistance to antibiotics)

International audienceStaphylococcus aureus is one of the major causes of dairy sheep mastitis. The S. aureus agr locus (accessory gene regulator) regulates the production of most staphylococcal exoproteins, including exoenzymes, toxins, surface proteins, and other virulence factors. S. aureus have four agr groups (alleles) determined by PCR. In this study, 46 S. aureus isolates, recovered in south-east of France, were also characterized by their properties of adherence to smooth surfaces, slime production and resistance to 10 antibiotics. For 46 S. aureus associated with dairy sheep mastitis (subclinical mastitis, clinical mastitis, environment of dairy sheep farm), 80% (37/46) belonged to agr group 3, 39% (18/46) were adherent (adherent, strongly adherent or with maximal adherence). For the same isolates, 26% (12/46) were slime producers (moderate or strong producers). All the 46 isolates were susceptible to oxacillin, except for two isolates including two sheep subclinical mastitis isolates. The dairy sheep subclinical mastitis isolates were for 79% (22/28), susceptible to nine other antibiotics tested

Outbreak of diarrhea related to Clostridium perfringens in a correctional facility: An epidemiologic investigation

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[i]Staphylococcus aureus[/i] proteins differentially produced in ewe gangrenous mastitis or ewe milk

Despite being one of the main pathogens involved in ruminant mastitis, little is known about what proteins Staphylococcus aureus does express, in vivo, during the infection. Here, two S. aureus strains were isolated from curds formed within the udder of two ewes suffering from gangrenous mastitis. Protein samples were prepared from cell fractions and were analyzed using 1D-LC MS/MS. Results were compared to 1D-LC MS/MS analysis of the same S. aureus strains grown in ewe milk. A total of 365 proteins were identified. Most of them were related to cellular metabolism, cellular division and stress response. Half of the proteins were found in both conditions but a substantial number were specifically found in in vivo conditions and gave indications about the active metabolic status and the stresses encountered by S. aureus within the cistern during a gangrenous mastitis