MMed (Clinical Microbiology), Faculty of Health Sciences, University of the Witwatersrand, 2009 / Introduction: The clinical laboratory is required to rapidly identify Staphylococcus aureus as a cause of bacteraemia, and in particular, to detect methicillin resistance amongst bacteraemic isolates, to facilitate prompt initiation of appropriate therapy which may directly impact on patient survival, and to allow for implementation of appropriate infection control measures. Hence, the laboratory needs to choose tests to detect methicillin-resistant S. aureus (MRSA) bacteraemia which are rapid, accurate, simple, cost-effective and appropriate for the setting. Primary study objective: To determine the accuracy of four phenotypic susceptibility tests to directly detect MRSA from blood culture specimens (BC) compared with detection of the mecA gene by the polymerase chain reaction (PCR) from S. aureus cultured from the same BC. Materials and Methods: BCs were selected from patients with incident, S. aureus bacteraemic episodes at two hospitals, during January and February 2006. S. aureus was identified by standard phenotypic tests, including the presence of a deoxyribonuclease (DNAse). Direct susceptibility tests (DST) were performed (oxacillin (1μg) and cefoxitin (30μg) disk diffusion (DD), oxacillin Etest® (AB bioMérieux) and CHROMagar®-MRSA (CHROMagar® Microbiology)), and repeated on stored cultures. Detection of nuc and mecA genes by PCR confirmed S. aureus and methicillin resistance respectively. The sensitivity and specificity of the DST were calculated with reference to the mecA PCR result, to fulfil the primary study objective. Results: During the two-month study period, 9,400 BC were submitted to the clinical laboratories at the 2 hospitals; S. aureus was isolated from 156 specimens. Of these, 89 BC from 89 incident cases were included in the study, and 65 were subjected to all tests, including PCR. Of the 65 nuc-positive S. aureus isolates from 65 BC, all were positive with the direct DNAse test, and 25 (38%) were mecA positive. Compared to PCR, sensitivity and specificity for the direct oxacillin DD, cefoxitin DD, oxacillin Etest® and CHROMagar®- MRSA was 100% and 90%, 98% and 100%, 100% and 100%, and 96% and 42% respectively. Discussion: In this study, we found that, compared to PCR for the nuc and mecA genes, the combination of a direct DNAse test and oxacillin Etest®, facilitated accurate detection of MRSA bacteraemia. The direct oxacillin Etest® result did not appear to be influenced by a non-standardised inoculum, in contrast to the other direct tests, and provided an oxacillin minimum inhibitory concentration. The direct cefoxitin DD test produced more accurate results than the direct oxacillin DD test, was easier to read and distinguished MRSA from MSSA with zone diameters clustering into more clearly defined susceptibility categories. Although the chromogenic agar performed well when used to identify methicillin resistance amongst cultured S. aureus isolates, it was apparent that this test, read at 24 hours, could not be used reliably as a DST. Since the Etest® is more costly than the DD test; its use should be reserved for BC from patients in “high-risk” hospital areas, e.g. intensive care units. The direct cefoxitin DD could be used for all BC positive for GPCC, and could be used without a direct identification test because of its lower cost; it is further recommended that the direct cefoxitin DD test replace the direct oxacillin test.

Séculos de relatos históricos dedicados à reverência por distintas civilizações emergem dos últimos 25 anos de estudos científicos sobre abelhas sem ferrão, espécies nativas eusociais de habitat tropical e subtropical, em contraste com a extensão das pesquisas voltadas aos atributos comerciais de Apis mellifera. Diferenças das abelhas melíferas incluem arquiteturas de ninhos peculiares, contendo a estrutura comum em cera e resinas vegetais, reforçadas com amálgamas de pólen, sementes ou so lo para produzir o geoprópolis, que compartilha a mesma atividade prevalente de própolis contra bactérias gram- positivas, atribuível a derivados terpênicos. O trabalho teve como objetivo empregar um esquema de técnicas aplicadas ao fracionamento bioguiado, para obtenção de compostos ativos contra Staphylococcus aureus de geoprópolis da abelha sem ferrão Melipona scutellaris. Entre os extratos etanólicos de seis lotes de geoprópolis coletados, aleatoriamente, de um meliponário da Bahia durante um ano, a variedade “marrom-escura” destacou-se pelo poder antiestafilocócico com a mesma atividade apresentada por sua fração acetato de etila (C IM 62,5-31,25 µg/mL), a qual revelou 6 compostos-alvo através da bioautografia, na medida em que conservou o perfil cromatográfico por CLAE-FR-DAD durante a etapa de purificação, motivando o fracionamento do extrato por cromatografia em coluna de sílica, que levou, em vista da baixa eficiência de separação verificada para a fração F1 selecionada (C IM 1,95- 0,98 μg/mL), às sub-frações SF2 (3,90-1,95 µg/mL) e SF3 (7,81-3,90 µg/mL) por CLAE-FR-DAD semi-preparativa. Apesar da resistência ao isolamento, os perfis de fragmentação dos compostos em frações, sub-frações e extratos de geoprópolis, determinados por CLAE-FR-ESI-TQ-EMn, revelaram componentes intimamente relacionados à estrutura comum de um núcleo diterpênico ligado a um resíduo de ácido cinâmico, coerente com as varreduras espectrais no UV homólogas e ao espectro de 1H-RMN da fração F2 (7,81-3,90 µg/mL), relativamente mais purificada. Amostras de geoprópolis consistiram, ao contrário, na variabilidade dos espectros de massas, ressaltando características das abelhas nativas, ao mimetizar as Apis mellifera, quanto aos comportamentos de coleta heterogêneos relacionados às fontes de resi nas disponíveis. Ensaios antibacterianos envolvendo bioautografias e determinações da concentração inibitória/bactericida mínima (CIM/CBM) por microdiluição e revelação com resazuri na biomonitoraram as etapas do fracionamento, enquanto triagens por CIM/CBM comporam o perfil antimicrobiano do extrato “marrom-escuro” e seus derivados mais ativos contra S. aureus, em que um painel de linhagens bacterianas patogênicas padronizadas delineou espécies gram- positivas, em especial de Micrococcus, Bacillus e Staphylococcus, inclusive MRSA, como altamente susceptíveis, em detrimento das gram-negativas, demonstrando o potencial promissor de geoprópolis como repositório de produtos naturais indeléveis aos desafios precursores de fármacos na terapêutica. / Long historical records of reverence by distinct ci vilizations emerge from the last 25 years of scientific studies on stingless bees, native eusocial species inhabiting the tropical and subtropical areas of the world, over extensive documentation devoted to the globally widespread Apis mellifera. Differences from the honeybees include peculiar nest strengthened arquictetures built in beeswax and plant resi ns, amalgamated with pollen, seeds or soil material to produce geopropolis, compositionally dissociated of traditional propolis, even sharing a prevailing activity against gram-positi ve bacteria, attributable to terpenic derivatives. A bioassay-guided fractionation technique was aimed to separate active compounds against Staphylococcus aureus in geopropolis from Melipona scutellaris, concerni ng its potential biological relevance. Among the ethanolic extracts of the six lots of geopropolis ramdomly collected from a meliponary settled in the Bahia State of Brazil, during a year, the “dark-brown” variety was highlighted by the same antistaphylococcal acti vity of ethyl acetate fraction (MIC 62,5-31,25µg/mL), which unveiled at least si x target compounds from bioautography in the purification step, even because their chromatographic profiles by RP-HPLC-PDA remained equivalent to the original extract, motivating its chromatographic fractionation by Low-Pressure Silica Gel Column Chromatography, followed by preparative RP-HPLC-PDA of the poorly resolved fraction F1 (MIC 1,95 – 0,98 μg/mL), yelding sub-fractions SF2 (3,90- 1,95 µg/mL) and SF3 (7,81-3,90 µg/mL). Although escaping from the isolation purpose, the fragmentation profiles of compounds in fractions, sub-fractions and extracts, determined by RP-HPLC-ESI-TQ-MSn, allowed propositions of components closely related to the structure of a diterpenic core bonded to a cinnamic acid group, consistent with the homologous UV spectral scans, associated to the 1H-NMR spectra of fraction F2 (7,81-3,90 µg/mL), relatively the most purified one available. Differing extracts consisted, in contrast, of the variability of mass spectra, suggesting mimics of Apis mellifera heterogeneous collecting behavior in the choice for resin sources by native bees. Antibacterial assays involving bioautography and resazurin microtitre plate-based minimum inhibitory, followed by bactericidal, concentrations (MIC/MBC) methods conducted the fractionation process, whilst the screening by MIC/MBC composed the antimicrobial profile of “dark brown” extract and its most active derivatives against S. aureus, displaying a panel of standardized human pathogenic bacterial strains, recongni zed as highly susceptible gram-positive species, in particular Micrococcus, Bacillus and Staphylococcus, includi ng the selected MRSA isolate, over the gram-negative ones, demonstrating the promising potential of geopropolis as a repository of natural products candidates to the challenges of drugs development in the course of health claims. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES

Bacteriophages (phages) are obligate intracellular parasites of bacteria that usually kill the bacterial host. Bacteriophage therapy is a recently revived approach for treating bacterial infection that relies on the traits of the phage lytic cycle. A lot of attention has been given to phage therapy with new research being published weekly and international conferences organised every year, bringing together the academic and industrial phage communities. However, despite this huge effort and considerable scientific interest there is still a great lack of understanding on how to use phage effectively and overcome the many obstacles in the near future. One of the main triggers for such interest was the increasing evidence of antibiotic resistance among human bacterial pathogens, which were once efficiently eliminated by drugs but are now causing alarmingly high levels of morbidity and mortality. Also, bacteria when causing a disease are able to produce highly protective biofilm communities. Biofilms are major causes of impairment of wound healing and two of the most common and aggressive wound pathogens are Staphylococcus aureus (Gram-positive) and Pseudomonas aeruginosa (Gram-negative), both displaying a large repertoire of virulence factors and reduced susceptibility to antibiotics. This work reports and explores the use of phages to target both S. aureus and P. aeruginosa pathogen biofilm producers. Firstly, isolation of promising phage candidates was performed and cocktails were established. Two phages (DRA88 and phage K) formed the cocktail to target S. aureus and six phages (DL52, DL54, DL60, DL62, DL64 and DL68) formed a cocktail to target P. aeruginosa. A thorough characterisation of each of the selected phages was performed, including their range of host infectivity and their genome sequences were analysed. The phage’s ability to infect and kill planktonic cultures was successfully studied and afterwards such ability was assayed on biofilms using an in vitro static biofilm system (microtitre-plate), followed by an in vitro dynamic biofilm system (The Modified Robbins Device). Both cocktails were shown to be effective in reducing and dispersing biofilms formed by the clinical strains showing them to be promising not only to combat topical bacterial infections (related to biofilm production), but also to control biofilms produced on the surfaces of medical devices, such as catheters. Finally, the phage cocktail’s ability to treat systemic infections caused by the two pathogens was assessed in an in vivo G. mellonella infection model. In the case of the P. aeruginosa infection, although the phages were not able to fully treat the larvae, the cocktail allowed a delay of larval death, caused by the infection. For the S. aureus infection, the cocktail did not show the same trend, but most likely the high bacterial cell numbers involved in the experiment interfered with a successful study on the phage cocktail. The phage mixture may form the basis of an effective treatment for infections caused by S. aureus and P. aeruginosa biofilms.

Thesis (M.A.)--Boston University / In view of the success of the Danish investigators Christensen and Kjems in inducing regressions of malignant tumors with streptococcus bacteriophage lysates, it was decided to test lysates of hemolytic Staphylococcus aureus upon malignancies in hamsters and mice. [TRUNCATED]

Staphylococcus aureus (S. aureus) is a significant pathogen that causes a wide variety of disease in humans and animals. Methicillin resistant S. aureus (MRSA) isolates carrying mecC, the gene that confers resistance to the antibiotic, have been isolated from humans but also from diverse animal species covering livestock, domestic and wild animals throughout Europe. Many of the known MRSA mecC isolates have been whole-genome sequenced by our group to gain insight into the evolution and epidemiology of these emerging lineages. For microbes and humans alike, iron is an essential cofactor in many biochemical reactions and S. aureus requires iron for colonisation and subsequent pathogenesis. The success of S. aureus is partly attributed to its ability to exploit the host iron pool. It does this through multiple iron uptake mechanisms, including at least two high-affinity iron scavenging siderophores (staphyloferrins A and B) and an iron-regulated surface determinant (Isd) pathway for haem-iron acquisition. Here I describe the identification of a novel locus encoding a siderophore-like non-ribosomal peptide synthetase (NRPS), directly downstream of the SCCmec insertion site in mecC S. aureus isolates. A homologous region was identified in Streptococcus equi 4047 (S. equi) which encodes a NRPS termed 'equibactin' that is involved in iron acquisition. I have therefore named the NRPS product 'staphylobactin' in MRSA, and the aim of this study was to determine the function of the staphylobactin biosynthesis cluster: is this region involved in iron acquisition and how might it be regulated? Analysis of the prevalence of isolates containing the staphylobactin locus showed it to be present in a large number of mecC strains in our collection but also identified homologues in other staphylococcus isolates. The region is highly conserved in all S. aureus isolates belonging to clonal complex (CC) 130 (broad host range lineage), suggesting that staphylobactin might impact on S. aureus's ability to infect a broad range of host species. The staphylobactin gene cluster contains 14 coding sequences, stbB-F, F1, G-M and O. Bioinformatic analysis results in predictions of domain and gene functions associated with iron acquisition. I hypothesized that staphylobactin might have been acquired to compensate for the lack of another siderophore, such as staphyloferrin B, but the staphyloferrin B biosynthesis cluster and transport is present in nearly all S. aureus strains, ruling out this model. Unlike the equibactin locus, however, the staphylobactin locus lacks a homolog for the iron-dependent regulator eqbA. Instead, expression of this locus appears to be regulated by MntR, a DtxR-like regulator. The staplylobactin gene cluster is flanked by direct repeats which suggest staphylobactin could have been gained by horizontal gene transfer. In order to study the role of the staphylobactin gene cluster, deletion mutants of MntR, the staphylobactin locus and staphyloferrins A and B, were generated using the pIMAY two step gene deletion procedure in the previously un-manipulated mecC S. aureus CC130 strains - a challenging protocol that required significant optimization given the difficulties with manipulating this bacterium. Analysis of the MntR mutant suggests that the staphylobactin operon is regulated by MntR, acting as a positive regulator, in an iron-dependent manner. By RT-PCR, I found that expression of the staphylobactin NRPS genes is increased when cultures are grown in the absence of iron, suggesting an involvement with iron acquisition. Genomic inactivation of the staphyloferrins resulted in a mutant severely incapacitated for growth in serum and transferrin as the sole iron source, and addition of iron reversed this phenotype. However, deletion of staphylobactin alone or in addition to the staphyloferrins, lacked an iron-dependent growth defect, and numerous assays failed to identify a clear role for staphylobactin in iron metabolism. Therefore, further experiments are needed to elucidate the function of this siderophore like NRPS. Analysis of the same sequenced CC130 mecC isolates from our strain collection in which the staphylobactin locus was found, led to the identification of a novel Von Willebrand (vwb) gene. In order to investigate possible reasons for these isolates to infect a wide range of host species, wild-type and vwb deletion mutant strains, along with the novel vwb expressed in lactococcus, were tested using a coagulation assay and were able to clot plasma from a broad range of host species. Thus the specificity of vWbp proteins can be used to infer the host specificity and evolutionary history of the S. aureus strains that harbour them. Although I was unable to generate definitive evidence revealing the biological role for the staphylobactin locus this study has generated valuable tools for further studies and thoroughly tested a number of hypotheses concerning its role in cation metabolism.

As interações que ocorrem entre as bactérias e amebas podem dar-se através de relações mútuas, onde ambos os organismos se beneficiam da associação ou parasitárias, em que um organismo se beneficia em detrimento do outro. Quando esses organismos compartilham o mesmo ambiente, pode resultar em algumas alterações, seja no crescimento dos organismos, nos padrões de adaptação, na morfologia, no seu desenvolvimento ou até mesmo na sua capacidade para sintetizar proteínas e outras substâncias. No presente estudo, avaliou-se a interação entre Acanthamoeba polyphaga e Staphylococcus aureus (MRSA) através de um modelo de cocultivo em diferentes tempos de incubação. A partir deste, 89% das células amebianas permaneceram viáveis após contato com a bactéria. O isolado bacteriano foi visualizado no interior da ameba através de microscopia confocal e de fluorescência em até 216 horas de cocultivo, sendo considerado um microrganismo resistente à ameba. O contato de A. polyphaga com S. aureus (MRSA) não demonstrou alteração fenotípica da ameba através dos testes fisiológicos de osmo e termotolerância. O lisado da cultura amebiana aumentou o crescimento de S. aureus (MRSA) nos diferentes tempos de incubação, porém houve diferença significativa apenas entre o controle e 96 horas de cocultivo. O crescimento de S. aureus (MRSA) foi inibido ao longo dos tempos de incubação pelo efeito do sobrenadante da cultura amebiana apresentando diferença significativa entre o controle e 96 horas de cocultivo, sugerindo-se que A polyphaga tenha secretado algum tipo de metabólito, que inibiu o crescimento da bactéria. A interação dos microrganismos não apresentou alterações significativas no perfil de susceptibilidade aos antibióticos testados. S. aureus (MRSA) permaneceu viável em cistos de A. polyphaga, reforçando a hipótese de que Acanthamoeba pode desempenhar um papel crucial na propagação de S. aureus (MRSA) na comunidade e ambiente hospitalar. O maior percentual de amebas encistadas deu-se em 96 horas de incubação quando cocultivadas com o isolado de S. aureus (MRSA), apresentando um aumento progressivo deste percentual a cada período de incubação. A partir disso, estudos devem intensificar-se para melhor compreender os mecanismos de virulência envolvidos na interação entre ambos os microrganismos. / The interactions that occur between bacteria and amoebae can give through mutual relations, where both organisms benefit from the association or parasitic in which one organism benefits at the expense of the other. When these organisms share the same environment, can result in some changes in the growth of organisms, in adaptation patterns, in morphology, development or even in their ability to synthesize proteins and other substances. In the present study, we evaluated the interaction between Acanthamoeba polyphaga and Staphylococcus aureus (MRSA) using a coculture model at different incubation times. From this, 89% of amoebic cells remain viable after contact with the bacteria. The bacterial isolate was visualized inside the amoeba through confocal microscopy and fluorescence for up to 216 hours of cocultivation, being considered a resistant microorganism to the amoeba. The contact of A. polyphaga with S. aureus (MRSA) showed no phenotypic changes of amoeba through physiologic osmo or thermotolerance tests. The lysate of amoebic culture increased the growth of S. aureus (MRSA) in the different incubation times, but there was a significant difference only between the control and 96 hours of cocultivation. The growth of S. aureus (MRSA) has been inhibited over the incubation times for the effect of amoebic culture supernatant showing a significant difference between the control and 96 hours of coculture, suggesting tha A. polyphaga has some kind of secreted metabolites inhibiting the growth of bacteria. The interaction of microorganisms showed no significant changes in the susceptibility profile of the tested antibiotics. S. aureus (MRSA) remained viable cysts of A. polyphaga, reinforcing the hypothesis that Acanthamoeba can play a crucial role in the spread of S. aureus (MRSA) in the community and hospital. The highest percentage of encysted amoebae occurred in 96 hours of incubation when cocultured with the isolate of S. aureus (MRSA), with a progressive increase in this percentage to each incubation period. From this, studies should be intensified to better understand the virulence mechanisms involved in the interaction between these two organisms.

Staphylococcus aureus, particularly the epidemic methicillin-resistant S. aureus (MRSA) USA300 strain, is a major cause of severe necrotizing lung, skin and systemic infection. Although these infections are generally attributed to the pathogen’s multiple toxins, exactly how S. aureus cause disease is not clearly defined. In this research, we sought to establish the role of necroptosis, a programmed form of necrosis, in the pathophysiology of S. aureus pneumonia, skin infection and sepsis. S. aureus, mainly through its multiple toxins, induced RIPK1/RIPK3/MLKL-mediated necroptosis in multiple host cells including human cell lines, freshly obtained alveolar macrophages, peripheral blood macrophages and epithelial cells. S. aureus toxin-associated pore-formation was essential for necroptosis, as cell death was blocked by exogenous K+ or dextran as well as by MLKL inhibition. To understand the role of necroptosis in S. aureus pneumonia, we used Ripk3-/- mice and mice treated with necrostatin-1s (Nec-1s), a potent inhibitor of RIPK1. Inhibition of necroptosis in a mouse model of pneumonia led to significantly improved outcome from S. aureus infection marked by increased bacterial clearance, preserved lung architecture, decreased inflammatory markers in the airway and retention of an anti-inflammatory macrophage population. In contrast, inhibiting necroptosis in vivo during skin infection led to worse outcome as determined by bacterial clearance and lesion sizes, which occurred in spite of the presence of neutrophils, macrophages and γδ T cells. Nec-1s-treated mice and Mlkl-/- mice had significantly larger lesions, increased cytokine response and more S. aureus recovered from the infected areas compared to control groups. We observed a similar outcome in Casp1/4-/- mice, which have limited ability to process IL-1β. Unlike Mlkl-/- mice, Ripk3-/- mice had improved outcome with increased bacterial clearance and decreased inflammation because of the effects of RIPK3 in activating the NLRP3 inflammasome and apoptosis during S. aureus skin infection. Casp1/4-/- immune cells showed a significant defect in their ability to kill S. aureus, whereas Mlkl-/- peritoneal exudate cells and bone marrow-derived macrophages did not. These results show that caspase-1 is essential for bacterial killing whereas necroptosis is necessary for regulating excessive inflammation. Similar to our findings in skin infection, inhibition of the executioner of necroptosis (using Mlkl-/- mice) or pyroptosis (using Casp1/4-/- mice) decreased survival in a mouse model of S. aureus sepsis. Ripk3-/- and wild type mice were equally resistant to S. aureus sepsis. Overall, these findings provide new insights into the complex roles of necrosome components in different tissues during S. aureus infection and may provide potential therapeutic targets to combat these infections.

O uso de metais na fabricação de implantes ortopédicos iniciou-se nas primeiras décadas do século XX. O aumento do uso de biomateriais implantáveis aumentam também os casos de infecção. A colonização da superfície do biomaterial pode ter início no momento da inserção do corpo estranho no organismo e geralmente é causada por microrganismos da microbiota da pele ou região adjacente ao implante. Este estudo teve por objetivo avaliar por métodos microbiológicos e microscópio eletrônico de varredura (MEV), a aderência bacteriana à superfície de aço inoxidável e liga de titânio de uso médico, bem como a molhabilidade da superfície destes metais. As bactérias usadas foram Staphylococcus epidermidis ATCC 12228 e Staphylococcus aureus ATCC 25923. Os discos de aço inoxidável (15,0 mm de diâmetro x 2,0 mm de espessura) e de liga de titânio (12,0 mm de diâmetro x 2,0 mm de espessura) foram inseridos, asséptica e separadamente, em tubos contendo 15,0 mL de caldo Mueller Hinton e 200,0 \'mü\'L de suspensão bacteriana da ordem de \'10 POT.8\' UFC/mL. Cada bactéria foi estudada individualmente. Os tubos foram incubados por 1, 6, 24, 48 e 72 horas sob agitação a 37 graus Celsius. Após os períodos de incubação, os discos foram retirados do caldo de cultura e submetidos ao banho de ultrassom em 5,0 mL de solução fisiológica 0,85% esterilizada. Deste líquido, foram realizadas diluições da ordem de \'10 POT.-1\' a \'10 POT.-4\' para a quantificação de células viáveis. Os valores foram expressos em UFC/mL. Para S. epidermidis sobre a liga de titânio, o número de células viáveis foi em 1 hora: 7,20 x \'10 POT.4\'; 6 horas: 3,90 x \'10 POT.6\'; 24 horas: 3,80 x \'10 POT.6\'; 48 horas: 9,70 x \'10 POT.6\' e 72 horas: 1,00 x \'10 POT.7\'. Sobre o aço inoxidável, o número de células viáveis foi em 1 hora: 3,00 x \'10 POT.3\'; 6 horas: 2,90 x \'10 POT.6\'; 24 horas: 3,20 x \'10 POT.6\'; 48 horas: 1,41 x \'10 POT.7\' e 72 horas: 1,88 x \'10 POT.7\'. Para S. aureus ) sobre a liga de titânio, o número de células viáveis foi em 1 hora: 2,00 x \'10 POT.3\'; 6 horas: 1,00 x \'10 POT.4\'; 24 horas: 3,10 x \'10 POT.4\'; 48 horas: 4,30 x \'10 POT.4\' e 72 horas: 5,80 x \'10 POT.3\'. Sobre o aço inoxidável, o número de células viáveis foi em 1 hora: 6,00 x \'10 POT.3\'; 6 horas: 2,00 x \'10 POT.3\'; 24 horas: 1,50 x \'10 POT.4\'; 48 horas: 3,20 x \'10 POT.5\' e 72 horas: 6,00 x \'10 POT.3\'. Ambas as superfícies metálicas foram caracterizadas como de média molhabilidade, onde a liga de titânio teve média \'+ OU -\' desvio padrão de 39,016 \'+ OU -\' 11,267 e o aço inoxidável 58,083 \'+ OU -\' 7,165. Tanto o S. aureus quanto o S. epidermidis aderiram às superfícies dos biomateriais estudados, como foi observado por meio de MEV. Com base nos resultados é possível concluir que os dois microrganismos são capazes de aderir a superfícies metálicas. Isto aumenta a preocupação quanto à patogênese das infecções relacionadas a implantes ortopédicos, uma vez que esses microrganismos estão presentes na pele humana e oferecem o risco de reações inflamatórias e infecção, promovendo a perda do implante para efetivar a cura. / The utilization of metals in the manufacture of orthopedic implants started in first decades of twentieth century. The increased use of implantable biomaterials increased also infection cases. Biomaterial surface colonization can start at the moment of foreign body insertion in the organism and is usually caused by microorganisms of skin microbiota or adjacent region to the implant. This study aimed to evaluate microbiological methods and scanning electron microscopy (SEM), the bacterial adhesion to surface of stainless steel and titanium alloy of medical use, as well as the surface wetability of these metals. The used bacteria were Staphylococcus epidermidis ATCC 12228 and Staphylococcus aureus ATCC 25923. The stainless steel (15,0 mm diameter x 2,0 mm thick) and titanium alloy (12,0 mm diameter x 2,0 mm thick) discs were inserted, aseptic and individually, into tubes containing 15,0 mL Mueller Hinton broth and 200,0 \'mü\'L of bacterial suspension with \'10 POT.8\' CFU/mL concentration. Each bacterium was individually studied. The tubes were incubated for 1, 6, 24, 48 and 72 hours under agitation at 37 Celsius degrees. After incubation periods, the discs were removed from culture broth and submitted to the ultrasound bath in 5,0 mL of sterile saline. From this liquid were realized dilutions of \'10 POT.-1\' to \'10 POT.-4\' to quantify the viable cells. Values were expressed in CFU/mL. S. epidermidis over titanium alloy viable cells number was in 1 hour: 7,20 x \'10POT.4\'; 6 hours: 3,90 x \'10 POT.6\'; 24 hours: 3,80 x \'10 POT.6\'; 48 hours: 9,70 x \'10 POT.6\' and 72 hours: 1,00 x \'10 POT.7\'. Over stainless steel viable cells number was in 1 hour: 3,00 x \'10 POT.3\'; 6 hours: 2,90 x \'10 POT.6\'; 24 hours: 3,20 x \'10 POT.6\'; 48 hours: 1,41 x \'10 POT.7\' and 72 hours: 1,88 x \'10 POT.7\'. To S. aureus over titanium alloy viable cells number was in 1 hour: 2,00 x \'10 POT.3\'; 6 hours: 1,00 x \'10 POT.4\'; 24 hours: 3,10 x \'10 POT.4\'; 48 hours: 4,30 x \'10 POT.4\' and 72 hours: 5,80 x \'10 POT.3\' and over stainless steel viable cells number was in 1 hour: 6,00 x \'10 POT.3\'; 6 hours: 2,00 x \'10 POT.3\'; 24 hours: 1,50 x \'10 POT.4\'; 48 hours: 3,20 x \'10 POT.5\' and 72 hours: 6,00 x \'10 POT.3\'. Both metal surfaces were characterized as medium wetability, where the contact angle of titanium alloy was mean \'+ OU -\' standard deviation 39,016 \'+ OU -\' 11,267 and stainless steel 58,083 \'+ OU -\' 7,165. Both S. aureus as S. epidermidis adhered to surfaces of biomaterials studied, as observed by SEM. Based on the results we concluded that two microorganisms are able to adhere to metal surfaces. This increases the concern about the pathogenesis of infections related to orthopedic implants, since these microorganisms are present in human skin and provide the risk of infection and inflammatory reactions, furthering implant loss to effective cure.

A infecção bacteriana é a principal complicação que um procedimento de artroplastia de quadril ou joelho pode apresentar. Mesmo após a incorporação de antibiótico (gentamicina) ao cimento ósseo, as taxas de infecções após este procedimento cirúrgico continuam gerando sérios prejuízos para o hospital e para o paciente. As principais bactérias envolvidas nas infecções relacionadas aos implantes ortopédicos são Pseudomonas aeruginosa, Staphylococcus aureus e Staphylococcus epidermidis. O objetivo deste trabalho foi avaliar a aderência e formação de biofilme de S. aureus, S. epidermidis e P. aeruginosa sobre o cimento ósseo polimetilmetacrilato (PMMA) com e sem antibiótico (gentamicina), de procedência nacional e internacional, por meio de microscópio eletrônico de varredura (MEV) e por cultura. Também, estimar quantitativamente as células viáveis recuperadas dos biofilmes formados. Foram produzidos discos de polimetilmetacrilato de 10,0 mm de diâmetro e 3,0 mm de espessura. Foram utilizadas cepas Pseudomonas aeruginosa - ATCC 27853, Staphylococcus epidermidis - ATCC 12228 e Staphylococcus aureus - ATCC 25932. Para este estudo foram utilizados corpos-de-prova de cimento ósseo de procedência nacional (BAUMER, CMM e BIOMECANICA) e internacional (BIOMET com gentamicina, BIOMET sem gentamicina e SIMPLEX). Biofilmes foram produzidos in vitro a partir da inoculação da suspensão bacteriana (\'10 POT.8\' unidades formadoras de colônia/mL) em Tryptic Soy Broth e incubados nos períodos de tempo de 1, 6, 24, 48, e 72 horas. Após os períodos de incubação os corpos-de-prova foram removidos do meio de cultura, lavados, sonicados e do sobrenadante realizadas diluições seriadas (\'10 POT.-1\' a \'10 POT.-5\'). A seguir, os corpos-de-prova foram preparados para observação por MEV. Os resultados de MEV mostraram bacilos e cocos aderidos e agrupados formando biofilme. Para P. aeruginosa: as contagens das células viáveis em média (UFC/mL) foram de 2,8 \'+ OU -\' 1,7 x \'10 POT.6\' (BAUMER), 1,7 \'+ OU -\' 0,9 x \'10 POT.6\' (BIOMECANICA), 1,7 \'+ OU -\' 0,7 x \'10 POT.6\' (CMM), 1,6 \'+ OU -\' 0,7 x \'10 POT.6\' (BIOMET sem gentamicina), 6,0 \'+ OU -\' 5,5 x \'10 POT.4\' (BIOMET com gentamicina) e 1,9 \'+ OU -\' 0,9 x \'10 POT.6\' (SIMPLEX); para S. epidermidis: 1,3 \'+ OU -\' 0,1 x \'10 POT.6\' (BAUMER), 1,5 \'+ OU -\' 0,2 x \'10 POT.6\' (BIOMECANICA), 2,3 \'+ OU -\' 1,7 x \'10 POT.6\' (CMM), 1,5 \'+ OU -\' 0,7 x \'10 POT.6\' (BIOMET sem gentamicina), 1,5 \'+ OU -\' 0,2 x \'10 POT.6\' (BIOMET com gentamicina) e 1,2 \'+ OU -\' 0,1 x \'10 POT.6\' (SIMPLEX); para S. aureus: 1,7 \'+ OU -\' 0,8 x \'10 POT.6\' (BAUMER), 1,6 \'+ OU -\' 0,7 x \'10 POT.6\' (BIOMECANICA), 1,4 \'+ OU -\' 0,6 x \'10 POT.6\' (CMM), 1,1 \'+ OU -\' 0,5 x \'10 POT.6\' (BIOMET sem gentamicina), 3,0 \'+ OU -\' 6,0 x \'10 POT.5\' (BIOMET com gentamicina) e 1,3 \'+ OU -\' 0,6 x \'10 POT.6\' (SIMPLEX), respectivamente. Os dados obtidos mostraram que o cimento ósseo de polimetilmetacrilato com e sem gentamicina não evitaram a aderência da Pseudomonas aeruginosa, Staphylococcus epidermidis e Staphylococcus aureus e formação de biofilme, como demonstrado pela MEV. Em conclusão, isto é um fator de risco para infecções. / The bacterial infection is the main complication of a procedure for hip or knee arthroplasty can present. Even after the addition of antibiotic (gentamicin) in the bone cement, the rates of infection after the surgical procedure continue causing serious damage to the hospital and the patient. The main bacteria involved in infections related to orthopedic implants are Pseudomonas aeruginosa, Staphylococcus aureus and Staphylococcus epidermidis. The objective of this study was to evaluate the adhesion and biolfilm formation of the S. aureus, S. epidermidis and P. aeruginosa on the bone cement polymethylmethacrylate (PMMA) with and without antibiotic (gentamicin) from national and international origin, by means scanning electron microscope (SEM) and by culture. Also, quantitatively estimate the viable cells recovered from biofilms formed. Discs of cement were produced from 10.0 mm in diameter and 3.0 mm thick. Strains used were Pseudomonas aeruginosa - ATCC 27853, Staphylococcus epidermidis - ATCC 12228 e Staphylococcus aureus - ATCC 25932. For this study we used coupons cement of national origin (Baumer, CMM and biomechanics) and international (BIOMET with gentamicin, BIOMET without gentamicin and SIMPLEX). Biofilms were produced in vitro from the inoculation of bacterial suspension (108 Colony-Forming Units/mL) in Tryptic Soy Broth and incubated for the time periods of 1, 6, 24, 48 and 72 hours. After the incubation periods of the coupons they were removed from the medium culture, washed, sonicated and serial dilutions of supernatant taken (\'10 POT.-1\' a \'10 POT.-5\'). Next, the coupons were prepared for observation by SEM. The results of SEM showed adherent cocci bacilli, and adhered to each other form a biofilm. For P. aeruginosa: the couting of viable cells on average (CFU/mL) were 2,8 \'+ OU -\' 1,7 x \'10 POT.6\' (BAUMER), 1,7 \'+ OU -\' 0,9 x \'10 POT.6\' (BIOMECANICA), 1,7 \'+ OU -\' 0,7 x \'10 POT.6\' (CMM), 1,6 \'+ OU -\' 0,7 x \'10 POT.6\' (BIOMET sem gentamicina), 6,0 \'+ OU -\' 5,5 x \'10 POT.4\' (BIOMET com gentamicina) e 1,9 \'+ OU -\' 0,9 x \'10 POT.6\' (SIMPLEX); para S. epidermidis: 1,3 \'+ OU -\' 0,1 x \'10 POT.6\' (BAUMER), 1,5 \'+ OU -\' 0,2 x \'10 POT.6\' (BIOMECANICA), 2,3 \'+ OU -\' 1,7 x \'10 POT.6\' (CMM), 1,5 \'+ OU -\' 0,7 x \'10 POT.6\' (BIOMET sem gentamicina), 1,5 \'+ OU -\' 0,2 x \'10 POT.6\' (BIOMET com gentamicina) e 1,2 \'+ OU -\' 0,1 x \'10 POT.6\' (SIMPLEX); para S. aureus: 1,7 \'+ OU -\' 0,8 x \'10 POT.6\' (BAUMER), 1,6 \'+ OU -\' 0,7 x \'10 POT.6\' (BIOMECANICA), 1,4 \'+ OU -\' 0,6 x \'10 POT.6\' (CMM), 1,1 \'+ OU -\' 0,5 x \'10 POT.6\' (BIOMET sem gentamicina), 3,0 \'+ OU -\' 6,0 x \'10 POT.5\' (BIOMET com gentamicina) e 1,3 \'+ OU -\' 0,6 x \'10 POT.6\' (SIMPLEX), respectively. The data showed that of polymethylmethacrylate bone cement with and without gentamicin did not prevent the adhesion of Pseudomonas aeruginosa, Staphylococcus epidermidis and Staphylococcus aureus and formation of biofilms, as demonstrated by SEM. In conclusion, this is risk factor for infections.

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