The Active Component of Aspirin, Salicylic Acid, Promotes Staphylococcus aureus Biofilm Formation in a PIA-dependent Manner

Aspirin has provided clear benefits to human health. But salicylic acid (SAL) -the main aspirin biometabolite- exerts several effects on eukaryote and prokaryote cells. SAL can affect, for instance, the expression of Staphyiococcus aureus virulence factors. SAL can also form complexes with iron cations and it has been shown that different iron chelating molecules diminished the formation of S. aureus biofilm. The aim of this study was to elucidate whether the iron content limitation caused by SAL can modify the S. aureus metabolism and/or metabolic regulators thus changing the expression of the main polysaccharides involved in biofilm formation. The exposure of biofilm to 2mM SAL induced a 27% reduction in the intracellular free Fe2+ concentration compared with the controls. In addition, SAL depleted 23% of the available free Fe2+ cation in culture media. These moderate iron-limited conditions promoted an intensificaron of biofilms formed by strain Newman and by S. aureus clinical isolates related to the USA300 and USA100 clones. The slight decrease in iron bioavailability generated by SAL was enough to induce the increase of PIA expression in biofilms formed by methicillin-resistant as well as methicillin-sensitive S. aureus strains. S. aureus did not produce capsular polysaccharide (CP) when it was forming biofilms under any of the experimental conditions tested. Furthermore, SAL diminished aconitase activity and stimulated the lactic fermentation pathway in bacteria forming biofilms. The polysaccharide composition of S. aureus biofilms was examined and FTIR spectroscopic analysis revealed a clear impact of SAL in a codY-dependent manner. Moreover, SAL negatively affected codY transcription in mature biofilms thus relieving the CodY repression of the ica operon. Treatment of mice with SAL induced a significant increase of S aureus colonization. It is suggested that the elevated PIA expression induced by SAL might be responsible for the high nasal colonization observed in mice. SAL-induced biofilms may contribute to S. aureus infection persistence in vegetarian individuals as well as in patients that frequently consume aspirin.Facultad de Ciencias Médica

Aromatic Compound-Dependent Staphylococcus aureus

Staphylococcus aureus nasal carriage is a risk factor for individuals suffering from trauma, surgical procedures, invasive devices, and/or decreased immunity. Recently, we demonstrated that artificial nasal colonization with an attenuated S. aureus mutant reduced by bacterial interference with the colonization of pathogenic strains of S. aureus. This could be an optional tool to diminish the rate of S. aureus infections in hospitalized patients. The aim of this study was to construct a safe ΔaroA mutant of S. aureus and to discriminate it from nasal colonizing and osteomyelitis S. aureus isolates by SmaI pulsed-field gel electrophoresis (PFGE) typing. The ΔaroA mutant, named RD17, exhibited an LD50 (3.2 × 106 colony-forming unit (CFU)) significantly higher than that of the parental strain (2.2 × 103 CFU). The colony number of the RD17 mutants recovered from nares of leukopenic mice was similar to that observed in the animals of the control group. Therefore, the ΔaroA mutant was demonstrated to be safe due to maintaining low growth levels in the nares regardless of immune status of the animals. PFGE typing allowed the unequivocal identification of the S. aureus and differentiation of aroA mutants in nasal colonizing and osteomyelitis isolates. This information could be important to discriminate endogenous infections from laboratory strains of S. aureus

The Active Component of Aspirin, Salicylic Acid, Promotes Staphylococcus aureus Biofilm Formation in a PIA-dependent Manner

Aspirin has provided clear benefits to human health. But salicylic acid (SAL) -the main aspirin biometabolite- exerts several effects on eukaryote and prokaryote cells. SAL can affect, for instance, the expression of Staphyiococcus aureus virulence factors. SAL can also form complexes with iron cations and it has been shown that different iron chelating molecules diminished the formation of S. aureus biofilm. The aim of this study was to elucidate whether the iron content limitation caused by SAL can modify the S. aureus metabolism and/or metabolic regulators thus changing the expression of the main polysaccharides involved in biofilm formation. The exposure of biofilm to 2mM SAL induced a 27% reduction in the intracellular free Fe2+ concentration compared with the controls. In addition, SAL depleted 23% of the available free Fe2+ cation in culture media. These moderate iron-limited conditions promoted an intensificaron of biofilms formed by strain Newman and by S. aureus clinical isolates related to the USA300 and USA100 clones. The slight decrease in iron bioavailability generated by SAL was enough to induce the increase of PIA expression in biofilms formed by methicillin-resistant as well as methicillin-sensitive S. aureus strains. S. aureus did not produce capsular polysaccharide (CP) when it was forming biofilms under any of the experimental conditions tested. Furthermore, SAL diminished aconitase activity and stimulated the lactic fermentation pathway in bacteria forming biofilms. The polysaccharide composition of S. aureus biofilms was examined and FTIR spectroscopic analysis revealed a clear impact of SAL in a codY-dependent manner. Moreover, SAL negatively affected codY transcription in mature biofilms thus relieving the CodY repression of the ica operon. Treatment of mice with SAL induced a significant increase of S aureus colonization. It is suggested that the elevated PIA expression induced by SAL might be responsible for the high nasal colonization observed in mice. SAL-induced biofilms may contribute to S. aureus infection persistence in vegetarian individuals as well as in patients that frequently consume aspirin.Facultad de Ciencias Médica

Induction of Cell-Mediated Immunity to Staphylococcus aureus in the Mouse Mammary Gland by Local Immunization with a Live Attenuated Mutant

The efficacy of intramammary (Ima) immunization with a live attenuated (la) Staphylococcus aureus mutant to protect the mouse mammary gland from infection has previously been established. The present study was aimed at evaluating whether Ima immunization with la-S. aureus can induce cell-mediated immune responses to the pathogen within the mammary gland. Mice were immunized by Ima route with la-S. aureus, and regional lymph node mononuclear cells were obtained thereafter. A higher expression of the interleukin-2 receptor was found on B and T cells from immunized mice when they were compared with control mice. Immunization with la-S. aureus induced strong proliferative responses to S. aureus. Moreover, significantly increased levels of gamma interferon (IFN-γ) were produced by CD4(+) T cells when lymphocytes from immunized mice, but not from control mice, were cultured in the presence of staphylococcal antigens. Moreover, a significant increase in the percentage of IFN-γ-producing CD4(+) and CD8(+) T cells was observed after S. aureus Ima challenge in immunized mice compared to challenged control mice. Our results demonstrated that Ima immunization with la-S. aureus induced primed lymphocyte populations capable of responding against staphylococcal antigens during in vitro stimulation, as well as during in vivo infection by S. aureus. CD4(+) and CD8(+) T cells appear to be the main lymphocyte subpopulations involved in this response. It is suggested that IFN-γ production induced by Ima immunization may play a pivotal role in the eradication of intracellular staphylococci

Attenuation and Persistence of and Ability To Induce Protective Immunity to a Staphylococcus aureus aroA Mutant in Mice

Staphylococcus aureus is the most important etiological agent of bovine mastitis, a disease that causes significant economic losses to the dairy industry. Several vaccines to prevent the disease have been tested, with limited success. The aim of this study was to obtain a suitable attenuated aro mutant of S. aureus by transposon mutagenesis and to demonstrate its efficacy as a live vaccine to induce protective immunity in a murine model of intramammary infection. To do this, we transformed S. aureus RN6390 with plasmid pTV1ts carrying Tn917. After screening of 3,493 erythromycin-resistant colonies, one mutant incapable of growing on plates lacking phenylalanine, tryptophan, and tyrosine was isolated and characterized. Molecular characterization of the mutant showed that the affected gene was aroA and that the insertion occurred 756 bp downstream of the aroA start codon. Complementation of the aroA mutant with a plasmid carrying aroA recovered the wild-type phenotype. The mutant exhibited a 50% lethal dose (1 × 10(6) CFU/mouse) higher than that of the parental strain (4.3 × 10(4) CFU/mouse). The aroA mutant showed decreased ability to persist in the lungs, spleens, and mammary glands of mice. Intramammary immunization with the aroA mutant stimulated both Th1 and Th2 responses in the mammary gland, as ascertained by reverse transcription-PCR, and induced significant protection from challenge with either the parental wild-type or a heterologous strain isolated from a cow with mastitis

Distinct phenotypic traits of Staphylococcus aureus are associated with persistent, contagious bovine intramammary infections

Abstract Staphylococcus aureus causing persistent, recurrent bovine intramammary infections are still a major challenge to dairy farming. Generally, one or a few clonal lineages are predominant in dairy herds, indicating animal-to-animal transfers and the existence of distinct pathotypic traits. The aim of this study was to determine if long term persistence and spreading of S. aureus are associated with specific phenotypic traits, including cellular invasion, cytotoxicity and biofilm formation. Mastitis isolates were collected over a 3-years period from a single dairy herd, resulting in two persistent subtypes, the high within-herd prevalent subtype ST9 (CC9)-methicillin-susceptible S. aureus (MSSA), designated HP/ST9, and the low within-herd prevalent subtype ST504 (CC705)-MSSA, designated LP/ST504. Characterization of the two different coexisting persistent subtypes showed that the following phenotypic traits are particularly associated with high within-herd prevalence: lack of capsular polysaccharide expression, high cellular invasiveness, low cytotoxicity and high biofilm/ poly-N-acetylglucosamine (PNAG) production, which may concomitantly contribute to the spreading of HP/ST9 within the herd. By contrast to HP/ST9, LP/ST504 is characterized by the formation of colony dendrites, which may help the bacteria to access deeper tissues as niches for persistence in single animals. Thus, within a single herd, two different types of persistence can be found in parallel, allowing longtime persistence of S. aureus in dairy cattle. Furthermore, this study indicates that ST9 (CC9)-MSSA strains, which are currently thought to have their primary reservoir in swine and humans, can also successfully spread to new hosts and persist in dairy herds for years

Salicylic Acid Diminishes Staphylococcus aureus Capsular Polysaccharide Type 5 Expression▿

Capsular polysaccharides (CP) of serotypes 5 (CP5) and 8 (CP8) are major Staphylococcus aureus virulence factors. Previous studies have shown that salicylic acid (SAL), the main aspirin metabolite, affects the expression of certain bacterial virulence factors. In the present study, we found that S. aureus strain Reynolds (CP5) cultured with SAL was internalized by MAC-T cells in larger numbers than strain Reynolds organisms not exposed to SAL. Furthermore, the internalization of the isogenic nonencapsulated Reynolds strain into MAC-T cells was not significantly affected by preexposure to SAL. Pretreatment of S. aureus strain Newman with SAL also enhanced internalization into MAC-T cells compared with that of untreated control strains. Using strain Newman organisms, we evaluated the activity of the major cap5 promoter, which was significantly decreased upon preexposure to SAL. Diminished transcription of mgrA and upregulation of the saeRS transcript, both global regulators of CP expression, were found in S. aureus cultured in the presence of SAL, as ascertained by real-time PCR analysis. In addition, CP5 production by S. aureus Newman was also decreased by treatment with SAL. Collectively, our data demonstrate that exposure of encapsulated S. aureus strains to low concentrations of SAL reduced CP production, thus unmasking surface adhesins and leading to an increased capacity of staphylococci to invade epithelial cells. The high capacity of internalization of the encapsulated S. aureus strains induced by SAL pretreatment may contribute to the persistence of bacteria in certain hosts

Antibodies to Capsular Polysaccharide and Clumping Factor A Prevent Mastitis and the Emergence of Unencapsulated and Small-Colony Variants of Staphylococcus aureus in Mice▿

The pathogenesis of Staphylococcus aureus infections is influenced by multiple virulence factors that are expressed under variable conditions, and this has complicated the design of an effective vaccine. Clinical trials that targeted the capsule or clumping factor A (ClfA) failed to protect the recipients against staphylococcal infections. We passively immunized lactating mice with rabbit antibodies to S. aureus capsular polysaccharide (CP) serotype 5 (CP5) or CP8 or with monoclonal antibodies to ClfA. Mice immunized with antibodies to CP5 or CP8 or with ClfA had significantly reduced tissue bacterial burdens 4 days after intramammary challenge with encapsulated S. aureus strains. After several passages in mice passively immunized with CP-specific antiserum, increasing numbers of stable unencapsulated variants of S. aureus were cultured from the infected mammary glands. Greater numbers of these unencapsulated S. aureus variants than of the corresponding encapsulated parental strains were internalized in vitro in MAC-T bovine cells. Furthermore, small-colony variants (SCVs) were recovered from the infected mammary glands after several passages in mice passively immunized with CP-specific antiserum. A combination of antibodies effectively sterilized mammary glands in a significant number of passively immunized mice. More importantly, passive immunization with antibodies to both CP and ClfA fully inhibited the emergence of unencapsulated “escape mutants” and significantly reduced the appearance of SCVs. A vaccine formulation comprising CP conjugates plus a surface-associated protein adhesin may be more effective than either antigen alone for prevention of S. aureus infections

Capsule-Negative Staphylococcus aureus Induces Chronic Experimental Mastitis in Mice

Staphylococcus aureus capsular polysaccharides (CP) have been shown to enhance staphylococcal virulence in numerous animal models of infection. Although serotype 5 CP (CP5) and CP8 predominate among S. aureus isolates from humans, most staphylococcal isolates from bovines with mastitis in Argentina are capsule negative. This study was designed to evaluate the effects of CP5 and CP8 expression on the pathogenesis of experimental murine mastitis. Lactating mice were challenged by the intramammary route with one of three isogenic S. aureus strains producing CP5, CP8, or no capsule. Significantly greater numbers of acapsular mutant cells were recovered from the infected glands 12 days after bacterial challenge compared with the encapsulated strains. Histopathological analyses revealed greater polymorphonuclear and mononuclear leukocyte infiltration and congestion in the mammary glands of mice infected with the encapsulated strains compared with the acapsular mutant, and the serotype 5 strain elicited more inflammation than the serotype 8 strain. In vitro experiments revealed that the acapsular S. aureus strain was internalized by MAC-T bovine epithelial cells in significantly greater numbers than the CP5- or CP8-producing strain. Taken together, the results suggest that S. aureus lacking a capsule was able to persist in the murine mammary gland, whereas encapsulated strains elicited more inflammation and were eliminated faster. Loss of CP5 or CP8 expression may enhance the persistence of staphylococci in the mammary glands of chronically infected hosts

Within-host evolution of bovine Staphylococcus aureus selects for a SigB-deficient pathotype characterized by reduced virulence but enhanced proteolytic activity and biofilm formation

Staphylococcus aureus is a major cause of bovine mastitis, commonly leading to long-lasting, persistent and recurrent infections. Thereby, S. aureus constantly refines and permanently adapts to the bovine udder environment. In this work, we followed S. aureus within-host adaptation over the course of three months in a naturally infected dairy cattle with chronic, subclinical mastitis. Whole genome sequence analysis revealed a complete replacement of the initial predominant variant by another isogenic variant. We report for the first time within-host evolution towards a sigma factor SigB-deficient pathotype in S. aureus bovine mastitis, associated with a single nucleotide polymorphism in rsbU (G368A → G122D), a contributor to SigB-functionality. The emerged SigB-deficient pathotype exhibits a substantial shift to new phenotypic traits comprising strong proteolytic activity and poly-N-acetylglucosamine (PNAG)-based biofilm production. This possibly unlocks new nutritional resources and promotes immune evasion, presumably facilitating extracellular persistence within the host. Moreover, we observed an adaptation towards attenuated virulence using a mouse infection model. This study extends the role of sigma factor SigB in S. aureus pathogenesis, so far described to be required for intracellular persistence during chronic infections. Our findings suggest that S. aureus SigB-deficiency is an alternative mechanism for persistence and underpin the clinical relevance of staphylococcal SigB-deficient variants which are consistently isolated during human chronic infections.Fil: Marbach H. University of Veterinary Medicine; AustriaFil: Mayer, Klaus Ulrich. University of Veterinary Medicine; AustriaFil: Vogl, Claus. University of Veterinary Medicine; AustriaFil: Lee, Jean Y.H.. University of Melbourne; AustraliaFil: Monk, Ian R.. University of Melbourne; AustraliaFil: Sordelli, Daniel Oscar. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Buzzola, Fernanda Roxana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Ehling-schultz, Monika. University of Veterinary Medicine; AustriaFil: Grunert, Tom. University of Veterinary Medicine; Austri