Protein Antigens Increase the Protective Efficacy of a Capsule-Based Vaccine against Staphylococcus aureus in a Rat Model of Osteomyelitis

Staphylococcus aureus is an invasive bacterial pathogen, and antibiotic resistance has impeded adequate control of infections caused by this microbe. Moreover, efforts to prevent human infections with single-component S. aureus vaccines have failed. In this study, we evaluated the protective efficacy in rats of vaccines containing both S. aureus capsular polysaccharides (CPs) and proteins. The serotypes 5 CP (CP5) and 8 CP (CP8) were conjugated to tetanus toxoid and administered to rats alone or together with domain A of clumping factor A (ClfA) or genetically detoxified alpha-toxin (dHla). The vaccines were delivered according to a preventive or a therapeutic regimen, and their protective efficacy was evaluated in a rat model of osteomyelitis. Addition of dHla (but not ClfA) to the CP5 or CP8 vaccine induced reductions in bacterial load and bone morphological changes compared with immunization with either conjugate vaccine alone. Both the prophylactic and therapeutic regimens were protective. Immunization with dHla together with a pneumococcal conjugate vaccine used as a control did not reduce staphylococcal osteomyelitis. The emergence of unencapsulated or small-colony variants during infection was negligible and similar for all of the vaccine groups. In conclusion, addition of dHla to a CP5 or CP8 conjugate vaccine enhanced its efficacy against S. aureus osteomyelitis, indicating that the inclusion of multiple antigens will likely enhance the efficacy of vaccines against both chronic and acute forms of staphylococcal disease.Fil: Lattar, Santiago Martín. 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: Noto Llana, Mariangeles. 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: Denoël, Philippe. GlaxoSmithKline Vaccines; BélgicaFil: Germain, Sophie. GlaxoSmithKline Vaccines; BélgicaFil: 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: Lee, Jean C.. Harvard Medical School; Estados UnidosFil: 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; Argentin

Rapid and reliable identification of Staphylococcus aureus capsular serotypes by means of artificial neural network-assisted Fourier-transform infrared spectroscopy

Staphylococcus aureus capsular polysaccharides (CP) are important virulence factors and represent putative targets for vaccine development. Therefore, the purpose of this study was to develop a high-throughput method to identify and discriminate the clinically important S. aureus capsular serotypes 5, 8, and NT (nontypeable). A comprehensive set of clinical isolates derived from different origins and control strains, representative for each serotype, were used to establish a CP typing system based on Fourier transform infrared (FTIR) spectroscopy and chemometric techniques. By combining FTIR spectroscopy with artificial neuronal network (ANN) analysis, a system was successfully established, allowing a rapid identification and discrimination of all three serotypes. The overall accuracy of the ANN-assisted FTIR spectroscopy CP typing system was 96.7% for the internal validation and 98.2% for the external validation. One isolate in the internal validation and one isolate in the external validation failed in the classification procedure, but none of the isolates was incorrectly classified. The present study demonstrates that ANN-assisted FTIR spectroscopy allows a rapid and reliable discrimination of S. aureus capsular serotypes. It is suitable for diagnostic as well as large-scale epidemiologic surveillance of S. aureus capsule expression and provides useful information with respect to chronicity of infection.Fil: Grunert, Tom. University of Veterinary Medicine; AustriaFil: Wenning, Mareike. Technische Universitat Munchen; AlemaniaFil: Barbagelata, María Sol. 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: Fricker, Martina. University of Veterinary Medicine; AustriaFil: 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 Schulz, Monika. University of Veterinary Medicine; Austri

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

The anti MRSA biofilm activity of Thymus vulgaris essential oil in nanovesicles

Background: Thymus vulgaris essential oil (T) could be an alternative to classical antibiotics against bacterial biofilms, which show increased tolerance to antibiotics and host defence systems and contribute to the persistence of chronic bacterial infections. Hypothesis: A nanovesicular formulation of T may chemically protect the structure and relative composition of its multiple components, potentially improving its antibacterial and antibiofilm activity. Study design: We prepared and structurally characterized T in two types of nanovesicles: nanoliposomes (L80-T) made of Soybean phosphatidylcholine (SPC) and Polysorbate 80 (P80) [SPC:P80:T 1:0.75:0.3 w:w], and nanoarchaeosomes (A80-T) made of SPC, P80 and total polar archaeolipids (TPA) extracted from archaebacteria Halorubrum tebenquichense [SPC:TPA:P80:T 0.5:0.50.75:0.7 w:w]. We determined the macrophage cytotoxicity and the antibacterial activity against Staphylococcus aureus ATCC 25,923 and four MRSA clinical strains. Results: L80-T (Z potential −4.1 ± 0.6 mV, ∼ 115 nm, ∼ 22 mg/ml T) and A80-T (Z potential −6.6 ± 1.5 mV, ∼ 130 nm, ∼ 42 mg/ml T) were colloidally and chemically stable, maintaining size, PDI, Z potential and T concentration for at least 90 days. While MIC 90 of L80-T was > 4 mg/ml T, MIC 90 of A80-T was 2 mg/ml T for all S. aureus strains. The antibiofilm formation activity was maximal for A80-T, while L80-T did not inhibit biofilm formation compared to untreated control. A80-T significantly decreased the biomass of preformed biofilms of S. aureus ATCC 25,923 strain and of 3 of the 4 clinical MRSA isolates at 4 mg/ml T. It was found that the viability of J774A.1 macrophages was decreased significantly upon 24 h incubation with A80-T, L80-T and T emulsion at 0.4 mg/ml T. These results show that from 0.4 mg/ml T, a value lower than MIC 90 and the one displaying antibiofilm activity, with independence of its formulation, T significantly decreased the macrophages viability. Conclusion: Overall, because of its lower MIC 90 against planktonic bacteria, higher antibiofilm formation capacity and stability during storage, A80-T resulted better antibacterial agent than T emulsion and L80-T. These results open new avenues to explode the A80-T antimicrobial intracellular activity.Fil: Perez, Ana Paula. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología; ArgentinaFil: Perez, Noelia Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología; ArgentinaFil: Suligoy Lozano, Carlos Mauricio. 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: Altube, María Julia. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: de Farias, Marcelo Alexandre. No especifíca;Fil: Portugal, Rodrigo Villares. No especifíca;Fil: Buzzola, Fernanda Roxana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. 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: Morilla, María José. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología; ArgentinaFil: Romero, Eder Lilia. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Photodynamic inactivation of planktonic and biofilm growing bacteria mediated by a meso-substituted porphyrin bearing four basic amino groups

Biofilm-associated diseases account for 80% of all infections in humans. Due to the emergence of antibiotic resistances, alternative therapies such as Photodynamic Inactivation (PDI) of microorganisms have emerged. Porphyrins with intrinsic positive charges have been proposed as successful photosensitizers (PSs) against microorganisms. We have recently designed the new synthetic porphyrin 5,10,15,20-tetrakis[4-(3-N,N-dimethylammoniumpropoxy)phenyl]porphyrin (TAPP) containing four basic amine groups in the periphery of the tetrapyrrolic macrocycle, which can acquire positive charges at physiological pH, thus favouring the interaction with biomembranes. Illumination of planktonic cultures of Staphylococcus aureus at 180 J/cm2 in the presence of 2.5 μM TAPP induced complete bacteria eradication. For the TAPP-PDI treatment of S. aureus biofilms, higher light fluences and PS concentrations were needed. Employing 20 μM TAPP and 180 J/cm2, around 3-log CFU reduction were obtained. In order to determine the efficacy of TAPP-PDI on Gram-negative bacteria, we performed planktonic and biofilm assays employing Pseudomonas aeruginosa. Much higher TAPP doses as compared to S. aureus were needed to achieve planktonic bacteria photosensitization (3-log CFU reduction at 20 μM TAPP and 180 J/cm2). On the other hand, high concentrations of TAPP were nontoxic to P. aeruginosa growing on biofilms, and employing 30 μM TAPP and 180 J/cm2 we obtained 3-log CFU reduction. The main conclusion of the present work is that TAPP is a promising and efficient PS capable of promoting photodynamic killing of both Gram-negative and -positive in planktonic bacteria, though more effectively in the latter. In addition, TAPP-PDI induces similar photoinactivation rates in both bacteria types growing on biofilms, with lower dark toxicity in the Gram-negative one.Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicada

Photodynamic inactivation of planktonic and biofilm growing bacteria mediated by a meso-substituted porphyrin bearing four basic amino groups

Biofilm-associated diseases account for 80% of all infections in humans. Due to the emergence of antibiotic resistances, alternative therapies such as Photodynamic Inactivation (PDI) of microorganisms have emerged. Porphyrins with intrinsic positive charges have been proposed as successful photosensitizers (PSs) against microorganisms. We have recently designed the new synthetic porphyrin 5,10,15,20-tetrakis[4-(3-N,N-dimethylammoniumpropoxy)phenyl]porphyrin (TAPP) containing four basic amine groups in the periphery of the tetrapyrrolic macrocycle, which can acquire positive charges at physiological pH, thus favouring the interaction with biomembranes. Illumination of planktonic cultures of Staphylococcus aureus at 180 J/cm2 in the presence of 2.5 μM TAPP induced complete bacteria eradication. For the TAPP-PDI treatment of S. aureus biofilms, higher light fluences and PS concentrations were needed. Employing 20 μM TAPP and 180 J/cm2, around 3-log CFU reduction were obtained. In order to determine the efficacy of TAPP-PDI on Gram-negative bacteria, we performed planktonic and biofilm assays employing Pseudomonas aeruginosa. Much higher TAPP doses as compared to S. aureus were needed to achieve planktonic bacteria photosensitization (3-log CFU reduction at 20 μM TAPP and 180 J/cm2). On the other hand, high concentrations of TAPP were nontoxic to P. aeruginosa growing on biofilms, and employing 30 μM TAPP and 180 J/cm2 we obtained 3-log CFU reduction. The main conclusion of the present work is that TAPP is a promising and efficient PS capable of promoting photodynamic killing of both Gram-negative and -positive in planktonic bacteria, though more effectively in the latter. In addition, TAPP-PDI induces similar photoinactivation rates in both bacteria types growing on biofilms, with lower dark toxicity in the Gram-negative one.Fil: Mamone, Leandro Ariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Investigaciones sobre Porfirinas y Porfirias. Universidad de Buenos Aires. Centro de Investigaciones sobre Porfirinas y Porfirias; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Ferreyra, Darío David. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Química; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Gándara, Lautaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Investigaciones sobre Porfirinas y Porfirias. Universidad de Buenos Aires. Centro de Investigaciones sobre Porfirinas y Porfirias; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Di Venosa, Gabriela Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Investigaciones sobre Porfirinas y Porfirias. Universidad de Buenos Aires. Centro de Investigaciones sobre Porfirinas y Porfirias; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Vallecorsa, Pablo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Investigaciones sobre Porfirinas y Porfirias. Universidad de Buenos Aires. Centro de Investigaciones sobre Porfirinas y Porfirias; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Saenz, Daniel Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Investigaciones sobre Porfirinas y Porfirias. Universidad de Buenos Aires. Centro de Investigaciones sobre Porfirinas y Porfirias; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Calvo, Gustavo Hernán. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Investigaciones sobre Porfirinas y Porfirias. Universidad de Buenos Aires. Centro de Investigaciones sobre Porfirinas y Porfirias; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Batlle, Alcira María del C.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Investigaciones sobre Porfirinas y Porfirias. Universidad de Buenos Aires. Centro de Investigaciones sobre Porfirinas y Porfirias; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; 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: Durantini, Edgardo Néstor. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Química; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Casas, Adriana Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Investigaciones sobre Porfirinas y Porfirias. Universidad de Buenos Aires. Centro de Investigaciones sobre Porfirinas y Porfirias; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; Argentin

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

Population Structure of Staphylococcus aureus from Remote African Babongo Pygmies

Staphylococcus aureus is a bacterium that colonizes humans worldwide. The anterior nares are its main ecological niche. Carriers of S. aureus are at a higher risk of developing invasive infections. Few reports indicated a different clonal structure and profile of virulence factors in S. aureus isolates from Sub-Saharan Africa. As there are no data about isolates from remote indigenous African populations, we conducted a cross-sectional survey of S. aureus nasal carriage in Gabonese Babongo Pygmies. The isolates were characterized regarding their susceptibility to antibiotic agents, possession of virulence factors and clonal lineage. While similar carriage rates were found in populations of industrialized countries, isolates that encode the genes for the Panton-Valentine leukocidin (PVL) were clearly more prevalent than in European countries. Of interest, many methicillin-susceptible S. aureus isolates from Babongo Pygmies showed the same genetic background as pandemic methicillin-resistant S. aureus (MRSA) clones. We advocate a surveillance of S. aureus in neglected African populations to control the development of resistance to antibiotic drugs with particular respect to MRSA and to assess the impact of the high prevalence of PVL-positive isolates