Study of the role of wall teichoic acids in the localization Staphylococcus aureus cell wall synthesis protein PBP4

The cell wall of Staphylococcus aureus is a highly complex network mainly composed of highly cross-linked peptidoglycan (PG) and teichoic acids (TAs), both important for the maintenance of the integrity and viability of bacteria. The penicillin binding proteins (PBPs), which catalyse the final stage of PG biosynthesis, are targets of β-lactam antibiotics and have been a key focus of antibacterial research. S. aureus has four native PBPs, PBP1-4 carried by both methicillin-sensitive (MSSA) and –resistant (MRSA) strains. PBP4 is required for the synthesis of the highly cross-linked PG and, as shown in recent studies, is essential for the expression of β-lactam resistance in community-acquired strains (CA-MRSA). This protein has a septal localization that seems to be spatially and temporally regulated by an unknown intermediate of the wall teichoic acids (WTA) biosynthesis pathway. Therefore, if WTA synthesis is compromised, PBP4 becomes dispersed throughout the entire cell membrane. The aim of this project was to identify the WTA precursor responsible for the septal recruitment of PBP4. In order to do so, inducible mutants of tarB and tarL genes in the background of NCTCPBP4-YFP were constructed allowing for the study of PBP4 localization in the presence and absence of these specific tar genes.With this work we were able to show that the absence of TarB or TarL leads to the delocalization of PBP4, indicating that TarL or a protein/WTA precursor whose localization/synthesis is dependent on TarL is responsible for the recruitment of PBP4.

Gold nanoprobes for assessing expression of critical genes in the infection pathway of MRSA

Staphylococcus aureus is an important opportunistic pathogen that can cause a wide variety of diseases from mild to life-threatening conditions. S. aureus can colonize many parts of the human body but the anterior nares are the primary ecological niche. Its clinical importance is due to its ability to resist almost all classes of antibiotics available together with its large number of virulence factores. MRSA (Methicillin-Resistant S. aureus) strains are particularly important in the hospital settings, being the major cause of nosocomial infections worldwide. MRSA resistance to β-lactam antibiotics involves the acquisition of the exogenous mecA gene, part of the SCCmec cassette. Fast and reliable diagnostic techniques are needed to reduce the mortality and morbidity associated with MRSA infections, through the early identification of MRSA strains. The current identification techniques are time-consuming as they usually involves culturing steps, taking up to five days to determine the antibiotic resistance profile. Several amplification-based techniques have been developed to accelerate the diagnosis. The aim of this project was to develop an even faster methodology that bypasses the DNA amplification step. Gold-nanoprobes were developed and used to detect the presence of mecA gene in S. aureus genome, associated with resistance traits, for colorimetric assays based on non-crosslinking method. Our results showed that the mecA and mecA_V2 gold-nanoprobes were sensitive enough to discriminate the presence of mecA gene in PCR products and genomic DNA (gDNA) samples for target concentrations of 10 ng/μL and 20 ng/μL, respectively. As our main objective was to avoid the amplification step, we concluded that the best strategy for the early identification of MRSA infection relies on colorimetric assays based on non-crosslinking method with gDNA samples that can be extracted directly from blood samples.

Study of in vivo interactions between penicillin-binding proteins of Staphylococcus aureus

Staphylococcus aureus (S. aureus) is a major human pathogen that has acquired resistance to practically all classes of β-lactam antibiotics, being responsible of Multidrug resistant S. aureus (MRSA) associated infections both in healthcare (HA-MRSA) and community settings (CA-MRSA). The emergence of laboratory strains with high-resistance (VRSA) to the last resort antibiotic, vancomycin, is a warning of what is to come in clinical strains. Penicillin binding proteins (PBPs) target β-lactams and are responsible for catalyzing the last steps of synthesis of the main component of cell wall, peptidoglycan. As in Escherichia coli, it is suggested that S. aureus uses a multi-protein complex that carries out cell wall synthesis. In the presence of β-lactams, PBP2A and PBP2 perform a joint action to build the cell wall and allow cell survival. Likewise, PBP2 cooperates with PBP4 in cell wall cross-linking. However, an actual interaction between PBP2 and PBP4 and the location of such interaction has not yet been determined. Therefore, investigation of the existence of a PBP2-PBP4 interaction and its location(s) in vivo is of great interest, as it should provide new insights into the function of the cell wall synthesis machinery in S. aureus. The aim of this work was to develop Split-GFPP7 system to determine interactions between PBP2 and PBP4. GFPP7 was split in a strategic site and fused to proteins of interest. When each GFPP7 fragment, fused to proteins, was expressed alone in staphylococcal cells, no fluorescence was detectable. When GFPP7 fragments fused to different peptidoglycan synthesis (PBP2 and PBP4) or cell division (FtsZ and EzrA) proteins were co-expressed together, fluorescent fusions were localized to the septum. However, further analysis revealed that this positive result is mediated by GFPP7 self-association. We then interpret the results in light of such event and provide insights into ways of improving this system.

Helicobacter pylori genotyping from positive clotests in patients with duodenal ulcer

Even though the seroprevalence of H. pylori may be high in the normal population, a minority develops peptic ulcer. Colonization of the gastric mucosa by more pathogenic vacA strains of H. pylori seems to be associated with enhanced gastric inflammation and duodenal ulcer. H. pylori genotyping from positive CLOtests was developed to determine the vacA genotypes and cagA status in 40 duodenal ulcer patients and for routine use. The pathogenic s1b/ m1/ cagA genotype was the most frequently occurring strain (17/42.5%); only two (5%) patients presented the s2/ m2 genotype, the less virulent strain. Multiple strains were also detected in 17 (42.5%) patients. Multiple strains of H. pylori colonizing the human stomach have been underestimated, because genotyping has been performed from cultures of H. pylori. We concluded that genotyping of H. pylori from a positive CLOtest had the advantages of reducing the number of biopsies taken during endoscopy, eliminating the step of culturing H. pylori, and assuring the presence of H. pylori in the specimen being processed.

Staphylococcus aureus em 2 (duas) rãs-touro (Rana catesbeiana) de um ranário em Belém, PA

RESUMOEfetuou-se análises bateriológicas em material proveniente de dois espécimes de rãs-touro com a finalidade de se verificar qual o possível agente responsável pela patologia manifestada em um ranário da cidade de Belém, Pará. Os resultados indicam a presença de Staphylococcus aureus.

Poly-N-acetyl glucosamine expression in Staphylococcus epidermidis biofilm cells affects the immune response and promotes tissue pathology in infected hosts

Staphylococcus epidermidis is a biofilm - forming bacterium and a leading etiological agent of nosocomial infections. The ability to establish biofilms on indwelling medical devices is a key virulence factor for this bacterium. Still, the influence of poly - N - acetyl glucosamine (PNAG), the major component of the extracellular biofilm matrix, in the host immune response has been scarcely studied. Here, t h is influence was assessed in mice challenged i.p. with PNAG - p roducing (WT) and isogenic - mutant lacking PNAG (M10) bacteria grown in biofilm - inducing conditions. Faster bacterial clearance was observed in the mice infected with WT bacteria than in M10 - infected counterparts , which w as accompanied by earlier neutrophil recruitment and higher IL - 6 production. Interestingly, in the WT - infected mice, but not in those infected with M10 , elevated serum IL - 10 was detected . To further study the effe ct of PNAG in the immune response, mice were primed with WT or M10 biofilm bacteria and subsequently infected with WT biofilm - released cells. WT - primed mice presented a higher frequency of splenic IFN - γ + and IL - 17 + CD4 + T cells, and more severe liver patho logy than M10 - primed counterparts. Nevertheless, T reg cells obtained from the WT - primed mice presented a higher suppressive function than those obtained from M10 - primed mice. This effect was abrogated when IL - 10 - deficient mice were similarly primed and infected indicating that PNAG promotes the differentiati on of highly suppressive T reg cells by a mechanism dependent on IL - 10. Altogether, these results provide evidence help ing explain ing the coexistence of inflammation and bacterial persistence often observed in biofilm - originated S. epidermidis infections

Compositional analysis of biofilms formed by Staphylococcus aureus isolated from food sources

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmicb. 2016.00390

Determination of the importance of iron acquisition for Staphylococcus epidermidis biofilm survival in human blood

Dissertação de mestrado em Bioengenharia

Characterization of microbial populations in a wastewater treatment plant focusing on Staphylococcus aureus and Pseudomonas aeruginosa

Dissertação de mestrado em Bioengenharia

Frecuencia de aislamiento de los géneros Clostridium y Staphylococcus de leche cruda bovina y leches en polvo

El uso del término Intoxicaciones Alimentarias está aplicado en forma genérica a todas aquellas enfermedades que se adquieren por consumo de alimentos o aguas contaminadas. Este término incluye las intoxicaciones causadas, entre otras, por toxinas elaboradas por proliferación bacteriana por S. aureus; C. perfringens; V. parahaemolyticus; E. coli; C. botulinum entre otras. La intoxicación alimentaria tiene una etiología predominante en su frecuencia de aislamiento. Generalmente se deben a cepas de estafilococos enterotóxicos, transmitida por consumo de leche cruda o productos lácteos. En cambio no existe mucha información relacionada con aquellas intoxicaciones alimentarias producidas por C. perfringens. De acuerdo a las observaciones preliminares es posible aislar C. perfringens de leche cruda bovina. (...) Este proyecto tiene como Objetivo General observar, desde el punto de vista epidemiológico, la frecuencia de aislamiento de los géneros Clostridium y Staphylococcus de dos productos lácteos importantes en la alimentación humana como son leche cruda de vaca y leche en polvo, expendida en forma comercial en los supermercados.

Estudio de los mecanismos inmunosupresores involucrados en la cronicidad de la infección intramamaria por staphylococcus aureus en bovinos. Immunosuppressive mechanisms involved in intrammary chronic infection by staphylococcus aureus in bovine.

La producción de productos lácteos conforma un complejo productivo de larga trayectoria en Argentina, con grandes transformaciones en el sector lechero en los últimos años. La provincia de Córdoba es una de las principales áreas de producción lechera, participando del 34.5 por ciento de la producción nacional. Esta provincia cuenta con un alto número de industrias, principalmente localizadas en la cuenca de Villa María, y concentra el 35 por ciento de los establecimientos lecheros del país. La Mastitis bovina (MB) es la principal causa de pérdidas económicas para el productor y la industria láctea a nivel mundial y regional, por lo cual se plantea la necesidad de mejorar la calidad higiénica y sanitaria de la leche a través de un mayor control de la mastitis. La MB es una inflamación de la glándula mamaria (GM) asociada a una infección bacteriana. Es la enfermedad más común y de mayor incidencia en el ganado lechero, siendo la principal causa de pérdidas económicas para la industria láctea a nivel mundial. Las medidas actuales de control de MB están basadas en prácticas de higiene apropiadas, reducción de la exposición ambiental al patógeno y terapia antibiótica del ganado, las cuales no son totalmente efectivas en el control de la infección. La defensa de la GM contra los patógenos causantes de MB depende de factores anatómicos, celulares y solubles, siendo la eficiencia de esos mecanismos la que determina la resistencia a nuevas infecciones. Los mediadores inmunes innatos y adquiridos de los tejidos y secreciones de la GM actúan en forma coordinada en la protección de la glándula contra enfermedades contagiosas. Staphylococcus aureus es el agente etiológico más importante en la mastitis. Esta bacteria evade la respuesta inmune inflamatoria mediante la inducción de mecanismos inmunosupresores, llevando a la patología a un curso crónico. S. aureus además puede colonizar el tejido epitelial y formar películas bacterianas conocidas como biofilms. Así, S. aureus adquiere más resistencia a la terapia antibiótica y a la acción del sistema inmunológico determinando la persistencia de la enfermedad. Nuestra hipótesis es que una respuesta inmune desarrollada en un microambiente particular de citoquinas, quimioquinas y células, podría contribuir a la evasión del patógeno y al desarrollo de una infección crónica. El polisacárido Quitosano (Q) presenta un efecto antibacteriano e inmunoestimulante en cultivos de células inmunes de ganado bovino, un efecto protectivo en modelos murinos de mastitis y actividad anti-biofilms de distintas cepas bacterianas. Por sus propiedades intrínsecas, este polisacárido es un candidato ideal en la regulación de las respuestas inmunológicas. El objetivo de este proyecto es caracterizar el microambiente local y sistémico, las señales inducidas por el microorganismo que promueven una falla inmunológica y caracterizar el efecto de Q sobre las respuestas generadas en la GM, mediante la realización de numerosos estudios in vivo e in vitro. Las estrategias para controlar la MB por S. aureus y disminuir el impacto de esta patología en la industria láctea, podrían orientarse a la manipulación del sistema inmunológico de la GM bovina a fin de incrementar los mecanismos de defensa naturales del huésped. Los resultados que se desprendan del proyecto permitirán adquirir conocimiento para el desarrollo de nuevas estrategias de inmunointervención a fin de controlar la MB por S. aureus y disminuir el impacto de esta patología en la industria láctea. Siendo la MB una patología relevante no solo en lo que hace a status sanitario animal, línea prioritaria definida en el Plan Estratégico Nacional de Ciencia, Tecnología e Innovación "Bicentenario 2006-2010", sino también a las implicancias económicas de esta problemática, y dado el desarrollo de la actividad lechera en la región y en el país, los resultados obtenidos podrían tener un importante impacto socioeconómico.

Untersuchungen zur Taxonomie, Epidemiologie, Genotypisierung und Immunpathologie von klinischen Staphylococcus-Isolaten

Magdeburg, Univ., Fak. für Naturwiss., Diss., 2009

Phäno- und genotypische Untersuchungen von Methicillin-sensiblen und -resistenten Staphylococcus aureus-Isolaten aus Panama

Magdeburg, Univ., Med. Fak., Diss., 2015

Microsatellite genotyping from faeces of Lontra longicaudis from southern Brazil

A genetic study of the neotropical river otter Lontra longicaudis (Olfers, 1818), which has an unknown conservation status, was carried out at the Taim Ecological Station and the margins of the Vargas stream, Rio Grande do Sul, southern Brazil. Faecal samples were collected, and DNA was extracted using a silica-guanidine method. Five microsatellite loci were amplified using PCR with heterologous primers previously described for Lutra lutra (Linnaeus, 1758). Sixteen faecal samples out of 29 from Taim and 11 out of 14 from Vargas stream margins contained enough DNA for genetic analysis. A total of 49 different alleles were found at both localities, from which 18 were exclusively found in individuals from Taim and 17 were exclusives from Vargas individuals. The most common allele was the same at both locations for three loci (Lut715, Lut733, and Lut818). A high level of genetic diversity was found at both sites (NeTaim=4.1, HoTaim=0.299, HeTaim=0.681; NeVargas=4.9, HoVargas=0.355, HeVargas=0.724), being higher at the Vargas stream site. A high and significant level of heterozygote deficiency was observed at most loci according to the χ2 test. The homogeneity χ2 test (P<0.001) showed that there were significant differences in the allele frequencies between the two locations. Genotyping for more than one locus was possible in 81.5% of samples, from which only 37% were possible to genotype for more than three loci. A low degree of relatedness was found among individuals from Taim (R=0.055±0.310), but an even lower value of relatedness was found at the Vargas site (R= -0.285±0.440). The significant degree of differentiation (I=0.890; F ST=0.059) found between Taim and Vargas individuals suggests that there is more than one population of otters in the southern extreme of Brazil, which probably are associated with the water body systems found in this region, the Mirim and the Caiuvá/Flores/Mangueira Lagoons. The high genetic diversity and low relatedness found at the Vargas stream, lead us to believe that the Vargas stream may be acting as a corridor between these water bodies for otter dispersion.