Characterization of a vraG Mutant in a Genetically Stable Staphylococcus aureus Small-Colony Variant and Preliminary Assessment for Use as a Live-Attenuated Vaccine against Intrammamary Infections

Abstract

Staphylococcus aureus is a leading cause of bovine intramammary infections (IMIs) that can evolve into difficult-to-treat chronic mastitis. To date, no vaccine formulation has shown high protective efficacy against S. aureus IMI, partly because this bacterium can efficiently evade the immune system. For instance, S. aureus small colony variants (SCVs) have intracellular abilities and can persist without producing invasive infections. As a first step towards the development of a live vaccine, this study describes the elaboration of a novel attenuated mutant of S. aureus taking advantage of the SCV phenotype. A genetically stable SCV was created through the deletion of the hemB gene, impairing its ability to adapt and revert to the invasive phenotype. Further attenuation was obtained through inactivation of gene vraG (SACOL0720) which we previously showed to be important for full virulence during bovine IMIs. After infection of bovine mammary epithelial cells (MAC-T), the double mutant (ΔvraGΔhemB) was less internalized and caused less cell destruction than that seen with ΔhemB and ΔvraG, respectively. In a murine IMI model, the ΔvraGΔhemB mutant was strongly attenuated, with a reduction of viable counts of up to 5-log10 CFU/g of mammary gland when compared to the parental strain. A complete clearance of ΔvraGΔhemB from glands was observed whereas mortality rapidly (48h) occurred with the wild-type strain. Immunization of mice using subcutaneous injections of live ΔvraGΔhemB raised a strong immune response as judged by the high total IgG titers measured against bacterial cell extracts and by the high IgG2a/IgG1 ratio observed against the IsdH protein. Also, ΔvraGΔhemB had sufficient common features with bovine mastitis strains so that the antibody response also strongly recognized strains from a variety of mastitis associated spa types. This double mutant could serve as a live-attenuated component in vaccines to improve cell-mediated immune responses against S. aureus IMIs.

Tomatidine and analog FC04–100 possess bactericidal activities against Listeria, Bacillus and Staphylococcus spp

“…NewbouldΔ hemB was generated from strain Newbould (ATCC 29740) by disrupting the hemB gene with the ermA cassette by homologous recombination [ 23 ]. Another SCV hemB mutant was similarly constructed from S. aureus ATCC 29213 [ 24 ]. SCVs from B. cereus , B. subtillis and L. monocytogenes were generated by growth in presence of a subinhibitory concentration of GEN.…”

Despite Antagonism in vitro, Pseudomonas aeruginosa Enhances Staphylococcus aureus Colonization in a Murine Lung Infection Model

“…Assessment of inflammation and infiltration of neutrophils during mono or coinfections of mouse lung tissues were evaluated by quantification of MPO activity using the o-dianisidine-H 2 O 2 method, as previously described (Côté-Gravel et al, 2016). Briefly, 10 µl of lung homogenate was mixed with a solution of o-dianisidine hydrochloride (167 µg/ml) (Sigma-Aldrich, Oakville, ON, Canada), 0.0005% H 2 O 2 (Sigma-Aldrich, Oakville, ON, Canada), 50 mM hexade-cyltrimethylammonium bromide (CTAB) and 50 mM phosphate buffer at pH 6.0, in a 96-well plate.…”

“…It was thus hypothesized that P. aeruginosa could exacerbate a pro-inflammatory response, which could help S. aureus colonization. MPO activity was therefore measured for a series of co-infections in the mouse since it was recently found to be a good indicator of inflammation (Côté-Gravel et al, 2016). S. aureus CF54A-L was used in all co-infections together with a variety of P. aeruginosa strains showing different levels of virulence and colonization.…”

Assessment of Mucosal and Systemic Immune Responses against Staphylococcus aureus in Ruminants

“…S taphylococcus aureus is a Gram positive bacterium and the most imperative animal and human pathogen responsible for a wide spectrum of morbidity and acute clinical infections, in addition to persistent chronic forms of diseases, like bacteremia, endocarditis, osteomyelitis, skin and soft tissue infections including mastitis in dairy cows and associated with enormous economic losses second to loses encountered in Orf virus infection in small ruminants (Zamri-Saad et al, 1999;Joshi and McNeely, 2013;Azhar et al, 2016;CoÃteÂ-Gravel et al, 2016). Amongst factors that can elucidate the botch of antibiotherapy and the proclivity to cause chronic infections, which could be ascribed to the pathogen's multidimensional virulence, largely to its abilities to weaken or evade the host immune responses by toxin secretion (Park et al, 2011;CoÃteÂ-Gravel et al, 2016), establishment of biofilm (Rice et al, 2007;Otto, 2013;CoÃteÂ-Gravel et al, 2016) and its persistence in nonphagocytic host cells, which could safeguard the pathogen from the attack of the host immune system and antibiotics (Brouillette et al, 2004;CoÃteÂ-Gravel et al, 2016). Additionally, commonness of S. aureus infections are becoming more bothersome with the development of compound antibiotic resistant strains (Chambers and Deleo, 2009;García-Álvarez et al, 2011;CoÃteÂ-Gravel et al, 2016).…”

“…Amongst factors that can elucidate the botch of antibiotherapy and the proclivity to cause chronic infections, which could be ascribed to the pathogen's multidimensional virulence, largely to its abilities to weaken or evade the host immune responses by toxin secretion (Park et al, 2011;CoÃteÂ-Gravel et al, 2016), establishment of biofilm (Rice et al, 2007;Otto, 2013;CoÃteÂ-Gravel et al, 2016) and its persistence in nonphagocytic host cells, which could safeguard the pathogen from the attack of the host immune system and antibiotics (Brouillette et al, 2004;CoÃteÂ-Gravel et al, 2016). Additionally, commonness of S. aureus infections are becoming more bothersome with the development of compound antibiotic resistant strains (Chambers and Deleo, 2009;García-Álvarez et al, 2011;CoÃteÂ-Gravel et al, 2016).…”