La présence de bactéries dans le sang, un milieu normalement stérile, peut avoir des conséquences graves voire fatales pour l’organisme atteint. Afin de diagnostiquer au plus tôt cette infection, appelée bactériémie, et ainsi administrer le traitement adéquat, il est nécessaire d’identifier les microorganismes isolés à partir du sang. Mais, la nature complexe de ce fluide biologique, associée à la faible charge bactérienne, parfois inférieure à 1 UFC par millilitre de sang ont des conséquences sur les méthodes pouvant être utilisées pour l’identification des bactéries. La plupart d’entre elles ont donc recours à une première étape, l’hémoculture, au cours de laquelle les microorganismes présents dans le prélèvement sanguin de volume important (20-30 mL) vont se multiplier. Leur croissance est facilitée par la dilution du sang dans des milieux de culture dédiés à cette étape particulière. C’est seulement ensuite que l’identification peut débuter. Elle nécessite encore entre 2 et 48 heures et parfois plus, selon les moyens à disposition et les microorganismes impliqués. Réduire considérablement le temps nécessaire à l’identification aurait pourtant des retombées bénéfiques à l’échelle du patient mais aussi plus globalement en réduisant les coûts associés à cette infection et en limitant la pression de sélection exercée par l’emploi d’antibiotiques à large spectre.Au cours de ce travail, l’évaluation d’une stratégie basée sur l’identification des bactéries lors de leur multiplication dans le milieu d’hémoculture est donc proposée. Elle repose sur l’observation en temps réel et sans marquage par Résonance Plasmonique de Surface par imagerie (SPRi) des interactions entre les bactéries et des ligands déposés à la surface d’un capteur. Dans un premier temps, des ligands alternatifs aux anticorps parmi lesquels figurent les aptamères, des protéines de l’immunité innée et la vancomycine sont testés. Suite à cette étude, les anticorps ont été retenus pour poursuivre ce travail. Leur emploi n’est cependant pas dénué de difficultés lorsqu’il s’agit de détecter spécifiquement Staphylococcus aureus, choisi comme l’un des modèles expérimentaux. En effet, la présence de protéine A chez cette bactérie est à l’origine d’interférences sur les immunoglobulines. Différentes stratégies pour s’affranchir de ces effets ont été évaluées, comme le clivage enzymatique des anticorps ou l’emploi d’anticorps de poule pour lesquels la protéine A n’a pas d’affinité. Ces essais aboutissent à des résultats encourageants en milieu de culture simple. L’ajout de sérum humain au milieu a soulevé de nouveaux problèmes pour la détection de cette bactérie. Les résultats montrent qu’en interagissant avec des constituants de l’échantillon sanguin, dont les anticorps, S. aureus devient indétectable par une biopuce à anticorps. Une discussion des moyens possibles pour lever cette inhibition est ensuite proposée. Des expériences de détection d’une autre bactérie, Salmonella enterica sérovar Enteritidis pour laquelle nous disposons d’un anticorps hautement affin et spécifique ont alors été entreprises afin de conclure sur l’employabilité du dispositif dans des conditions proches d’une hémoculture. Des interférences affectant différentiellement les anticorps selon leur point isoélectrique ont ainsi été mises en évidences et l’implication de l’anticoagulant (polyanéthole sulfonate de sodium, SPS) présent dans les milieux d’hémoculture a été démontrée. La résolution partielle de ce problème a finalement permis la détection de 1 UFC.mL-1 de sang dans 32 mL au total démontrant ainsi la possibilité de détecter spécifiquement une bactérie dans des conditions proches d’une hémoculture. / The presence of bacteria in the blood, a normally sterile environment, can cause dramatic consequences for an organism. In order to diagnose this infection, called bacteremia, the identification of the microorganism present in blood must be performed. Furthermore, proper diagnosis enables the administration of a suitable antibiotic therapy. Blood complexity as well as the low bacterial load, usually lower than 1 CFU.mL-1, make the diagnosis of this infection quite challenging. Indeed, most identification methods begin only after the blood culture turns positive due to their insufficient sensitivity. For this they require incubation of a large blood sample volume (20 – 30 mL) in specific culture media that allows bacterial growth above their detection limit. Therefore, its increases considerably the time of diagnosis, which usually takes between 2 and 48 hours and sometimes even more time after blood culture positivity depending on the method and the microorganism present in blood. A reduction of the time required for identification would have a positive impact for both the patient and the healthcare systems by reducing selective pressure on resistant bacteria and hospitalization costs by giving proper treatment faster.In this work, the evaluation of a new strategy based on the identification of bacteria during their multiplication in the blood culture is presented. This method is based on Surface Plasmon Resonance imaging (SPRi) which enables real time and label-free measurements of interactions occurring between bacteria and specific probes. Alternative ligands like aptamers, innate immune proteins and vancomycin have been tested. Following this study antibodies have been chosen as the major specific probes in this work. Nonetheless, the presence of the staphylococcal protein A leads to false-positive results in all immunoglobulin G (IgG). Enzymatic cleavage to remove the constant fragment of antibody where protein A interacts and the use of chicken antibodies (IgY) for which protein A has no affinity have been evaluated. Both methods allow to get rid of protein A interactions in pure culture media. But the presence of human serum in the media results in the total loss of signal. Our results show that interactions between blood components and staphylococcal proteins exposed at the bacterial surface, including the interactions between protein A and circulating antibodies, are responsible for this phenomenon. Solutions to alleviate this inhibition are discussed and tested. Detection experiments of another bacterial model, Salmonella enterica serovar Enteritidis in blood culture media are presented. The crucial role played by the anticoagulant Sodium Polyanethole Sulfonate in non-specific interactions on antibodies is demonstrated. These interactions leading to a total loss of specificity for some antibodies are influenced by the isoelectric point (pI) of the probes which interact with this anionic compound and then attract blood components. After the partial resolution of this issue, we show the feasibility of detecting less than one bacteria per blood milliliter in a total volume of 32 milliliters, conditions close to real blood culture.

In einer sächsischen Milchviehanlage mit etwa 1800 Milchkühen, deren Tankmilchzellzahl, infolge vermehrten Auftretens von Euterinfektionen mit S. aureus als Leitkeim längerfristig über 300.000 Zellen/ml aufwies, wurden zwei Vakzinen eingesetzt. Die Erwartungshaltung lautete, dass mit den Impfungen die Inzidenz- und Prävalenzraten von S. aureus- und KNS-bedingten Mastitiden bei Färsen und bei Kühen bis zur Geburt und auch danach sinken. Es wurde vor allem erwartet, dass bei den geimpften Tieren die Zellzahlen langfristig erniedrigt bleiben und sich folglich die Eutergesundheit durch die Vakzinationen verbessert. Anhand der Gesamtgemelkszellzahlen (GZZ) der letzten drei Milchleistungsprüfungen (MLP) a. p. und der zytobakteriologischen Befunde einer Beprobung auf Viertelebene wurden die Kühe (n=416) in Statusgruppen eingeteilt. In Statusgruppe 2 befanden sich eutergesunde Kühe (n=112). Tiere (n=146) mit moderat erhöhten Viertel- (VZZ) und GZZ, die auf mindestens einem Viertel bakteriologisch positiv waren, wurden in Statusgruppe 3 zusammengefasst. Die Statusgruppe 4 bildeten Kühe (n=158), die durch stark erhöhte GZZ und VZZ charakterisiert waren und ggf. bakteriologisch positiv waren. Färsen (n=181) wurden in Statusgruppe 1 zusammengefasst. Alle Tiere mussten klinisch gesund sein, Färsen sollten eutergesund erscheinen. Als Impfstoffe wurden Startvac® (HIPRA Deutschland GmbH, Düsseldorf), eine kommerzielle Vakzine gegen S. aureus, KNS, Escherichia coli und coliforme Keime, sowie eine bestandsspezifische Vakzine (Bestvac Rind Mastitis®, IDT Biologika GmbH, Dessau-Rosslau) basierend auf S. aureus-Isolaten aus Mastitismilchen des Bestandes eingesetzt. Nach dem Zufallsprinzip wurden die Tiere innerhalb der Statusgruppen den Impfgruppen oder der Kontrollgruppe zugeteilt. Die erste Vakzination wurde einen Tag nach dem Trockenstellen vorgenommen (bei Färsen an vergleichbaren Trächtigkeitstagen), die zweite Vakzination erfolgte ca. 31 Tage vor dem errechneten Kalbedatum. Um den 53. Tag p. p. fand die dritte Impfung statt. Zytobakteriologische Beprobungen aller Tiere auf Viertelebene wurden am Tag 5 sowie am Tag 52 p. p. vorgenommen. Außerdem wurden während der Laktation die monatlichen MLP-Daten sowie jene zu tierärztlichen Behandlungen der in der Studie befindlichen Kühe sowie Abgänge und Abgangsursachen erfasst. Innerhalb der Statusgruppen gab es zwischen den Vakzinationsgruppen und den Kontrolltieren bezogen auf die VZZ zu den Beprobungszeitpunkten 5 und 52 sowie auf die GZZ aus den MLPs der gesamten Laktation keine nennenswerten Unterschiede. Die Erregerprävalenzen zu den genannten Zeitpunkten nebst deren Verlauf und jene der zytobakteriologischen Diagnosen, erbrachten nur punktuell signifikante Unterschiede, die in der Summe aber keine anhaltende Tendenz erkennen ließen, die auf das bessere Abschneiden einer Vakzinationsgruppe im Vergleich zur Kontrollgruppe hindeuten würde. Zum gleichen Ergebnis gelangen die Auswertungen der Inzidenzraten, klinische Mastitisdaten und jene der Abgangsursachen im Anschluss an die Vakzinationen. Zusammenfassend hatte der Einsatz der bestandsspezifischen Vakzine Bestvac Rind Mastitis® sowie des Impfstoffes Startvac® mit EU-Zulassung bezogen auf die Zellzahlenentwicklung, die Inzidenz- und Prävalenzraten von S. aureus und KNS, die Behandlungsdauer und den Schweregrad von Mastitiden sowie die Heilungsraten im Vergleich zur Placebo-Gruppe in keiner der Statusgruppen erkennbare positive Effekte auf die Eutergesundheit.

L’analyse microbiologique pour confirmer l’absence de bactéries dans des échantillons biologiques normalement sains, comme le sang, est une routine dans de nombreux laboratoires. En effet, la présence de bactéries dans le sang, appelée bactériémie, peut avoir des conséquences très graves, voire mortelles pour le patient. Le protocole standard pour la détection des bactériémies repose jusqu’ici sur l’enrichissement des échantillons sanguins prélevés sur les patients lors de l’hémoculture, afin d’obtenir une population suffisante pour analyse. La lenteur de ce procédé retarde ainsi de parfois plusieurs jours le diagnostic et donc l’adaptation du traitement antibiotique administré au patient. Ces dernières décennies, des techniques comme l’identification par spectrométrie de masse ou les analyses moléculaires, ont permis de diminuer le délai requis pour identifier les pathogènes en cause. Dans ce contexte, l’emploi de biocapteurs est également une alternative. Ce travail propose d’inclure des sondes à large spectre dans un capteur optique par imagerie SPR (résonance de plasmons de surface). Ce système est déjà développé pour la reconnaissance spécifique de pathogènes au cours de leur croissance dans le sang. Les nouveaux ligands proposés et évalués sont les peptides antimicrobiens (PAM). Ces courts peptides cationiques et amphiphiles, présentent l’avantage d’un large spectre d’interaction couplé à une haute stabilité (chimique, thermique et séchage) comparativement aux anticorps employés jusqu’ici. Leur immobilisation sur des prismes SPRI permet d’évaluer simultanément l’affinité de plusieurs PAM à la même souche bactérienne. Les biocapteurs ainsi préparés ont permis de détecter des souches pathogènes d’Escherichia coli et Staphylococcus aureus en milieu de culture simple, comme en plasma et en sang dilué au milieu d’hémoculture. Le système obtenu permet la détection des pathogènes présents à une concentration initiale de l’ordre de 1 UFC.ml-1, en moins de 24 heures et quel que soit le milieu. Enfin, la mise en place d’analyses statistiques multidimensionnelles a abouti à une classification cohérente des espèces ciblées en milieu simple, comme en sang. Ces résultats montrent le potentiel de ce système pour parvenir à développer un biocapteur à large spectre capable à la fois de détecter mais aussi d’identifier par affinité croisée des pathogènes bactériens. / Microbiological analysis to confirm the absence of bacteria in normally sterile biological samples, such as blood, is routine in many laboratories. The presence of bacteria in blood, called bacteremia, can have very serious, and even fatal consequences for the patient. So far, the standard protocol for their detection has been based on the enrichment of blood samples collected from patients, thanks to blood culture, in order to obtain a sufficient population for analysis. These procedures are time consuming which sometimes lead to delays in diagnosis and subsequent adaptation of antibiotic treatments by several days. In recent decades, techniques such as mass spectrometry identification or molecular analyses have reduced the time required to identify the pathogens involved. In this context, the use of biosensors is another promising alternative. This work proposes to include wide spectrum probes in an optical sensor using SPR imaging (surface plasmon resonance). This system is already developed for the specific recognition of pathogens during their growth in the blood. The new ligands we propose to evaluate are antimicrobial peptides (AMP). These short, cationic and amphiphilic peptides have the advantage of having a broad spectrum of interaction with bacteria, coupled with high stability (chemical, thermal and drying), especially compared to the antibodies used so far in this technique. Their immobilization on SPRI prisms allows the simultaneous evaluation of the affinity of several AMP to the same bacterial strain. The biosensors based on AMP were able to detect pathogenic strains of Escherichia coli and Staphylococcus aureus in simple culture medium, such as plasma and diluted blood in blood culture medium. The system obtained allows the detection of pathogens present at an initial concentration of about 1 CFU.ml-1, in less than 24 hours and in all assayed media. Finally, the implementation of multidimensional statistical analyses has resulted in a consistent classification of targeted species, in simple culture medium, such as blood. These results show the potential of this system to develop a wide-spectrum biosensor capable of both detecting and cross-referencing bacterial pathogens.

Le traitement antibiotique local de l'infection osseuse par le polyméthacrylate de méthyle (PMMA), chargé de gentamicine ou de tobramycine, montre actuellement des limites. Ses inconvénients sont liés à la non résorbabilité du PMMA et à la rétention d'une grande partie de l'antibiotique intégré au PMMA. L’association fréquente à l’infection de pertes de substance osseuse a favorisé la recherche de vecteurs d’antibiothérapie locale, alternative au PMMA, parmi les substituts de comblement osseux résorbables et ostéoconducteurs. Les ciments phosphocalciques (CPC) pourraient devenir parmi les plus performants dans cette utilisation. Ils sont biocompatibles et offrent avec le Dicalcium Phosphate-ß-Tricalcium Phosphate (DCPD-ß-TCP), un CPC, la possibilité d'obtention d'un mélange DCPD-ß-TCP-gentamicine à une température de 43°C n'altérant pas l'antibiotique, contrairement aux céramiques phosphocalciques qui sont fabriquées par frittage à très haute température. Le but de notre travail était de tester in vitro (élution d’antibiotique) et in vivo (essai de traitement d'ostéite expérimentale) le DCPD-ß-TCP-gentamicine comme alternative possible au PMMA-gentamicine. [...] / Local antibiotic treatment of osteomyelitis is based on the use of gentamicin- (or tobramycin-) loaded polymethylmethacrylate (PMMA). These two aminoglycosides are effective against most cultured orthopedic microorganisms, including Staphylococcus aureus, the most frequent cause of infection. The extensive use of PMMA as a Local Antibiotic Delivery System (LADS) has various disadvantages. Firstly, only a small proportion (about 5 to 17%) of the antibiotic is released by the cement (trapping effect). Secondly, the most significant problem is that PMMA is not resorbable and presents a physical obstacle to osteogenesis. A second surgical operation is therefore always required to remove the PMMA and to fill the cavity caused by bone loss with a bone graft or a synthetic substitute. Several absorbable synthetic substitutes, such as calcium phosphate ceramics, calcium sulfate, and polymers of polylactic-polyglycolic acids, have been investigated as antibiotic carriers. These synthetic substitutes are largely underused as LADS in clinical practice. Polymers are not perfectly biocompatible, and ceramics provide a burst release of antibiotics as a consequence of their manufacturing techniques (Antibiotic adsorption onto the carrier, after sintering of the carrier at high temperature, 1000-1200°C). We have developed a possible alternative to gentamicin loaded-PMMA for local treatment of osteomyelitis in the form of novel calcium phosphate cement (CPC): dicalcium phosphate dihydrate-β-tricalcium phosphate (DCPD-β-TCP). The biocompatibility of such a cement has been demonstrated experimentally and has been clinically confirmed for the treatment of burst fractures and for filling bone cavities in osteoporotic fractures. DCPD-ß-TCP is made in granules from 2 to 3 mm in diameter to avoid the superficial ―creeping substitution‖ observed when DCPD-β-TCP is used as a cement block. [...]

No presente estudo, um conjunto de três experimentos foram conduzidos com o objetivo de avaliar a ocorrência de Staphylococcus aureus e Listeria monocytogenes em três lacticínios (A, B e C) localizados na região sudeste do Brasil de dezembro 2013 a abril de 2015 (Experimento 1), a eficiência do tratamento com ácido peracético (APA) e jato de plasma a frio (PF) para inativar os isolados em diferentes tempos (Experimento 2) e a capacidade dos isolados produzir biofilmes na superfície de poliestireno e de aço inoxidável, juntamente com inativação e remoção de células aderidas pelo APA (Experimento 3). No Experimento 1, foram analisadas amostras de leite e queijo, superfícies com e sem contato com alimentos. L. monocytogenes foi isolada em apenas uma amostra (0,3%, N = 349) de ralo no laticínio B, enquanto seis (1,7%, n = 349) S. aureus foram isolados de luvas de manipuladores em laticínio A, salmoura no laticínio B e superfície do queijo, utensílio, bota e mão esquerda de trabalhador no lacticínio C. Apesar das incidências desses dois agentes patogênicos de origem alimentar nos lacticínios avaliados foram baixo, sua presença também indica a necessidade de controle estratégias para impedir a sua persistência e contaminação cruzada. No Experimento 2, tratamento com APA (0,5%) e jato de PF foram aplicados diretamente sobre suspensões de isolados de S. aureus e L. monocytogenes. A inativação bacteriana (aproximadamente de 7 ciclos log) foi alcançada em 120 seg. com o tratamento com APA para todos os isolados, enquanto que o tratamento com plasma a frio reduziu aproximadamente 2 ciclos log nas superficies. Outros estudos usando tratamentos de plasma a frio mais longos são necessários para a total descrição da cinética desta tecnologia para a inativação de importantes patógenos de origem alimentar. No Experimento 3, o tratamento com APA (0,5%) em diferentes tempos (0-controle, 15, 30, 60 e 120 seg.) foi avaliada para a remoção de células aderidas de quatro isolados de S. aureus e um isolado de L. monocytogenes em microplacas de poliestireno, assim como para a inativação de biofilmes dos isolados em aço inoxidável. O tratamento com APA removeu (p<0,05) células aderidas de todos os isoladoas estudados S. aureus da superfície, sem diferenças (p> 0,05) no indice de formação de biofilmes nos tempos de tratamento. No entanto, nenhum efeito (p> 0,05) foi observado em células aderidas de L. monocytogenes. A microscopia de epifluorescência mostrou que todas as bactérias testadas foram parcialmente e completamente inativadas após 15 seg e 30 seg. respectivamente. Os resultados indicam um potencial para a utilização de APA contra biofilmes formados por S. aureus e L. monocytogenes, e da necessidade de novos estudos com a PF para determinar os parâmetros ideais para a inativação dos patógenos de origem alimentar. / In the present study, a set of three experiments were conducted aiming to evaluate the occurrence of Staphylococcus aureus and Listeria monocytogenes in three dairy plants (A, B and C) from Southeast region of Brazil from December 2013 to April 2015 (Experiment 1), the efficiency of peracetic acid (PAA) and cold plasma (CP) jet treatment to inactivate the isolates at different times (Experiment 2) and the ability of the isolates to produce biofilms on polystyrene and stainless steel surface, along with inactivation and removal of biofilms by PAA (Experiment 3). In Experiment1, samples of milk and cheese, food contact surfaces and non-food contact were analyzed. L. monocytogenes was isolated in only one sample (0.3%, N=349) of drain sponge swab in dairy plant B, while 6 (1.7%, N=349) S. aureus strains were isolated from handlers\' glove in dairy plant A, brine in dairy plant B and cheese surface, cheese utensil, worker\'s boot and worker\'s left hand in dairy plant C. Although the incidences of those two food-borne pathogens in the dairy plants evaluated were low, their presence also indicates the need for control strategies to prevent their persistence and cross-contamination. In Experiment 2, PAA (0.5%) and CP jet treatment were applied directly on suspensions of S. aureus and L. monocytogenes strains. Reduction of bacterial load (nearly 7 log cycles) was achieved with 15 sec. of PAA treatment of all strains, whereas CP treatment reduced approximately 2 log cycles after 2 min. Hence, plasma treatment has a potential for reducing the bacterial load on surfaces, although further studies using longer CP treatment times are necessary to fully describe the kinetics of this technology for inactivation of important food pathogens. In Experiment 3, PAA (0.5%) treatment at different times (0-control, 15, 30, 60 and 120 sec.) was evaluated for removing of adherent cells of 4 strains of S. aureus and one strain of L. monocytogenes on polystyrene plates, as well as for inactivation of biofilms of those strains on stainless steel. PAA treatment removed (p<0.05) all the S. aureus cells from the surface, with no difference (p>0.05) in the reduction of the biofilm-forming index at the treatment times. However, no effect (p>0.05) was observed on L. monocytogenes adhered cells. Epifluorescence microscopy showed that all bacterial strains tested were partially and completely inactivated after 15 sec. and 30 sec., respectively. Results indicate a potential use of PAA against biofilms formed by S. aureus and L. monocytogenes, and the need of further studies with CP to determinate the ideal parameters for inactivation of food-borne pathogens.

Submitted by Celso Magalhaes (celsomagalhaes@ufrrj.br) on 2017-10-03T16:25:38Z No. of bitstreams: 1 2016 - Viviane Figueira Marques.pdf: 1883187 bytes, checksum: 9996d5ea41ecd4f93424c808c4d82ba6 (MD5) / Made available in DSpace on 2017-10-03T16:25:38Z (GMT). No. of bitstreams: 1 2016 - Viviane Figueira Marques.pdf: 1883187 bytes, checksum: 9996d5ea41ecd4f93424c808c4d82ba6 (MD5) Previous issue date: 2016-03-02 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Staphylococcus spp. plays an important role in the etiology of bovine mastitis. Staphylococcus aureus is considered the most relevant specie due to the production of virulence factors such as ?slime?, which is required for biofilm formation. This study aimed to detect the phenotypic expression of the biofilm formation in 20 S. aureus isolates from bovine mastitis, to detect and quantify the expression of genes involved in its production and regulation, as well as to detect the phenogenotypic resistance to beta-lactam in order to evaluate the possible relation between biofilm production and antimicrobial resistance. The isolates were characterized by MALDI-TOF and phenogenotypic identification assays. Also they were submitted to the phenotypic tests to evaluate biofilm production and the susceptibility to beta-lactams. Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC) and Minimum Inhibitory Concentration in the Biofilm (MICB) were determined to three isolates presenting distinct biofilm production. Futherly, a PCR for the detection of ?slime? production genes (icaA and icaD), Bap protein (bap), beta-lactamase (blaZ) and protein altered penicillin-binding (mecA). Also, the Agr system was typified (agr I, agr II, agr III and agr IV) and its regulator (agr RNAIII) was detected. Scanning Electron Microscopy (SEM) was performed to determine the most suitable time interval for biofilm analysis. Real-time PCR (qPCR) was performed at the chosen times to quantify the expression of icaA, icaD and hld genes in the three studied isolates. All 20 isolates were biofilm producers and most presented icaA and icaD genes. Only one isolate presented the bap gene. The agr gene type II presented a prevalence of 70%. The SEM showed gradual changes in bacterial arrangement during the biofilm formation along the phases of the growth curve. The peak was reached at the stationary phase. Transcriptional analysis revealed increased expression of ica genes at 8 h of growth and of hld at 24 h. However, for the N-365 strain the ica expression was of low yield. For this study, the penicillin resistance was related to the production of beta-lactamase otherwise the high MBC detected for cefoxitin may be associated to biofilm protection, evidentiated by the fact that the isolates have MICB values higher than MICs tested for planktonic cells / Staphylococcus spp. tem papel importante na etiologia da mastite bovina. Staphylococcus aureus ? considerada a esp?cie mais relevante devido ? produ??o de fatores de virul?ncia, tais como ?slime?, o que favorece a forma??o do biofilme. O presente trabalho teve por objetivo detectar a express?o fenot?pica da forma??o de biofilme em 20 isolados de S. aureus oriundos de mastite bovina, detectar e quantificar a express?o dos genes envolvidos na sua produ??o e regula??o, al?m de detectar a resist?ncia fenogenot?pica aos beta-lact?micos para avalia??o da poss?vel rela??o da produ??o de biofilme com a resist?ncia antimicrobiana. Os isolados foram caracterizados atrav?s de testes fenogenot?picos e MALDI-TOF, submetidos ?s provas fenot?picas de detec??o da forma??o de biofilme e avalia??o da suscetibilidade aos beta-lact?micos. A Concentra??o Inibit?ria M?nima (CIM), Concentra??o Bactericida M?nima (CBM) e a Concentra??o Inibit?ria M?nima no Biofilme (CIMB) foram determinadas para tr?s isolados selecionados com base na varia??o da intensidade da produ??o de biofilme. Posteriormente, todos os isolados foram submetidos ? t?cnica de PCR para detec??o dos genes de produ??o de ?slime? (icaA e icaD), prote?na Bap (bap), beta-lactamase (blaZ) e prote?na ligante de penicilina alterada (mecA). Al?m de detec??o do sistema regulador Agr (agr RNAIII) e da tipifica??o do sistema Agr (agr I, agr II, agr III e agr IV). Foi realizada Microscopia Eletr?nica de Varredura (MEV) para determinar o intervalo de tempo mais adequado para a an?lise do biofilme. A PCR em tempo real (qPCR) foi realizada nos tempos selecionados para quantificar a express?o dos genes icaA, icaD e hld em tr?s isolados com produ??o variada de biofilme. Todos os isolados foram produtores de biofilme e a maioria apresentou os genes icaA e icaD. Apenas um isolado apresentou o gene bap. O gene agr tipo II mostrou preval?ncia de 70%. A MEV mostrou mudan?as graduais no arranjo bacteriano durante a forma??o de biofilme ao longo das fases da curva de crescimento que atingiu seu pico de forma??o na fase estacion?ria. A an?lise transcricional evidenciou maior express?o dos genes ica no tempo de 8 h de crescimento e hld em 24 h. Contudo, a cepa N-365 mostrou baixa produ??o dos genes ica. Para este estudo, a resist?ncia ? penicilina foi relacionada com a produ??o de beta-lactamase, enquanto a elevada CBM detectada para cefoxitina pode estar associada ? prote??o que o biofilme oferece, epis?dio evidenciado pelo fato dos isolados apresentarem valores de CIMB superiores aos CIMs testados para as c?lulas planct?nicas.

La pyocyanine (PYO) est une phénazine de couleur bleu-vert, produite spécifiquement par la bactérie pathogène opportuniste Pseudomonas aeruginosa (Pa). La toxicité aérobie de la PYO envers les cellules de mammifères, les levures et les bactéries a été décrite de longue date, mais la compréhension des mécanismes d'action est encore lacunaire, en particulier en conditions de limitation en O2 (conditions rencontrées dans le contexte infectieux). De plus, il a récemment été montré que la PYO peut apporter des effets bénéfiques pour la souche productrice en hypoxie. Au cours de ce travail, nous avons réexaminé les effets de la PYO sur un large panel de bactéries dont son propre producteur (Pa) ainsi que sur un modèle cellulaire eucaryote Saccharomyces cerevisiae exposées à différentes tensions en O2. Nos données suggèrent que la toxicité aérobie de la PYO envers S. cerevisiae est multifactorielle, impliquant à la fois une interaction avec le complexe III de la chaîne respiratoire et l'induction d'un stress oxydatif. Pour la première fois, nous avons mis en évidence une toxicité de la PYO exacerbée en anaérobiose chez un eucaryote (S. cerevisiae). Le mécanisme d'action impliquerait le PYO radical. Nous avons également montré que la PYO peut inhiber la croissance aérobie et anaérobie des microorganismes concurrents, plus particulièrement S. aureus en bloquant le complexe III de la chaîne respiratoire. A l'inverse, la PYO peut stimuler la respiration de Pa surtout dans les conditions mimant le contexte infectieux (hypoxie, vie ralentie). Le complexe III et/ou les oxydases terminales cbb3 serait impliqué favorablement. En conclusion, la PYO jouerait à la fois un rôle de poison hypoxique mais aussi un rôle de navette redox bénéfique pour la survie et la virulence de Pa en hypoxie.

No presente estudo, um conjunto de três experimentos foram conduzidos com o objetivo de avaliar a ocorrência de Staphylococcus aureus e Listeria monocytogenes em três lacticínios (A, B e C) localizados na região sudeste do Brasil de dezembro 2013 a abril de 2015 (Experimento 1), a eficiência do tratamento com ácido peracético (APA) e jato de plasma a frio (PF) para inativar os isolados em diferentes tempos (Experimento 2) e a capacidade dos isolados produzir biofilmes na superfície de poliestireno e de aço inoxidável, juntamente com inativação e remoção de células aderidas pelo APA (Experimento 3). No Experimento 1, foram analisadas amostras de leite e queijo, superfícies com e sem contato com alimentos. L. monocytogenes foi isolada em apenas uma amostra (0,3%, N = 349) de ralo no laticínio B, enquanto seis (1,7%, n = 349) S. aureus foram isolados de luvas de manipuladores em laticínio A, salmoura no laticínio B e superfície do queijo, utensílio, bota e mão esquerda de trabalhador no lacticínio C. Apesar das incidências desses dois agentes patogênicos de origem alimentar nos lacticínios avaliados foram baixo, sua presença também indica a necessidade de controle estratégias para impedir a sua persistência e contaminação cruzada. No Experimento 2, tratamento com APA (0,5%) e jato de PF foram aplicados diretamente sobre suspensões de isolados de S. aureus e L. monocytogenes. A inativação bacteriana (aproximadamente de 7 ciclos log) foi alcançada em 120 seg. com o tratamento com APA para todos os isolados, enquanto que o tratamento com plasma a frio reduziu aproximadamente 2 ciclos log nas superficies. Outros estudos usando tratamentos de plasma a frio mais longos são necessários para a total descrição da cinética desta tecnologia para a inativação de importantes patógenos de origem alimentar. No Experimento 3, o tratamento com APA (0,5%) em diferentes tempos (0-controle, 15, 30, 60 e 120 seg.) foi avaliada para a remoção de células aderidas de quatro isolados de S. aureus e um isolado de L. monocytogenes em microplacas de poliestireno, assim como para a inativação de biofilmes dos isolados em aço inoxidável. O tratamento com APA removeu (p<0,05) células aderidas de todos os isoladoas estudados S. aureus da superfície, sem diferenças (p> 0,05) no indice de formação de biofilmes nos tempos de tratamento. No entanto, nenhum efeito (p> 0,05) foi observado em células aderidas de L. monocytogenes. A microscopia de epifluorescência mostrou que todas as bactérias testadas foram parcialmente e completamente inativadas após 15 seg e 30 seg. respectivamente. Os resultados indicam um potencial para a utilização de APA contra biofilmes formados por S. aureus e L. monocytogenes, e da necessidade de novos estudos com a PF para determinar os parâmetros ideais para a inativação dos patógenos de origem alimentar. / In the present study, a set of three experiments were conducted aiming to evaluate the occurrence of Staphylococcus aureus and Listeria monocytogenes in three dairy plants (A, B and C) from Southeast region of Brazil from December 2013 to April 2015 (Experiment 1), the efficiency of peracetic acid (PAA) and cold plasma (CP) jet treatment to inactivate the isolates at different times (Experiment 2) and the ability of the isolates to produce biofilms on polystyrene and stainless steel surface, along with inactivation and removal of biofilms by PAA (Experiment 3). In Experiment1, samples of milk and cheese, food contact surfaces and non-food contact were analyzed. L. monocytogenes was isolated in only one sample (0.3%, N=349) of drain sponge swab in dairy plant B, while 6 (1.7%, N=349) S. aureus strains were isolated from handlers\' glove in dairy plant A, brine in dairy plant B and cheese surface, cheese utensil, worker\'s boot and worker\'s left hand in dairy plant C. Although the incidences of those two food-borne pathogens in the dairy plants evaluated were low, their presence also indicates the need for control strategies to prevent their persistence and cross-contamination. In Experiment 2, PAA (0.5%) and CP jet treatment were applied directly on suspensions of S. aureus and L. monocytogenes strains. Reduction of bacterial load (nearly 7 log cycles) was achieved with 15 sec. of PAA treatment of all strains, whereas CP treatment reduced approximately 2 log cycles after 2 min. Hence, plasma treatment has a potential for reducing the bacterial load on surfaces, although further studies using longer CP treatment times are necessary to fully describe the kinetics of this technology for inactivation of important food pathogens. In Experiment 3, PAA (0.5%) treatment at different times (0-control, 15, 30, 60 and 120 sec.) was evaluated for removing of adherent cells of 4 strains of S. aureus and one strain of L. monocytogenes on polystyrene plates, as well as for inactivation of biofilms of those strains on stainless steel. PAA treatment removed (p<0.05) all the S. aureus cells from the surface, with no difference (p>0.05) in the reduction of the biofilm-forming index at the treatment times. However, no effect (p>0.05) was observed on L. monocytogenes adhered cells. Epifluorescence microscopy showed that all bacterial strains tested were partially and completely inactivated after 15 sec. and 30 sec., respectively. Results indicate a potential use of PAA against biofilms formed by S. aureus and L. monocytogenes, and the need of further studies with CP to determinate the ideal parameters for inactivation of food-borne pathogens.

Background: Bacterial contamination of surfaces with methicillin-resistant Staphylococcus aureus (MRSA) is a serious problem in the hospital environment and is responsible for significant nosocomial infections. The pathogenic contaminants form biofilms, which are difficult to treat with routine biocides. Thus, a continuous search for novel disinfection methods is essential for effective infection control measures. This demonstration of a novel technique for the control of virulent pathogens in planktonic form as well as in established biofilms may provide a progressive alternative to standard methodology. Methods: We evaluated a novel technique of normal atmospheric nonthermal plasma known as floating-electrode dielectric-barrier discharge (FE-DBD) plasma against a control of planktonic and biofilm forms of Escherichia coli, S aureus, multidrug-resistant methicillin-resistant S aureus (MRSA) -95 (clinical isolate), -USA300, and -USA400, using widely accepted techniques such as colony count assay, LIVE/DEAD BacLight Bacterial Viability assay, and XTT (2,3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) assay. Results: Exposure of free living planktonic forms of E coli, S aureus, and MRSA were rapidly inactivated by DBD plasma. Approximately 107 bacterial cells were completely (100%) killed, whereas 108 and 109 were reduced by approximately 90% to 95% and 40% to 45%, respectively, in less than 60 seconds (7.8 J/cm2) and completely disinfected in ≤120 seconds. In established biofilms, the susceptibility of MRSA USA400 was comparable with USA300 but less susceptible than MRSA95 (clinical isolate), S aureus, and E coli (P < .05) to FE-DBD plasma, and plasma was able to kill MRSA more than 60% within 15 seconds (1.95 J/cm2). The killing responses were plasma exposure-time dependent, and cell density dependent. The plasma was able disinfect surfaces in a less than 120 seconds. Conclusion: Application of DBD plasma can be a valuable decontamination technique for the removal of planktonic and biofilm-embedded bacteria such as MRSA -USA 300, -USA 400, methicillin-sensitive S aureus (MSSA), and E coli, the more common hospital contaminants. Of interest, E coli was more resistant than S aureus phenotypes.

Bacteria are known to adhere to surfaces, which allows for the formation of biofilms, possibly causing a surge in hospital-offset infections, perilous diseases, and in some cases, death. Although certain bacteria are present in the natural flora of the human skin, some present extreme clinical significance due to the ability to transmit and adhere, and can be resistant to antibiotics. They also evolve over time to survive in harsh environmental conditions. Current research reveals that design of plastic surfaces containing submicron structures, is becoming a popular approach to tackle issues concerning infection transmission, with inspiration being derived from biomimetics and self-cleaning surfaces, such as the surface of a gecko skin, and the hydrophobic wax layer of forest leaves. Main barriers to adoption include that these surfaces alone are difficult to manufacture on 3D products, expensive to fabricate on a large scale and do not last long when subjected to environmental wear. Replication of nano-scale ridges was carried out using micro-injection, and the various samples were characterised using a range of tools to determine physical and biomechanical parameters. The sample surfaces were then cultured with the pathogenic bacterium Staphylococcus aureus under several environmental conditions, and the results were statistically analysed to reveal that anti-fouling LIPSS (laser induced periodic surface structures) ridges perform better to reduce bacteria cell-substrate adhesion, when compared to flat surfaces, or surfaces containing dual structures (anti-fouling ridges combined with anti-wear walls). It was therefore demonstrated that nanotextured polymeric surfaces with hydrophobic characteristics have exceptional non-fouling properties, preventing S. aureus, a very significant bacterial strain, from initial adhesion, a critical primary mechanism in its ability to proliferate. Collectively, the findings of this study strongly support the literature, suggesting that the bacteria struggle to adhere onto polymeric topography with increased water contact angles and simple nanostructures. However, the addition of certain anti-wear micro-features increased bacterial adhesion, reducing the efficacy of the non-fouling nanostructures from preventing biofilm formation.