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Made available in DSpace on 2021-06-25T12:39:56Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-01-01. Added 1 bitstream(s) on 2021-07-15T14:36:34Z : No. of bitstreams: 1 S0036-46652021000100208.pdf: 315461 bytes, checksum: 46e0bb1594d398e0f052e0429b6d98da (MD5) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Nasal carriage of Staphylococcus aureus by healthcare workers is of great clinical importance as it facilitates the contamination of medical devices and cross-transmission. However, studies regarding the epidemiology and dissemination of S. aureus and Methicillin-resistant S. aureus (MRSA) within the Primary Health Care in Brazil are scarce. The current study aimed to detect and characterize S. aureus and MRSA strains from the nasal cavities of 63 healthcare working in primary health care units in order to determine the prevalence of S. aureus and MRSA, biofilm formation and resistance profile of these isolates. PCR reactions were performed for detecting mecA, icaA and ical) genes. The phenotypic antimicrobial susceptibility was assessed by the disk diffusion method and biofilm formation by the Congo Red Agar (CRA) method. The MRSA isolates were typed for the Staphylococcal Cassette Chromosome mec (SCCmec). The prevalence of nasal carriage of S. aureus was 74.6%. of which 72.3% were MRSA carrying SCCmec type I (24.4%), III (34.1%), IV (36.6%). Two (4.9%) isolates presented a non-typeable cassette by the performed technique. The antimicrobial susceptibility evaluation evidenced penicillin resistance in 66.1% of S. aureus, erythromycin resistance in 49.2%. while 37.3% were resistant to oxacillin, 28.8% to cefoxitin, 5.1% to levofloxacin and 5.1% to clindamycin. All isolates were biofilm producers and 96.6% of the strains contained the ica biofilm-forming genes (icaA and/or ical)). We have demonstrated a high prevalence of S. aureus and MRSA carriage among health care working in Primary health Care units, the presence of SCCmec types I, III and IV, in addition to their high ability to form biofilm, factors that possibly contribute to the dissemination and persistence of these pathogens within the primary care services. These observations highlight the importance of broadening the perspective of Health Care-Associated Infections prevention, including all health care levels, which are currently little explored. In addition, the dynamics and resistance mechanisms of S. aureus transmission still need to be further clarified to enable the implementation of more effective prevention measures. Univ Oeste Paulista, Rua Jose Bongiovani 700, BR-19050920 Presidente Prudente, SP, Brazil Univ Estadual Paulista, Dept Ciencias Quim & Biol, Botucatu, SP, Brazil Inst Lauro Souza Lima, Dept Patol, Bauru, SP, Brazil Univ Estadual Paulista, Dept Ciencias Quim & Biol, Botucatu, SP, Brazil FAPESP: 2017/01104-5

Resumen: El biofilm es definido como una comunidad microbiana altamente estructurada, compleja, inmersas dentro de una matriz de biopolímeros y adheridas a un sustrato o interfase inerte o viviente. Las bacterias dentro del biofilm están protegidas del ataque del sistema inmune del hospedero, de los antibióticos y otros agentes antimicrobianos. Staphylococcus aureus es una bacteria Gram positiva considerada como un patógeno oportunista, ya que hace parte del microbioma humano pero tiene la capacidad de causar infecciones. Esta bacteria genera un gran impacto en la salud pública mundial, ya que es el cuarto microorganismo recuperado de infecciones de pacientes hospitalizados. Adicionalmente, S. aureus tiene la capacidad de formar biofilm sobre diferentes superficies, incluyendo implantes médicos. El biofilm en S. aureus es producido por el operón ica, el cual está conformado por los genes icaA, icaB, icaC e icaD, que codifican las enzimas relacionadas con la producción del polisacárido de adhesión intercelular (PIA), componente principal de la matriz extracelular del biofilm en este microorganismo. En trabajos realizados previamente en el Laboratorio de Genética Molecular Bacteriana (LGMB) de la Universidad el Bosque se determinó la existencia de una región dentro del gen icaA altamente conservada en varias especies de Staphylococcus spp y que además contiene una secuencia palindrómica. Además existen indicios de proteínas que se unen de manera específica a esta secuencia palindrómica. El objetivo de este estudio fue identificar y caracterizar una proteína de unión a esta secuencia palindrómica del gen icaA y determinar su posible participación en la formación del biofilm en S. aureus. A partir de ensayos de retardamiento en gel y un proceso de purificación basado en cromatografía de proteínas de alta resolución se logró establecer la existencia de dos proteínas que reconocen esta secuencia palindrómica, SarX y una proteína hipotética, previamente no descrita. Ya se ha determinado que SarX tiene la habilidad para unirse al gen icaA. Sin embargo, la proteína hipotética correspondió a un ortólogo de la proteína YheA de Bacillus subtilis y además contiene un dominio denominado com_ylbF, el cual también está presente en las proteínas YlbF y YmcA de esta misma bacteria. Adicionalmente, se realizó un análisis estructural “in silico” de la proteína YheA, y de las proteínas YlbF y YmcA de S. aureus y B. subtilis. Modelos tridimensionales (3D) de las tres proteínas fueron construidos usando los programas I-TASSER y Quark. A partir de este análisis dos nuevas características de YheA, YlbF y YmcA fueron encontradas: una estructura 3D altamente conservada, a pesar de sus bajos porcentajes de identidad 16% y la presencia de un motivo putativo específico de cada proteína localizado dentro del dominio com_ylbF. Esta proteína YheA tiene un alto contenido de glutamina (13,2%), a nivel general y en su motivo putativo (QQKQMQ), lo cual sugiere que este aminoácido podría estar participando en la función de la proteína. Basados en estos hallazgos, se sugiere renombrar YheA como proteína rica en glutamina o Qrp (por su nombre en inglés Glutamine-rich protein) en S. aureus. Usando ensayos de mutagénesis dirigida, la deleción del gen yheA/qrp, que codifica para la proteína Qrp/YheA, produce una disminución en la formación del biofilm y un aumento en la hemólisis de eritrocitos por S. aureus. Estos resultados señalan que S. aureus posee una nueva familia de proteínas que contienen el dominio com_ylbF, de las cuales una de ellas, Qrp/YheA, está participando en la formación de biofilm a través de una interacción, directa o indirecta, con una secuencia palindrómica presente dentro del gen icaA. Abstract: The biofilm is a complex and high structured microbial community, which is immersed within a matrix composed of biopolymers with ability to adhere to inert or living substrates. The bacteria within the biofilm are protected the host defenses, antibiotics and other antimicrobial compounds. Staphylococcus aureus is a Gram positive bacterium, an opportunistic pathogen that generates a high clinic impact in the public health due to that it is the fourth bacterium recovered from infections in hospitalized patients. In addition, S. aureus has the ability to form biofilm on different substrates, including medical implants. The biofilm in S. aureus is produced by ica operon, which is composed for the icaA, icaB, icaC and icaD genes, encoding the enzymes responsible for the Polysaccharide Intercellular Adhesin (PIA) synthesis, principal biopolymer of the biofilm. In previous studies performed in the Laboratorio de Genetica Molecular Bacteriana of the Universidad El Bosque, a high conserved region was identified within icaA gene of the several Staphylococcal species, and interestingly, this region contained a palindromic sequence. There are experimental evidences that suggest the existence of proteins that recognize specifically this palindromic sequence. For these reasons, the objective of this study was to identify and to characterize one protein that recognizes this palindromic sequence and to establish whether it participates in the biofilm formation in S. aureus. Using Electrophoretic Mobility Shift Assay (EMSA) and FPLC-based protein purification, we achieve to identify two proteins that recognize this palindromic sequence, SarX and a hypothetical protein, which has not been previously described. It is known that SarX protein has the ability for binding to icaA gene. However, the hypothetical protein was an ortholog of the YheA protein of Bacillus subtilis and it contains the com_ylbF domain, which has also been identified in the proteins YlbF and YmcA in this same bacterium. Then, we performed an “in silico” structural analysis of the YheA protein, as well as the YlbF and YmcA. Three-dimensional (3D) models of these three proteins, in both S. aureus and B. subtilis, were built using the I-TASSER and Quark programs. From these analysis two new and important characteristics for YheA, YlbF and YmcA were found: a highly conserved 3D structure (despite their low amino acid identity, 16%) and the presence of a putative conserved motif located in the central region of the domain, which could be involved in its function. The YheA protein has a high content of glutamine (13.2%), even within its putative motif (QQKQMQ), which suggests that this amino acid could to be participating in the protein function. Based on these findings, we suggest renaming YheA as glutamine-rich protein (Qrp) in S. aureus. Interestingly, the yheA/qrp gene deletion in the S. aureus genome produces a decrease in the biofilm formation but an increase in the red blood cells hemolysis. Our results indicate that S. aureus possesses a new protein family that contain the com_ylbF domain, and that least one of these, Qrp/YheA, is participating in the biofilm formation through either direct or indirect interaction with a palindromic sequence located within the icaA gene. Doctorado

Staphylococcus aureus es un patógeno oportunista de gran importancia clínica por su capacidad de causar infecciones agudas y crónicas. Este microorganismo tiene una extraordinaria capacidad de adquirir resistencia a los antibióticos, dificultando su tratamiento. Adicionalmente, S. aureus puede formar biofilm (o biopelícula) por medio del operon ica, el cual está conformado por los genes icaA, icaD, icaB e icaC. Estudios previos analizando el gen icaA permitieron encontrar una secuencia palindrómica (SP) en su interior, la cual se encuentra altamente conservada en diferentes especies de Staphylococcus spp. Debido al reconocido papel de las secuencias palindrómicas en diversos procesos celulares, el objetivo de este trabajo fue determinar e identificar alguna proteína que se uniera a ésta secuencia palindrómica. Para lograrlo, se realizó un proceso de purificación de proteínas de unión a esta SP por medio de cromatografía líquida rápida de proteínas (FPLC por sus siglas en inglés). El seguimiento de la proteína de unión (DNA-proteína) se realizó por medio de la técnica de cambio en la movilidad de la electroforesis (EMSA por sus iniciales en inglés) utilizando un oligonucleótido marcado que simulaba esta SP. Después del proceso de purificación, la proteína fue identificada a través de espectrometría de masas (MALDI-TOF). Una proteína hipotética de 114 aminoácidos fue identificada in S. aureus y un análisis bioinformático permitió establecer una alta homología con la proteína YheA de Bacillus subtilis y la presencia de un dominio YlbF. Este dominio es característico de las proteínas YheA, YlbF y YmcA, las cuales son reguladores transcripcionales relacionados con la formación del biofilm en Bacillus subtilis. Este estudio permitió la identificación de una nueva proteína (YheA), la cual puede estar involucrada en la regulación de la formación de biofilm en Staphylococcus aureus a través de la unión al gen icaA. Abstract: Staphylococcus aureus is an opportunistic pathogen of great clinical importance for its ability to cause acute and chronic infections. Staphylococcus aureus has an extraordinary ability to acquire resistance to antibiotics, making more difficult to treat its infections. In addition, this bacterium can form bioflm through the ica operon, which is constituted by the icaA, icaD, icaB, and icaC genes. A previously analysis of icaA gene has allowed to find an internal palindromic sequence (PS), which is highly conserved among different species of Staphylococcus spp. As it is known, the palindromic sequences play an important role between several cellular processes, in this sense, the aim of this study was to identify if any protein recognizes and binds to this PS. The identification process consisted of the following steps: first, purification by fast liquid chromatography of proteins (FLPC), the target protein was followed by electrophoretic mobility shift assay (EMSA) using a labeled oligonucleotide that mimicked this PS and second, target protein was identified by mass spectrometry (MALDI-TOF). A hypothetical protein of Amino acids 114 was identified in S. aureus and a bioinformatic analysis allowed to establish a high homology with the YheA protein from Bacillus subtilis and the presence of an YlbF domain. This domain is characteristic of YheA, YlbF, and YmcA proteins, which are transcriptional regulators related to the formation of the biofilm in Bacillus subtilis. This study allowed the identification of a protein new (YheA), which may be involved in the regulation of biofilm formation in Staphylococcus aureus through binding to the icaA gene. Maestría

Made available in DSpace on 2014-06-11T19:32:55Z (GMT). No. of bitstreams: 0 Previous issue date: 2005-12-13Bitstream added on 2014-06-13T19:22:47Z : No. of bitstreams: 1 bernardi_aca_dr_arafcf.pdf: 1525508 bytes, checksum: c65f0bdce77e95dac615f5ee33dd6287 (MD5) Universidade Estadual Paulista (UNESP) Os Staphylococcus coagulase-negativa, particularmente, os Staphylococcus epidermidis são a causa mais freqüente de infecções relacionadas ao cateter por sua habilidade em aderir a uma superfície e entre si (aderência intercelular) formando biofilme em multicamadas sobre superfícies de polímeros. O objetivo do presente estudo foi avaliar cepas hospitalares de Staphylococcus coagulasenegativa isoladas de cateteres intravenosos, quanto à resistência a oxacilina, produção de slime, aderência ao poliestireno, habilidade de formar biofilme sobre superfícies abióticas (cateter esterilizado) e a presença de genes icaAD. Na presente pesquisa, a presença de icaA e icaD foi determinada pelo método PCR, em uma coleção de 27 amostras Staphylococcus coagulase-negativa (10 Staphylococcus epidermidis, 4 S. haemolyticus, 2 S. hominis, 2 S. lugdunensis, 1 S. saprophyticus, 1 S. schleiferi, 2 S. xylosus e 4 S. warneri). Os genes icaAD foram detectados em dez cepas S. epidermidis... Coagulase-negative Staphylococcus, particularly, Staphylococcus epidermidis are frequent cause of infections associated with catheters and is attributed to the attachment ability on a surface and each other (intercellular adhesion) forming a multilayered biofilm on polymeric surfaces. The objective of the present study was to evaluate coagulase-negative Staphylococcus strains isolated from intravenous catheters by oxacillin resistance, slime production (qualitative method) and spectrophotometric assay (quantitative method), ability to form biofilm on abiotic surfaces (steriled catheter) and the presence of icaAD genes. In the present study icaA and icaD were determined by PCR method, in a collection of 27 coagulasenegative Staphylococcus (10 Staphylococcus epidermidis, 4 S. haemolyticus, 2 S. hominis, 2 S. lugdunensis, 1 S. saprophyticus, 1 S. schleiferi, 2 S. xylosus and 4 S. warneri). The icaA genes were detected in nine S. epidermidis and icaD in ten. The slime-producing ability was determined by culture on Congo red agar plates in which slime-producing strains formed black colonies in 10 S. epidermidis, 4 S.haemolyticus, 4 S. warneri, 2 S. xylosus and 1 S. chromogenes, while nonslimeformingones develop red colonies. The quantitative assay of coagulase-negative Staphylococcus was observed in 19 strains, including: 10 S. epidermidis, 3 S.haemolyticus, 3 S. warneri, 2 S. xylosus, 1 S. chromogenes. The ability of coagulasenegative Staphylococcus to form biofilm embedded in an amorphous substance wasobserved by scanning electronic microscope on abiotic surface in 10 S. epidermidis,3 S. haemolyticus, 2 S. hominis, 2 S. lugdunensis, 1 S. saprophyticus, 1 S. schleiferi,2 S. xylosus and 3 S. warneri. The oxacillin resistance was observed in 9 strains S.epidermidis, 3 S. haemolyticus, 3 S. warneri, 1 S. xylosus and 1 S. chromogenes. All strains of staphylococci were susceptible... (Complete abstract, click eletronic address below)