Dissertations / Theses on the topic 'Staphylococcus'

Darbo tikslas: nustatyti koaguliazei teigiamų stafilokokų paplitimą tarp gyvūnų augintinių. Darbo uždaviniai: 1. išskirti koaguliazei teigiamus stafilokokus iš gyvūnų augintinių nosies ertmės; 2. išskirti koaguliazei teigiamus stafilokokus iš gyvūnų augintinių tiesiosios žarnos; 3. identifikuoti išskirtas stafilokokų padermes; 4. įvertinti įvairių veiksnių įtaką stafilokokų paplitimui; 5. nustatyti išskirtų stafilokokų atsparumą antimikrobinėms medžiagoms. Darbo apimtis – 50 puslapių. Šiame darbe yra 6 lentelės bei 14 paveikslų. Magistro darbą sudaro 4 dalys. Pirmojoje dalyje apžvelgiami literatūros šaltiniai susiję su analizuojama tema, išskiriant koaguliazei teigiamų stafilokokų virulentiškumo veiksnius, atsparumą antimikrobinėms medžiagoms, sukeliamas ligas ir šių ligų gydymą. Aptariamas Staphylococcus aureus bei Staphylococcus pseudintermedius paplitimas ir paplitimą įtakojantys veiksniai. Antrojoje dalyje nurodyti tyrimo metodai, kuriais remiantis gauti duomenys tyrimų analizei. Trečiojoje dalyje analizuojami gauti tyrimo rezultatai pagal iškeltus uždavinius. Rezultatai pateikiami atsižvelgiant į statistinių duomenų patikimumą. Ketvirtoji dalis skirta literatūros apžvalgos ir tyrimo rezultatų skirtumų ir panašumų palyginimui. Tyrimo metu iš gyvūnų augintinių nosies ertmės ir tiesiosios žarnos išskirti Staphylococcus aureus bei Staphylococcus pseudintermedius. Nustatyta, kad koaguliazei teigiamų stafilokokų paplitimas gyvūnų augintinių tarpe priklauso nuo gyvūnų... [toliau žr. visą tekstą]
The The goal of the study: to determine prevalence of coagulase positive staphylococci in companion animals. The aim of the study: 1. to isolate coagulase positive staphylococci in nasal cavity of companion animals; 2. to isolate coagulase positive staphylococci in rectum of companion animals; 3. to identificate the isolated strains of staphylococci; 4. to evaluate risk factors for prevalence of staphylococci; 5. to determine antibiotic resistance in isolated staphylococci. The master study consists of 50 pages. It includes 6 tables and 14 pictures. The master study consist of 4 major chapters. The first chapter is dedicated to review of literature that is related with analized topic. This part includes coagulase positive staphylococci virulence factors, antibiotic resistance, diseases caused by staphylococci and treatment use. Furthermore chapter contains review of the prevalence and risk factors influenced the prevalence of Staphylococcus aureus and Staphylococcus pseudintermedius. The second chapter introduce with materials and methods, that were used in the research at this master study. In the third chapter the results of the research are presented. The results are presented according to the statistical reliability. The fourth chapter is the resemblance and similarity comparision of the literature review and master study research. In this master study Staphylococcus aureus and Staphylococcus pseudintermedius were isolated from nasal cavity and rectum of companion... [to full text]

Les staphylocoques coagulase-negatifs (scn), bacteries commensales de la peau et des muqueuses sont frequemment isoles au cours d'infections graves principalement chez des patients immunodeprimes ou porteurs de materiel etranger. L'absence de systeme de lysotypie des scn permettant de caracteriser ces souches pathogenes et de les differencier des couches isolees comme contaminants, nous a conduit a isoler nos propres bacteriophages. Deux lots de phages ont ete isoles de s. Epidermidis et s. Saprophyticus et evalues dans un premier temps comme marqueurs epidemiologiques en terme d'activite lytique, de reproductibilite et de pouvoir discriminant. Le nombre de souches typables reste faible: 23% avec les phages de s. Epidermidis et 8% avec les phages de s. Saprophyticus. Cependant nos sytemes presentent une bonne specificite et reproductibilite. L'etude au microscope electronique a montre que ces phages appartenaient au groupe morphologique b1 (queue longue et non contractile, tete isometrique) defini par ackermann et eisenstark et a la famille des siphoviridae. Trois sous-groupes ont pu etre constitues en fonction des dimensions: -phages de s. Epidermidis, -phages de s. Saprophyticus, morphotype i phages de s. Saprophyticus morphotype ii. Une correlation etroite entre la morphologie, la structure antigenique et les caracteres des acides nucleiques est observee pour les phages de s. Saprophyticus. Les phages de s. Epidermidis presentent quant a eux une heterogeneite au niveau des adn (27 a 79% d'homologie). A l'issue de ce travail, deux nouvelles especes de phages de staphylococcus saprophyticus sont proposees: une premiere representee par les phages 1139, 1314, 1259 (type morphologique ii), la deuxieme representee par les phages 1154a et 1405 (type i) est originale par la petite taille des virions

Staphylococcus is a significant cause of human infection and mortality, worldwide. Currently, there are greater than 60 taxa within Staphylococcus, and nearly all are pathogenic. The collective potential for virulence among species of Staphylococcus heightens the overall clinical significance of this genus and argues for a thorough understanding of the evolutionary relationships among species. Within Staphylococcus, aureus is the most common cause of human infection, where nasal carriage of this bacterium is a known risk factor for autoinfection. The predisposition to infection by nasal carriers of S. aureus, and the ease with which strains are transferred between individuals, suggests that nasal carriage is a major vector for the transmission of virulent strains throughout the community. This hypothesis, however, has not been assessed in any great detail to identify the genetic relationships between clinical isolates of S. aureus and those strains being carried asymptomatically throughout the community. Also lacking within this field is a unified and robust estimate of phylogeny among species of Staphylococcus. Here, we report on a highly unified species phylogeny for Staphylococcus that has been derived using multilocus nucleotide data under multiple Bayesian and maximum likelihood approaches. Our findings are in general agreement with previous reports of the staphylococcal phylogeny, although we identify multiple previously unreported relationships. Regardless of methodology, strong nodal support and high topological agreement was observed with only minor variations in results between methods. Based on our phylogenetic estimates, we propose that Staphylococcus species can be evolutionarily clustered into 15 groups, and six species groups. In addition, our more defined phylogenetic analyses of S. aureus revealed strong genetic associations between both nasal carriage strains and clinical isolates. Genetic analyses of hypervariable regions from virulence genes revealed that not only do clinically relevant strains belong to identical genetic lineages as the nasal carriage isolates, but they also exhibited 100% sequence similarity within these regions. Our findings indicate that strains of S. aureus being carried asymptomatically throughout the community via nasal colonization are genetically related to those responsible for high levels of infection and mortality. Due to nasal carriage of S. aureus being a risk factor for autoinfection, standardized preoperative decolonization has become a major consideration for the prevention of nosocomial infection. Toward this end, we have identified the macrocyclic ?-defensin analogue RC-101 as a promising anti-S. aureus agent for nasal decolonization. RC-101 exhibited bactericidal effects against S. aureus in both epithelium-free systems, and ex vivo models containing human airway epithelia. Importantly, RC-101 exhibited potent anti-S. aureus activities against all strains tested, including USA300. Moreover, RC-101 significantly reduced the adherence, survival, and proliferation of S. aureus on human airway epithelia without any noted cellular toxicity or the induction of a proinflammatory response. Collectively, our findings identify RC-101 as a potential preventative of S. aureus nasal colonization.
ID: 030646199; System requirements: World Wide Web browser and PDF reader.; Mode of access: World Wide Web.; Thesis (Ph.D.)--University of Central Florida, 2011.; Includes bibliographical references (p. 140-159).
Ph.D.
Doctorate
Molecular Biology and Microbiology
Medicine
Biomedical Sciences

Staphylococcus xylosus et Staphylococcus carnosus sont utilisés comme ferments de salaisons. S. Xylosus est fréquemment isolé dans l'environnement des ateliers de transformation alimentaire alors que S. Carnosus l'est rarement. Ainsi, nous avons étudié la capacité de ces deux espèces à former des biofilms sur divers supports abiotiques. Les souches de S. Carnosus sont hydrophiles, adhèrent à des supports hydrophiles mais ne forment pas de biofilms. Certaines souches de S. Xylosus sont hydrophiles, d'autres moyennement hydrophobes et elles forment des biofilms quelque soit le support. S. Xylosus C2a, choisie comme souche d'étude, forme des biofilms denses avec des agrégats intercelluaires séparés par des canaux et englués dans une matrice constituée de polysaccharides dont la synthèse ne semble pas liée au gène icaA. Nous avons mis en evidence les gènes atl et bap codant des protèines de surface impliquées dans la formation de biofilm chez S. Aureus. Pour étudier l'ensemble des protéines de surface impliquées, nous avons développé une méthode d'analyse des protéines pariétales et membranaires. Un total de 101 protéines a été identifié dont 51 sont prédites comme protéines de surface et seulement 9 sont cmmunes aux deux fractions. La comparaison des profils protéiques des fractions pariétales, membranaires et intracellulaires a révélé une expression différentielle de 115 protéines dont 74 sont surexprimées en mode sessile et 41 en planctonique. Cette étude a révélé la modification de nombreuses voies métabolliques. Leur analyse permettra de mieux appréhender les mécanismes mis en jeu par S. Xylosus lors de sa croissance en biofilm

Šio darbo tikslas - Staphylococcus aureus ir Staphylococcus pseudintermedius išskyrimas iš gyvūnų augintinių, jų savininkų ir antimikrobinių medžiagų atsparumo nustatymas. Darbo uždaviniai: 1. Išskirti stafilokokus iš gyvūnų augintinių. 2. Išskirti stafilokokus iš gyvūnų augintinių savininkų. 3. Identifikuoti koaguliazei teigiamus stafilokokus. 4. Įvertinti įvairių veiksnių įtaka šunų nosies ertmės ir tiesiosios žarnos mikroflorai. 5. Įvertinti įvairių veiksnių įtaka gyvūnų augintinių savininkų nosies ertmės mikroflorai. 6. Nustatyti atsparumą antimikrobinėms medžiagoms. Iš viso paimti 123 mėginiai, iš jų 82 mėginiai paimti iš šunų laikomų namuose ir lauke, ir 41 mėginys iš jų savininkų. Mėginiai mikrobiologiniam tyrimui buvo imami į transportines terpes TRANSWAB® (naudojama pernešimo terpė aerobams ir anaerobams išskirti (Amies, Liofilchem, Italija)). Mikroorganizmų išskirta iš 37 (30,1 proc.) mėginių. Po vieną mikroorganizmų rūšį nustatyta 33 (89,1 proc.) mėginiuose. Po dvi mikroorganizmų rūšis (mišri infekcija), nustatyta 4 (10,9 proc.) mėginiuose. Iš šunų nosies ertmės ir tiesiosios žarnos paimto 41 mėginio mikroorganizmai buvo išskirti iš 27 (59,3 proc.). Staphylococcus pseudintermedius sudarė 32,4 proc. išskirtų mikroorganizmų, Staphylococcus aureus – 12,2 proc., Bacillus spp. – 14,7 proc. Iš šunų savinikų nosies ertmės paimto 41 mėginio mikroorganizmai buvo išskirti iš 16 (55,9 proc.) mėginių. Staphylococcus aureus sudarė 36,5 proc. išskirtų mikroorganizmų... [toliau žr. visą tekstą]
The objective of the research: Staphylococcus aureus and Staphylococcus pseudintermedius isolation from a pet and their owners, and determine the resistance of isolates to antimicrobial agent. Tasks of the research: 1. To isolate staphylococci from pet. 2. To isolate staphylococci from pet owners. 3. To identify coagulase-positive staphylococci. 4. Evaluate the influence of canine nasal and rectal flora. 5. Evaluate the influence of the pet owners nasal cavity flora. 6. To determine the resistance of isolates to antimicrobial agent. 41 samples of dogs and 41 from humans were investigated. Total take 123 samples, including 82 samples were taken from dogs kept in the home and outdoors, and 41 samples of their owners. Samples for microbiological analysis were collected in transport medium TRANSWAB ® (used to transport medium for aerobes and anaerobes release (Amies, Liofilchem, Italy)). Microorganisms were identified in 37 (30.1 percent.) samples. Single type of microorganisms were identified in 33 (89.1 percent) samples. Two types of microorganisms (mixed infection) were identified in 4 (10.9 percent) samples. In dogs, nasal and rectal were taken 41 samples. Microorganisms were identified in 27 (59.3 percent) samples. Staphylococcus pseudintermedius accounted 32.4 percent of identified microorganisms, Staphylococcus aureus – 12.2 percent, Bacillus spp. – 14.7 percent. Dogs owners nasal were taken 41 samples. Microorganisms were identified in 16 (55.9 percent) samples... [to full text]

Staphylococcus xylosus et staphylococcus carnosus sont deux especes a gram-positif, anaerobies facultatives, utilisees comme ferments dans les saucissons. Ils modulent la nature et le niveau des composes volatils issus de l'oxydation des acides gras. Leurs superoxyde dismutases (sods) et leurs catalases ont ete caracterisees afin de determiner leur pouvoir antioxydant. S. Xylosus c2a possede une seule sod proche des mnsods. Le gene codant (sod) a ete clone et sequence et deux promoteurs fonctionnels ont ete identifies en amont. L'expression du gene sod, etudiee a l'aide de fusions transcriptionnelles sod-lach, n'est pas modifiee par le paraquat ou le manganese, mais, elle est induite en phase stationnaire dans un milieu complexe. L'analyse du mutant sod, revele un role protecteur de la sod face a un stress oxydant. La sequence nucleotidique du gene kata de s. Xylosus c2a, codant une catalase proche des catalases monofonctionnelles, a ete determinee. L'expression du gene kata, etudiee a l'aide d'une fusion transcriptionnelle kata-lach, est induite, en phase stationnaire, par l'oxygene et le peroxyde d'hydrogene (h 2o 2). Elle est reprimee par le fer et surtout par le manganese. La presence d'une boite perr putative en amont du gene suggere une possible regulation par perr. Le mutant kata garde une activite catalase elevee revelant que kata n'est pas la principale catalase chez s. Xylosus c2a. En revanche, kata est essentiel pour une resistance optimale a l'h 2o 2 s. Xylosus c2a inhibe l'oxydation de l'acide linoleique. Sa sod et sa catalase kata contribuent a limiter cette oxydation. S. Carnosus 833 semble posseder une seule sod et une seule catalase dont les conditions de synthese semblent proches de celles de s. Xylosus c2a. La sod de s. Carnosus 833 presente de fortes similitudes de sequence avec celle de s. Xylosus c2a.

Staphylococcus aureus est l’un des principaux agents étiologiques des infections suppuratives superficielles et profondes ainsi que des syndromes liés à l’action de toxines. Paradoxalement, cette bactérie est un agent commensal qui est présent sur la peau ainsi que dans les cavités nasales notamment. Cela permet de considérer cette bactérie comme un organisme colonisant commensale. Les bases génétiques expliquant la différence entre une bactérie pathogène et une bactérie commensale reste inconnues. En utilisant la technique Optical Maps sur des souches de S. aureus isolées de plaies de pieds diabétiques avec différents niveau de virulence, nous avons pu montrer l’existence d’un prophage insérés dans le génome des souches colonisantes et absent des souches infectantes. Le phage, nommé ROSA, est localisé dans un hotspot d’insertion de phage NM2. Il est aussi localisé en amont du locus isd qui est requis pour l’assimilation du fer essentiel à la bactérie dans sa phase pathogène. Le phage ROSA inactive la voie isd en dérégulant l’activité du régulateur transcriptionnel majeur Fur en absence de fer. Il réduit aussi la virulence de ces souches sur les 2 modèles de virulence (Le ver C. elegans et le Zebrafish). L’expulsion du phage ROSA restaure la régulation du locus isd par Fur et la production de sidérophores en absence de Fer, la formation du biofilm et la virulence des souches. La mutation du gène Fur nous a permis de déduire que le phage ROSA affectait les bactéries de manière indépendante de Fur. Enfin, nous avons étudié la prévalence des souches colonisantes sur les plaies de pieds diabétiques. Nous avons observé que 20% des souches présentait l’insertion ROSA et 89% appartenait au complexe clonal CC8. Les souches colonisantes, avec leur niveau bas de virulence, devraient faire l’objet de détection dans le but de rationnaliser l’utilisation des antibiotiques et ainsi lutter contre l’apparition de bactéries multirésistantes aux antibiotiques
Staphylococcus aureus is an opportunistic bacterium capable of causing a wide range of severe diseases when it gains access to underlying tissues. Paradoxically, this causative pathogen is a common inhabitant of the skin microflora and colonizes the nares and other human mucosa, and as such, may be considered as a commensal colonizing organism. The genetic basis for the differences in pathogenic/colonizing potential is unknown. By performing optical maps comparisons of a collection of S. aureus strains of defined virulence potential isolated from diabetic foot ulcers at different stages, we brought to light a prophage present in colonizing-causing bacteria. The phage, namely ROSA, was localized in a hotspot region NM2 near the locus isd, the main iron surface determinant that transport iron across the bacterial wall. It induces a deregulation of the activity of the transcriptional regulator Fur involving the biofilm formation of the bacteria in response to low iron environment. It reduced also significantly the virulence of the strain in two in vivo models (the nematode C. elegans and the zebrafish). The expulsion of the phage restored the regulation of the locus isd, the siderophore production, the biofilm formation and the virulence of the strain. The mutation of the fur gene within the colonizing strain enabled us to determine that the phage ROSA affect the the bacteria in a Fur-independent manner. Finally we determined the prevalence of these colonizing strains in skin and soft tissue infections (diabetic foot ulcers). We observed that 20% (39/195) of the strains harboured this insertion and 89% belonged to the clonal complex CC8. This colonizing strain by its low virulence potential must be detected in the aim to contribute to a sounder use of antibiotic treatment, an important point in front of the increase of multidrug resistant bacteria

O presente relatório teve como base o estágio curricular em Clínica e Cirurgia de bovinos de aptidão leiteira realizado na Associação de Jovens Agricultores Micaelenses. A primeira parte deste relatório inclui a compilação da casuística acompanhada, seguida da segunda parte que corresponde à revisão bibliográfica sobre a mastite bovina. A terceira e última parte refere-se ao estudo desenvolvido em 100 explorações leiteiras da ilha de São Miguel, Açores. Do total das amostras de leite do tanque recolhidas nas explorações, 59% foram positivas a Staphylococcus aureus, 100% a Staphylococcus coagulase-negativos, 75% foram positivas a Escherichia coli e 35% a outros coliformes. Alguns dos fatores de risco em estudo, tais como a utilização de luvas e o pré-dipping, foram significativamente associados à prevalência dos agentes acima citados no leite do tanque. Verificou-se ainda uma influência significativa do Staphylococcus aureus na contagem de células somáticas no leite do tanque (P <0,001); DAIRY CATTLE MEDICINE AND SURGERY Risk factors associated with the bulk-milk prevalence of Staphylococcus spp. and coliforms in dairy farms of São Miguel Island, Azores ABSTRACT: This report concerns to the academic internship in dairy cattle Medicine and Surgery performed at Associação de Jovens Agricultores Micaelenses. The first part of the report includes the compilation of accompanied casuistry, followed by the literature revision on bovine mastitis. The third and last part refers to a study in 100 dairy farms of São Miguel Island, Azores - Portugal. From the total bulk tank milk samples collected in the study dairy farms, 59% were positive to Staphylococcus aureus, 100% to coagulase-negative Staphylococci, 75% were positive to Escherichia coli and 35% to other coliforms. Several risk factors in study, such as gloves use and pré-dipping, were significantly associated with the prevalence of the agents mentioned above in bulk tank milk. Moreover, the study revealed a significant influence of Staphylococcus aureus in somatic cells count in bulk tank milk (P <0,001).

Osmoregulation in Staphylococcus aureus has been studied. Glycine betaine was found to act as an osmoprotectant by stimulating specific growth rate and salt tolerance of osmotically stressed S. aureus cells. The accumulation of this compatible solute was accomplished via two constitutive Na⁺ dependant transport systems, a high-affinity system (Km = 3μM; Vmax = 26nmol. min^-1 mg total protein^-1) and a low-affinity system (Km = 133μM; Vmax = 155 nmol. min^-1 mg total protein^-1). The high-affinity system is specific for glycine betaine and its activity is not greatly stimulated by osmotic pressure. The low-affinity sytem transports proline and proline analogues and its activity is stimulated by increases in external osmolarity. Proline transport is achieved via two similar systems. Through transposon mutagenesis it was demonstrated that the low-affinity glycine betaine transport system and the low-affinity proline transport system are the same system. The low-affinity system is the major system responsible for the accumulation of glycine betaine. This is the more important system for the osmoregulation strategy of S. aureus. All three transport systems have been demonstrated to be subject to feedback regulation. The internal compatible solute concentration dictates the level of activity of the transport systems. A mutant has been isolated that lacks the low-affinity system and the high-affinity proline transport system.

Ce travail a cherché à établir l'importance du réservoir méconnu des porteurs de Staphylococccus aureus résistant à la méticilline (SARM) dans l'épidémiologie hospitalière de cette bactérie multirésistante aux antibiotiques, la plus fréquente en France. Il montre au travers d'enquêtes prospectives, que la prévalence et l'incidence du SARM sont beaucoup plus elévées que ne laissent penser les surveillances basées sur des prélèvements à visée clinique. Il établit des facteurs de risque de portage de SARM, et suggère enfin que la stratégie de dépistage et précautions " contact " est efficace. Le contrôle de la dissémination manuportée du SARM doit inclure une stratégie raisonnée de dépistage des porteurs
Our research aimed to determine the significance of unknown carriage of methicillin-resistant Staphylococcus aureus in the epidemiology of this multiply-resistant strains in the hospital setting. Through prospective observational studies of MRSA carriage, we demonstrated that incidence and prevalence of MRSA at hospital admission are much higher than that estimated by clinical isolates only. We established parameters associated with MRSA carriage, and suggested that active surveillance screening and contact precautions are valuable in the ICU setting. Controlling the hospital spread of MRSA should include a judicious strategy for screening MRSA

Staphylococci strains are among the most widespread multidrug-resistant nosocomial pathogens in Canada. Rapid and accurate identification and epidemiological typing of methicillin-resistant S. aureus (MRSA) and its discrimination from coagulase-negative staphylococci (CNS) and glycopeptide-intermediate S. aureus (GISA) are crucial for appropriate therapy and for monitoring and limiting intra- and inter-hospital spread of epidemic MRSA strains. Although pulsed-field gel electrophoresis and polymerase chain reaction methods for the identification of MRSA are reliable, they are technically demanding, time-consuming and inappropriate for routine clinical diagnosis. Moreover, no reliable method exists for discrimination of epidemic MRSA from sporadic MRSA and from GISA strains. The objective of the research described in this thesis was to investigate whether Fourier transform infrared (FTIR) spectroscopy could be used to distinguish MRSA from methicillin-susceptible S. aureus, borderline oxacillin-resistant S. aureus (BORSA), CNS, including methicillin-resistant CNS, and GISA. The application of FTIR spectroscopy for epidemiological typing of Canadian epidemic MRSA (CMRSA) strains as well as their discrimination from sporadic MRSA was also assessed. FTIR spectra were recorded from intact stationary-phase cells grown on Universal Medium (UM™) and deposited and dried on a ZnSe optical window, normalized, and converted to first-derivative spectra. Various chemometric approaches were employed to cluster the different phenotypes of staphylococci species and to subtype five CMRSA strains based on the similarity of their infrared spectral fingerprints in narrow spectral regions selected by visual inspection and by employing a singularvalue decomposition (SVD) algorithm. Pairwise separation of MRSA from MSSA, BORSA, CNS, MRCNS, and GISA was accomplished by using principal component analysis (PCA), self-organizing maps (SOM), and the K-nearest neighbors (KNN) algorithm. These chemometric techniques were also successfully employed for epidemiological typing of the five CMRSA strains and their discrimination from sporadic MRSA strains using a combination of different optimal spectral regions selected by SVD. These results demonstrate that FTIR spectroscopy has considerable potential as a rapid method for the identification of different phenotypes of staphylococci and epidemiological typing of MRSA.

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Os estafilococos coagulase negativos emergem como importantes patógenos nosocomiais, frequentemente isolados em bacteremias humanas, fato intimamente relacionado com o aumento do uso de dispositivos médicos ao longo dos últimos anos. Sua capacidade de aderir a superfícies poliméricas e produzir biofilmes constitui o principal fator de virulência associado a estes dispositivos. A fim de minimizar esta possibilidade, antissépticos são largamente utilizados para desinfecção de pele e mucosas na rotina hospitalar, no entanto, tem-se observado uma diminuição da susceptibilidade bacteriana a estes agentes. A diminuição da susceptibilidade dos microrganismos a antissépticos, associados a sua capacidade de formar biofilmes tem elevado a morbidade, mortalidade, período de internação e os custos relativos ao cuidado com a saúde. No estudo aqui apresentado, foram analisadas 81 cepas de Staphylococcus epidermidis resistentes a meticilina (MRSE) e 55 cepas de Staphylococcus haemolyticus resistentes a meticilina (MRSHa), todas resistentes a meticilina, isoladas de pacientes internados em hospital Universitário localizado na cidade de Niterói-RJ nos anos de 2008 e 2010. A determinação do perfil de susceptibilidade aos biocidas triclosan, clorexidina e cloreto de benzalcônio mostrou que frente ao triclosan, as amostras apresentaram as menores taxas de susceptibilidade bacteriana. Papaína não apresentou atividade antibacteriana. Entretanto, foi capaz de reduzir expressivamente a formação de biofilme (p<0,06) em ambas as espécies, mostrando-se a mais eficiente entre os produtos analisados. Foi capaz também de desintegrar biofilmes maduros formados por Staphylococcus epidermidis. Os biocidas não levaram à redução significante de biofilmes, exceto a clorexidina, que foi capaz de reduzir a formação do biofilme pelas MRSE. Foi possível verificar que cepas de MRSHa tratadas com cloreto de benzalcônio têm 40% menos probabilidade de formar biofilme se comparada às tratadas com triclosan e clorexidina. A análise estatística nos mostrou que a expressão do gene qacA/B influenciou significativamente a formação de biofilme e confirmou a relação existente entre os valores de concentração mínima inibitória (CMI) e a formação de biofilme (p< 0,001)
Coagulase negative staphylococci have emerged as important nosocomial pathogens, commonly isolated in human bacteremia, a fact closely related to the increased use of medical devices over the last few years. Its ability to adhere to polymer surfaces and produce biofilms is the main virulence factor associated with these devices. To minimize this possibility, antiseptics are widely used for disinfection of skin and mucosa in routine hospital, however, there has been an increase in bacterial resistance to these agents. Decreased susceptibility of microorganisms to antiseptics, associated with its ability to form biofilms has high morbidity, mortality, length of stay and costs related to health care. In the study presented here, we analyzed 81 strains of methicillin-resistant Staphylococcus epidermidis (MRSE) and 55 strains of methicillin-resistant Staphylococcus haemolyticus (MRSHa), all meticillin- resistant, isolated from patients hospitalized at University Hospital in the city of Niterói, Rio de Janeiro in 2008 and 2010. The determination of the susceptibility profile of biocides triclosan, chlorhexidine and benzalkonium chloride demonstrated that front of triclosan, the strains have the biggest rates of bacterial susceptibility. Papain showed no antibacterial activity. However, it was able to significantly reduce biofilm formation (p <0.06) in both species, being the most efficient among the analyzed products. It was also able to degrade established biofilms formed by Staphylococcus epidermidis. Biocides showed no significant reduction of biofilms, except chlorhexidine, which was able to reduce biofilm formation by MRSE. We noticed that strains MRSHa treated with benzalkonium chloride are 40% less likely to form biofilm compared to those treated with chlorhexidine and triclosan. Statistical analysis showed that the gene expression qacA/B was significantly influential to biofilm formation and confirmed positive relationship between the values of MIC and biofilm formation (p <0.001)

Orientadores : Thales Rocha de Mattos Filho, Francisco Carlos Groppo
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
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Doutorado
Farmacologia, Anestesiologia e Terapeutica
Doutor em Odontologia

A nivel mundial la piodermia es una de las enfermedades de la piel más diagnosticada en caninos. Entre los agentes involucrados se encontraba Staphylococcus intermedius, el que se creía el principal agente de las piodermias caninas; sin embargo, en el año 2005 fue reclasificado en 3 especies: S. intermedius, S. pseudintermedius y S. delphini. Posteriormente, estudios en diferentes países reportaron que el Staphylococcus pseudintermedius es en realidad el agente bacteriano más frecuentemente aislado en piodermias, seguidamente surgieron los primeros reportes de aislados resistentes a meticilina. Por otro lado, Staphylococcus aureus, patógeno importante en medicina humana, se aísla con más frecuencia en muestras de piodermias caninas, siendo estos potenciales reservorios para reinfecciones en humanos de cepas resistentes a antibióticos. Por ello, este estudio buscó determinar la presencia S. pseudintermedius y S. aureus en 141 aislados de Staphylococcus sp. provenientes de casos de piodermia canina del periodo 2016 - 2018. El ADN de los 141 aislados fue extraído y analizado mediante PCR-RFLP, el cual consistió en la amplificación del gen pta y la digestión con la enzima MboI. Obteniendo que los aislados de Staphylococcus sp. fueron identificados como Staphylococcus pseudintermedius en un 87.9%, 12.1% como otros Staphylococcus sp. y no se detectó Staphylococcus aureus. Concluyéndose que el Staphylococcus más frecuentemente involucrado en piodermias caninas es el Staphylococcus pseudintermedius; sin embargo, no se debe omitir el rol potencial que puede cumplir Staphylococcus aureus como patógeno en caninos.
Universidad Nacional Mayor de San Marcos (Lima). Vicerrectorado de Investigación y Posgrado
Tesis

Nasal carriage of Stephvtococcue aureus is associated with increased risk of infection in humans. Several factors are known to affect carriage including host genetics and S. aureus immune evasion. However while members of the microbial community have been shown to affect S. aureus nasal colonisation individually, relatively little work has been done to understand how and why nasal microbial community as a whole, affects carriage of S. aureus. Here, the cultivable bacteria from 60 anterior nares communities were sampled and identified to species level using apiSTAPH and 16S rRNA gene sequencing. The taxa distributions across communities revealed negative associations of S. aureus with the following taxa: S. capitis, Corynebacterium propinquum, C. macgin/eyi, Enterobacter aerogenes, S. epidermidis, Micrococcus sp., Bacillus sp., C. acco/ens, S. schleiferi and Gemella haemo/ysans. Since toxin-mediated interference can affect community composition, nasal isolates were screened for their ability to inhibit growth of S. aureus on a solid medium. Overlaying this inhibition data onto community taxa distributions revealed that negative associations between S. aureus and S. epidermidis, S. capitis, C. propinquum, C. acco/ens and a Micrococcus sp. were potentially driven by toxin- mediated interference competition. Moreover novel negative associates were found between S. aureus and an inhibitory subset of Micrococcus /uteus and S. hominis. By also measuring the cumulative inhibition of entire natural communities, it was possible to show that S. aureus was less frequent in highly inhibitory microbial communities. The quorum sensing mechanism, encoded by the agr locus, and biofilm formation have been proposed to play an important role in nasal colonisation of S. aureus. Therefore to further investigate community dynamics, S. capitis, S. epidermidis and corynebacteria isolates were assayed for their ability to interfere with Agr signaling and biofilm formation. No evidence was obtained to indicate that biofilm interference by these species affected the distribution of S. aureus across communities. By contrast, S. epidermidis isolates that interfered with Agr signaling were significantly more likely to coexist with S. aureus, and S. capitis isolates interfering with Agr signaling were significantly less likely to coexist with S. aureus. In theory, toxin-mediated interference competition can act both to protect producers against invasion, and, conversely, to promote the invasion of producers into an occupied niche. An experimental ecology approach was used to show that S. aureus is less likely to invade an inhibitor-producing S. epidermidis population than a non-inhibitor-producing population, especially on a spatially-structured medium. Furthermore, inhibitor-producing populations of S. epidermidis invade more successfully than non-inhibitor-producers, although they do not displace the S. aureus resident due to evolution of toxin resistance. There is also evidence of eo- evolution where inhibitor-producing strains of S. epidermidis can evolve stronger inhibitory activity when invading sensitive S. aureus populations that evolve resistance. These findings could impact the future treatment of S. aureus infections and help to control nasal carriage.

Notre travail porte sur une bacterie pathogene, staphylococcus aureus, qui intervient dans les infections humaines et animales. Le polysaccharide (ps) de s. Aureus etant un compose faiblement immunogene, son immunogenicite peut-etre nettement augmentee par le couplage a une proteine porteuse. L'originalite de notre etude reside dans l'utilisation d'un ps capsulaire et d'une proteine, l'hemolyse alpha, issus de la meme bacterie, ce qui devrait augmenter les proprietes antigeniques du conjugue. Nous avons donc forme des composes glycoproteiques en couplant par la methode au glutaraldehyde le ps de deux souches humaines aux hemolysines alpha des souches correspondantes. Apres avoir observe l'immunogenicite et l'antigenicite de conjugues produits a partir de composes partiellement purifies nous avons isole puis purifie les ps et les fractions hemolytiques et/ou antigeniques des deux souches de s. Aureus. L'etude structurale a permis d'identifier le ps 31 a un polymere d'acide aminogalacturonique lie en 1->4 et le ps 8914 a un polymere d'acide aminomannuronique lie en 1->3. Parmi les differents conjugues (ps-fraction proteique) obtenus, le produit couple homologue (ps-f42,5 kda) 8914 et le produit couple heterologue ps31-f42,5 kda 8914 se sont reveles les plus immunogenes et antigeniques mettant en evidence l'efficacite de la fraction proteique f42,5 de s. Aureus 8914 en tant que proteine porteuse. Les deux serums obtenus a partir de ces conjugues induisent une resistance contre s. Aureus 8914 chez la souris, celle-ci semble maximale dans le cas du serum heterologue. L. Reynaud-rondier, a. Voiland and g. Michel (1991). Fems microbiology immunology 76 193-200

Susceptibility of 112 strains of Staphylococcus aureus obtained from Dameron Hospital, Stockton, California was tested with 18 antimicrobials . The MIC method was used with the following antimicrobials : tetracycline, oxacillin, penicillin, ampicillin, vancomycin, cefazolin, erythromycin, clindamycin, gentamycin, rifampin, trimethoprimsulfamethoxazole, chloramphenicol, and cefotaxime . The standard Kirby-Bauer disc diffusion method was used to test neomycin, tobramycin, and amikacin . Methicillin, oxacillin, and nafcillin were tested with a modified Kirby-Bauer method, which included the addition of a 4% salt supplement to the media, incubation at 32C, and readings at both 24 and 48 hours. Comparing results of this study with those of Hall (1975), suggested that resistance to the following antibiotics has increased: penicillin, ampicillin, erythromycin, neomycin, gentamycin, methicillin, oxacillin, nafcillin, cefazolin, and clindamycin . Resistance to tetracycline has decreased. No resistance to chloramphenicol or vancomycin was encountered in either study . Of the 112 strains studied, 13 . 4% were susceptible to all antibiotics tested. Twelve patterns of resistance were identified : 0 . 9% were resistant to neomycin only, 1.8% to erythromycin only, 63.9% to both penicillin and ampicillin, and 20 . 0% were multiply- resistant . Nine patterns of multiple-resistance were found, involving a minimum of three antibiotics and a maximum of nine . Three MRSA strains were identified from out-patient isolates; no in-patient isolates were methicillin-resistant . The study suggests that MRSA strains are not a problem at Dameron Hospital, but identification of this group would be more accurate if incubation of the MIC panels is maintained for at least 24 hours at ~35C . It was found that the MIC method of antimicrobial susceptibility testing is more reliable than the Kirby-Bauer disc diffusion method for detection of methicillin-resistance. Problems involved in identification of heteroresistant staphylococci are discussed .

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Conselho Nacional de Desenvolvimento Científico e Tecnológico
A cadeia produtiva de carne suína está susceptível a diferentes fontes de contaminação microbiológica em diferentes etapas, desde a produção primária até o processamento de produtos finais. Considerando o contexto atual de comércio internacional de produtos, o controle de eventuais perigos microbiológicos deve ser efetivo, visando a inocuidade dos produtos finais e segurança do consumidor. Em relação a carne suína, Staphylococcus coagulase positiva (SCP) possui grande importância por serem importantes indicadores das condições de manipulação, e também por indicarem a presença de S. aureus, que na cadeia produtiva de suínos possui relevância pela possibilidade de carrear genes de resistência a diferentes antimicrobianos nos produtos finais e consumidores. O objetivo desse estudo foi rastrear a contaminação por SCP e S. aureus resistentes a antimicrobianos na cadeia produtiva de carne suína. Duas granjas de criação de suínos (ciclo completo) e um frigorífico foram selecionados para coleta de 603 amostras ao longo da cadeia produtiva de carne suína (1 - Granjas: baia de terminação, n = 18; 2 - Abate: carcaça após sangria, n = 90; carcaça após chamuscamento, n = 90; carcaça após evisceração, n = 90; carcaça após lavagem, n = 90; 3 - Processamento: faca limpa, n = 27; faca durante processamento, n = 27; mãos limpas, n = 27; mãos durante processamento, n = 27; mesa limpa, n = 27; mesa durante processamento, n = 27; 4 - Produtos finais: costela, n = 18; paleta, n = 18; pernil, n = 18; linguiça, n = 9), que foram submetidas a enumeração de SCP, e posterior isolamento e identificação de S. aureus. A contaminação por SCP foi inferior a 2 log UFC/cm2 ou g em 512 (84,9%) amostras, entre 2 e 3 logs UFC/cm2 ou g em 52 (8,6%) amostras, entre 3 e 4 logs UFC/cm2 ou g em 6 (1,0%) amostras, e superior a 4 log UFC/cm2 ou g em 33 (5,5%) amostras. Contagens superiores a 4 log UFC/cm2 ou g foram observadas em baias de terminação, carcaças após evisceração, carcaças após lavagem, facas limpas, mesas limpas, costela e linguiças. A enumeração de SCP foi possível em 197 (32,7%) amostras, e as contagens médias variaram entre 1,0 log UFC/cm2 (mesa durante processamento) e 5,2 logs UFC/cm2 (baia de terminação). Considerando as diferentes etapas de processamento, não foram observadas diferenças significativas entre as amostras obtidas no ambiente de processamento e nos produtos finais (p > 0,05). Durante o abate, as contagens médias de SCP obtidas nas carcaças após sangria foram superiores quando comparadas as etapas posteriores (p > 0,05). Um total de 315 colônias de SCP foi selecionado e caracterizado quanto ao perfil bioquímico e presença do gene femA para identificação de S. aureus. Assim, 246 isolados de S. aureus foram identificados e submetidos a caracterização de seus perfis de resistência em relação a 11 antimicrobianos, por testes de susceptibilidade e pela pesquisa de genes relacionados a resistência. Considerando os resultados fenotípicos, 90,7% dos isolados de S. aureus apresentaram resistência a sulfamethoxazole (SUL), 87,0% a ciprofloxacina (CIP), 67,9% a penicilina (PEN), 49,6% a eritromicina (ERI), 40,2% a oxacilina (OXA), 40,2% a clindamicina (CLI), 29,3% a rifampicina (RIF), 28,5% a cloranfenicol (CLO), 21,1% a tetraciclina (TET), 16,3% a vancomicina (VAN) e 6,5% a gentamicina (GEN). Apenas 15 isolados foram susceptíveis a todos os antimicrobianos testados (isolados obtidos de baias de terminação, carcaças, pernil e linguiça), 7 a apenas um antimicrobiano (CIP, SUL ou TET), e os demais resistentes simultaneamente a 2 a 10 diferentes antimicrobianos; o perfil de resistência a PEN-ERI-CIP-SUL foi o que apresentou maior frequência entre os isolados de S. aureus (n = 37). Em relação aos genes relacionados a resistência a diferentes antimicrobianos, 74,0% dos isolados de S. aureus apresentaram resultados positivos para blaZ (PEN), 8,1% para femB (OXA), e 3,7% para tetK (TET); não foram observados resultados positivos para vanA (VAN), mecA e mecC (OXA). Entre os isolados de S. aureus, 60 não apresentaram nenhum dos genes pesquisados, 164 apresentaram apenas um dos genes isolados (blaZ, femB e tetK), 15 apresentaram simultaneamente os genes femB-blaZ, 4 os genes blaZ-tetK, e 3 os genes femB-blaZ-tetK. Apenas em relação a TET foi verificado que o teste de susceptibilidade foi equivalente a presença de blaZ (p = 0,147), e ausência de equivalência de resultados para OXA (femA, femB), VAN (vanA) e TET (tetK) (p < 0,05). Os resultados obtidos demonstram a relevância de SCP como indicadores de manipulação e higiene na cadeia produtiva de carne suína, assim como a presença de S. aureus com resistência a diferentes antimicrobianos, alertando para a necessidade de monitoramento por metodologias fenotípicas e moleculares.
The pork production chain is susceptible to different sources of microbiological contamination at different stages, from primary production to the processing of final product. Regarding the current context of international trade in products, the control of possible microbiological hazards must be effective, aiming at the safety of final products and consumer. In relation to pork, Staphylococcus coagulase positive (SCP) is relevant because they are important indicators of the manipulation condition and also because they indicate the presence of S. aureus, which in the production chain of pigs has relevance due to the possibility of carrying resistance genes to different antimicrobials in the final products and consumers. The objective of this study was to track the contamination by SCP and S. aureus resistant to antimicrobial in the pork production chain. Two pig farms (complete cycle) and a refrigerator were selected to collect 603 samples through the pork production chain (1 - Farms: termination bay, n = 18; 2 - Slaughter: carcass after bleeding, n = 90; carcass after scorching, n = 90; carcass after evisceration, n = 90; carcass after washing, n = 90; 3 - Processing: clean knife, n = 27; knife during processing, n = 27; clean hands, n = 27; hands during processing, n = 27; clean table, n = 27; table during processing, n = 27; 4 - Final products: rib, n = 18; palette, n = 18; leg, n = 18; sausage, n = 9) which were submitted to SCP enumeration, subsequent isolation and identification of S. aureus. The SCP contamination was less than 2 log CFU / cm2 or g in 512 (84.9%) samples, between 2 and 3 log CFU / cm2 or g in 52 (8.6%) samples, between 3 and 4 CFU logs / cm2 or g in 6 (1.0%) samples, and greater than 4 log CFU / cm2 or g in 33 (5.5%) samples. Counts greater than 4 log CFU / cm2 or g were observed in termination bins, carcasses after evisceration, carcasses after washing, clean knives, clean tables, rib and sausages. SCP enumeration was possible in 197 (32.7%) samples, and the mean counts varied between 1.0 log CFU / cm2 (table during processing) and 5.2 log CFU / cm2 (termination bay). Considering the different steps of the process, no significant differences were observed between the obtained samples in the processing environment and in the final products (p> 0.05). During slaughter, the SCP mean counts obtained on carcasses after bleeding were higher when compared to the later stages (p> 0.05). A total of 315 SCP colonies were selected and characterized in relation to the biochemical profile and the presence of the femA gene for identification of S. aureus. Thus, 246 isolates of S. aureus were identified and submitted to characterization of their resistance profiles in relation to 11 antimicrobials, by susceptibility tests and by the search of genes related to resistance. Taking into account the phenotypic results, 90.7% of S. aureus isolates showed resistance to sulfamethoxazole (SUL), 87.0% to ciprofloxacin (CIP), 67.9% to penicillin (PEN), 49.6% to erythromycin (IRI), 40.2% to oxacillin (OXA), 40.2% to clindamycin (CLI), 29.3% to rifampicin (RIF), 28.5% to chloramphenicol (CLO), 21.1% to tetracycline (TET), 16.3% vancomycin (VAN) and 6.5% gentamycin (GEN). Only 15 isolates were susceptible to all antimicrobials tested (isolates obtained from finishing bays, carcasses, shanks and sausage), 7 to only one antimicrobial (CIP, SUL or TET), and the other resistent simultaneously to 2 to 10 different antimicrobials; the resistance profile to PEN-ERI-CIP-SUL was the most frequent among S. aureus isolates (n = 37). Regarding the genes related to resistance to different antimicrobials, 74.0% of S. aureus isolates presented positive results for blaZ (PEN), 8.1% for femB (OXA), and 3.7% for tetK (TET); no positive results were observed for vanA (VAN), mecA and mecC (OXA). Among the S. aureus isolates, 60 did not present any of the studied genes, 164 had only one of the isolated genes (blaZ, femB and tetK), 15 simultaneously presented the femB- blaZ genes, 4 the blaZ-tetK genes, and 3 femB-blaZ-tetK genes. Only in relation to TET, the susceptibility test was equivalent to the presence of blaZ (p = 0.147), and no equivalence of results for OXA (femA, femB), VAN (vanA) and TET (tetK) (p < 0.05). The results obtained demonstrate the relevance of SCP as indicators of handling and hygiene in the pork production chain, as well as the presence of S. aureus with resistance to different antimicrobials, highlighting the need for monitoring by phenotypic and molecular methodologies.

The aim of this paper was to evaluate vaccine efficacy under field conditions against Staphylococcus aureus and Coagulase Negative Staphylococci (CNS) intramammary infections (IMI) in dairy cows. During the 21-month duration of the study, 1,156 lactations in 809 cows were identified in two herds. At the beginning of the trial, all cows that were due to calve were vaccinated until approximately 50% of cows in the milking herd were vaccinated (~6 months). At that point in time (50% vaccination coverage was reached), cows were systemically random assigned to be vaccinated or left as negative controls. Cows that were vaccinated got the 1st injection 45 days (+ 3 days) before the expected parturition date, the 2nd injection 35 days (5 weeks) thereafter (+ 3 days), corresponding to 10 days before the expected parturition date and 3rd vaccination was done at 52 days (+ 3 days) after the actual parturition date. Cure rate, risk of new infections, prevalence, incidence and duration of infections were analyzed. The farms showed a large difference in management to control infection during the study. In conclusion vaccination reduced the basic reproduction ratio of Staph. aureus by approximately 45% and for CNS by approximately 35%. Vaccination is a valuable tool in improving the incidence and prevalence of staphylococcal infection in herds. Vaccine utilization will need to be combined with excellent milking procedures, culling of known infected cattle and other management procedures to further reduce incidence and duration of infection.

Realizou-se um trabalho experimental com o objectivo de obter informação sobre a evolução do crescimento de Staphylococcus aureus. Foram utilizadas duas estirpes de Staphylococcus aureus, uma isolada a partir de rissóis de frango e uma estirpe de referência, a ATCC n. 9213, estas estirpes foram sujeitas a 3 valores de pH diferentes (que representam os valores de pH que é possível, ou seja pH 4, 5,5 e 7, a 3 valores de concentração de NaCl, nomeadamente, 0,5%, 7% e 15%. A temperatura de desenvolvimento será de 7°C, 37°C e 50aC. Utilizaram-se dois métodos para avaliar o crescimento de Staphylococcus aureus, ao longo do tempo, nomeadamente o Método Turbidímétrico e o Método de contagem de unidades formadoras de colónias (método das diluições sucessivas). ABSTRACT: Carried out experimental work in order to obtain information on the evolution of the growth of Staphylococcus aureus. We used two strains of Staphylococcus aureus, a strain isolated from a chicken patties and one reference strain, ATCC Nº 29213, these strains were subjected to 3 different pH values (which represent the values of pH it is possible, or is pH 4, 5.5 and 7, the 3 values of NaCI concentration, namely, 0.5%, 7% and 15%. The growth temperature is 7 °C, 37°C and 50ºC. We used two methods to evaluate the growth of Staphylococcus aureus, over time, including the turbidimetric method and the method of counting colony forming units (method of successive dilutions).

Methicillin-resistant (MRSA) and multidrug-resistant (MDR) Staphylococcus aureus, and the serotype (ST) 398 have been associated with human and livestock infections, being also detected in retail meat. The aim of this study was to determine the prevalence and molecular types of S. aureus strains from animals, retail raw meat, deli meat, and humans, determining the genetic similarity between the strains. A two-step selective enrichment followed by selective plating were used to isolate S. aureus from animals (n=167), retail raw meat (n=145), and deli meat (n=46). In addition, S. aureus from healthy people (n=550) was isolated by culture method. Positive isolates and MRSA isolates from clinical cases (n=108) were subjected to multiplex PCR (16S rRNA, mecA, and PVL genes), molecular typing and antimicrobial susceptibility testing. In addition, a real-time PCR assay was developed in order to decrease the time of detection of target genes of S. aureus in animal and meat samples, comparing the results with the standard culture/PCR method. The prevalence of S. aureus was 34.7% in animals, 47.6% in meat, and 13.0% in deli meat. The mecA gene was detected in S. aureus isolated from five pork meat samples and exhibited penicillin resistance. The ST398 was found in sheep, pigs, and pork meat. The S. aureus nasal carriage in healthy people was 7.6%. A total of 105 MRSA strains (97.2%) from clinical cases harbored the mecA gene and 11 (10.2%) the PVL gene. The rate of MDR was 70% in humans. A genetic similarity between strains from animals and meat, and from humans and meat was observed. Total agreement between the culture/PCR method and real-time PCR for detection of S. aureus was 68.9 to 97.8% (k=0.68-0.88), and the mecA gene, 86.7 to 98.7% (k=0-0.49). Therefore, the real-time PCR assay may be recommended as a rapid method for the detection of S. aureus, with confirmation of MRSA using the standard culture method. The presence of emerging S. aureus strains in the meat production chain and the genetic similarity between strains of different origin, suggests the contamination of meat, and a potential risk of transmission to humans.
Dean?s Office, College of Agriculture, Food Systems and Natural Resources, North Dakota State University

Nous avons souhaité étudier l'épidémiologie du portage de staphylocoques à coagulase négative résistants à la méticilline (SCNRM) dans la communauté, et leur rôle de réservoir de Staphylococcal Cassette Chromosome mec (SCCmec) dans cet environnement. La prévalence du portage nasal de C-SCNRM était comprise entre 11% et 50% dans les différentes populations étudiées. Ce portage était un phénomène dynamique, à l'échelle individuelle comme à celle d'une population donnée. Staphylococcus epidermidis RM (SERM) représentait 32% à 78% des isolats de C-SCNRM : le typage des souches de SERM par MLST et MLVA suggérait l'émergence fréquente de nouvelles souches par transfert horizontal de SCCmec. Les SCCmec présents chez les C-SCNRM étaient polymorphes. SCCmec IV, majoritaire chez les souches communautaires de SARM (C-SARM), était la cassette la plus fréquente chez les C-SERM. Les séquences des SCCmec IV portés par les C-SERM étaient identiques à >99% à celles décrites chez certains clones épidémiques de C¬SARM. L'élément génétique Arginine Catabolic Mobile Element (ACME), identifié chez 68% des C-SERM, pourrait faciliter leur persistance dans la flore cutanéo-muqueuse. Enfin, l'imp_pct de l'antibiothérapie ambulatoire sur le portage nasal de C-SCNRM a été analysé chez 479 patients de médecine générale. L'acquisition de C-SCNRM était plus fréquente chez les patients traités par pénicillines que chez ceux exposés aux fluoroquinolones, aux macrolides ou à la pristinamycine. Ces travaux apportent des données originales sur la dissémination massive des SERM et des autres SCNRM dans la communauté, et sur leur rôle de réservoir dynamique de SCCmec dans cet environnement
Our objectives were to appraise the epidemiological patterns of methiillin-resistant coagulase-negative staphylococci (MRCNS) carriage in the community and to assess the role of MRCNS in the dissemination of Staphylococcal Cassette Chromosome mec (SCCmec) elements in this environment. Rates of MRCNS nasal carriage ranged from 11% to 50% (6 distinct geographic regions). Chronic comorbidities and living with other persons at home were associated with an increased risk of carriage. MR Staphylococcus epidermidis (MRSE) (from 32% to 78% of MRCNS isolates) were characterized by MLST and MLVA: a marked diversity was observed, suggesting that new strains commonly emerge in the community. SCCmec elements were highly polymorphic. SCCmec IV, the leading type in community-acquired MRSA (CA-MRSA), was the most frequent cassette in MRSE. Complete sequences of SCCmec IV from MRSE were >99% identical to those described in epidemic clones of CA-MRSA. These findings strongly suggest that CA-MRSE are a potential source of SCCmec IV for S. Aureus in the community. The Arginine Catabolic Mobile Element (ACME) found in 68% of CA-MRSE strains may esse their persistence in the skin and mucosal microbiota. The impact of antimicrobials on CA-MRCNS carriage was investigated in 479 primary care patients. CA-MRCNS acquisitions were more frequent in patients receiving penicillinase-resistant penicillins than in those treated by fluoroquinolones, macrolides or pristinamycin. These results point out that MRSE and other MRCNS are widely disseminated in the community, and form a dynamic reservoir of SCCmec elements in this environment

Introdução: a colonização de indivíduos com HIV/aids por microrganismos patogênicos tem sido associada a maior risco de morbidade e mortalidade, principalmente quando esse microrganismo é o Staphylococcus aureus. Identificar precocemente esta condição permite implementar medidas preventivas do adoecimento a ele relacionado, em nível individual e coletivo. Objetivo: avaliar a prevalência de colonização por Staphylococcus aureus em indivíduos com HIV/aids internados em um hospital escola. Metodologia: estudo de corte transversal, tendo como sujeito pessoas vivendo com HIV/aids, internadas em duas unidades especializadas em HIV/aids de um Hospital Escola do município de Ribeirão Preto- SP. Todos os preceitos éticos foram criteriosamente respeitados. No período de Agosto/2011 a Julho/2012, todos os indivíduos internados foram abordados e para aqueles que aceitaram participar, procedeu-se a coleta de amostra de saliva e secreção nasal, além da coleta de dados sociodemográficos, clínicos e imunológicos, obtidos por meio do prontuário e entrevista individual. As amostras foram encaminhadas e processadas pelo Laboratório de Microbiologia e Sorologia da instituição em estudo. Foram semeadas em meios de cultura ágar sangue e manitol, e após, transferidas para o sistema automatizado Vitek® 2 (BioMérieux(TM)), por meio dos cartões GP Test Kit Vitek® 2, para bactérias gram-positivas. Foram empregados cartões AST-P585 para avaliar a sensibilidade dos Staphylococcus aureus meticilina resistente (MRSA) aos antibióticos. Os dados foram armazenados em planilhas do Microsoft Office Excel 2011 for Mac e organizados por meio do software Statistical Package for the Social Sciences (SPSS), versão 17.0 for Windows. Resultados: De 229 indivíduos com HIV/aids internados nas unidades, 169 constituíram os sujeitos desta pesquisa, dos quais 57,4% eram do sexo masculino, 39,6% apresentaram idade de 40 a 49 anos e 45% tinham o primeiro grau completo. Foram obtidas 338 amostras (169 de secreção nasal e 169 de saliva). A prevalência de colonização por Staphylococcus aureus foi identificada em 20,4% das amostras, com 21,7% de resistência à oxacilina, sendo em secreção nasal 66,7% e em saliva 33,3%. Apresentaram contagem de linfócitos T CD4 abaixo de 200 células/mm3 60,0% dos indivíduos com MRSA nasal e 80,0% estavam em uso de antimicrobianos. Em 40,0% dos indivíduos com MRSA na saliva carga viral foi igual ou superior a 500.001 cópias/mL, e 80,0% destes também usavam antimicrobianos, MRSA nasal e saliva foi identificado em 60,0% dos indivíduos que não estavam em uso de antirretroviral. Conclusão: a prevalência de colonização por Staphylococcus aureus em indivíduos com HIV/aids foi predominante em secreção nasal, com baixa contagem de linfócitos T CD4, com história de internação prévia, uso de antimicrobiano e ausência do uso de antirretroviral, podendo representar importante fonte de infecção.
Introduction: colonization by pathogenic microorganisms in individuals with HIV/AIDS has been associated with increased risk of morbidity and mortality, especially when that organism is Staphylococcus aureus. Early identification of this condition allows implementing preventive measures of illness related to it, both individually and collectively. Objective: to evaluate the prevalence of Staphylococcus aureus colonization in individuals with HIV/AIDS in a teaching hospital. Method: cross-sectional study; the subjects were people living with HIV/AIDS and hospitalized in two specialized HIV/AIDS care units of a Teaching Hospital in the city of Ribeirão Preto. All ethical principles were carefully observed. In the period from August 2011 to July 2012, all subjects hospitalized were approached and, for those who agreed to participate, the collection of saliva and nasal discharge sample was performed, in addition to collecting sociodemographic, clinical and immunological data, obtained through medical record and individual interviews. The samples were forwarded and processed by the Laboratory of Microbiology and Sorology of the institution. They were seeded in blood agar and mannitol-salt-agar culture medium, and thereafter, transferred to the automated system Vitek® 2 (BioMérieux(TM)) through Vitek® 2 Test Cards for Gram-positive bacteria. AST-P585 cards were used to assess the sensitivity of methicillin-resistant Staphylococcus aureus (MRSA) to the antibiotic. Data were stored in spreadsheets of Microsoft Office Excel 2011 for Mac and organized by the Statistical Package for the Social Sciences (SPSS) version 17.0 for Windows. Results: of the 229 individuals with HIV/AIDS hospitalized in the units, 169 were the subjects in this study, of whom 57.4% were male, 39.6% were aged from 40 to 49 years, and 45% had completed elementary school. 338 samples were collected (169 of nasal discharge and 169 of saliva). The prevalence of Staphylococcus aureus colonization was identified in 20.4% of samples, with 21.7% of oxacillin resistance, being 66.7% in nasal discharge and 33.3% in saliva. 60.0% of individuals with MRSA in nasal had lymphocytes T CD4 count below 200 cells/mm3 , and 80.0% were taking antimicrobials. In 40.0% of the individuals with MRSA in saliva, the viral load was equal or higher than 500.001 copies/mL, and 80.0% of these also used antimicrobials; MRSA in nasal and in saliva were detected in 60.0% of individuals who were not taking antiretroviral. Conclusion: the prevalence of Staphylococcus aureus colonization in individuals with HIV/AIDS was prevalent in nasal discharge, had lymphocytes T CD4 low count, with a history of previous hospitalization, antimicrobial use and the absence of antiretroviral use, and it may represent an important source of infection.

Molecular imaging provides a less invasive means for studying bacterial infections in vivo compared to traditional techniques. However, molecular imaging of infection is currently limited due the lack of suitable imaging tracers. The most widely adopted approach for studying Staphylococcus aureus infections utilises a recombinant bioluminescent strain. However, several environmental requirements and signal attenuation through host tissue render this approach unsuitable for several applications. In order to provide an alternative research tool, the use of SNAP-tag, CLIP-tag and HaloTag was explored. Once expressed, these tags specifically bind fluorescent ligands. The tags were expressed via an inducible plasmid based system, and engineered to covalently attach to the cell surface of S. aureus. The function of the tags was validated in vitro by fluorescence imaging. SNAP-tag was subsequently incorporated into the genome of bioluminescent S. aureus Xen29. This thesis presents the first demonstration of visualising SNAP-tag expressing bacteria in vivo by fluorescent imaging. Potential applications for SNAP-tag imaging were then investigated; such as screening antimicrobials. Imaging of SNAP-tag expressing S. aureus was shown to provide additional information about the infection site compared to bioluminescence imaging alone. Novel [99mTc] SNAP-tag ligands were then developed and evaluated to provide a means for nuclear imaging of SNAP-tag. In addition, tools for infection diagnosis by nuclear imaging currently rely on targeting host immune responses rather than the bacteria directly; resulting in a high false positive rate. In order to develop a tracer with clinical potential to detect infection and not inflammation, the bacterial Universal Hexose Phosphate Transporter (UHPT) was selected as a target. [18F]FDG is an analogue of glucose and is widely used within nuclear medicine. [18F]FDG was phosphorylated, making it a substrate for UHPT. Validations in vitro suggested this probe may be a good tool for specific S. aureus detection; however in vivo biodistribution rendered it an unsuitable candidate.

Staphylococcus aureus is a bacterial pathogen of major health concern. Furthermore, the emergence of methicillin-resistant S. aureus (MRSA) strains limit the number of treatment options. Iron is essential for S. aureus survival and growth; however, the majority of iron in the human body is stored as hemoglobin (Hb) in erythrocytes. Hb released from lysed erythrocytes is quickly captured by serum haptoglobin (Hp) and the complex is degraded in the liver where released iron is then recycled. To date, the Iron Surface Determinant (Isd) system is the only heme utilization pathway identified in S. aureus. IsdB is the primary receptor at the cell surface that binds Hb to extract heme. Heme is then transferred to IsdA or IsdC, which then relay it to other Isd proteins for internalization. IsdB contains two NEAT (NEAr Iron Transporter) domains, and both domains and the intervening linker are needed for efficient heme uptake from Hb. In the first part of my study, the molecular mechanism of heme transfer from Hb to IsdB was investigated using site-directed mutagenesis. Residues in the heme-binding pocket were identified by inspection of a crystal structure of the complex of IsdB and Hb. The Y440F/Y444F (YFYF), E354A, M363L and S361A variants were found to be deficient in heme transfer. In particular, spectroscopic analysis of the YFYF mutant mixed with Hb provided evidence of a trapped heme transfer intermediate similar to that observed in the crystal structure of the wild-type protein. In the second part of my study, Hp was found to inhibit heme transfer from Hb to IsdB in a concentration-dependent manner and heme transfer was completely blocked when Hp was added to Hb above the binding stoichiometry determined by structural and solution studies. Moreover, Hb mixed with excess Hp did not support growth of S. aureus on iron-restrictive media. This study gained insight into the heme transfer mechanism between Hb and IsdB and revealed a novel role of Hp in blocking heme uptake by S. aureus.
Science, Faculty of
Microbiology and Immunology, Department of
Graduate

The Gram-positive bacterium Staphylococcus aureus is an extremely successful opportunistic bacterium capable of causing a wide range of hospital-acquired and community-acquired infections, and is becoming increasingly virulent and resistant to antibiotics. In order to investigate this pathogen, various methods have been used to analyse the pathogenic behaviour including genomics, transcriptomics and proteomics. S. aureus expresses approximately 55-70 lipoproteins with only about half with known functions. Little is known about the biochemical functions of many individual lipoproteins and their proteomics has not been investigated in detail. Lipoproteins have a broad ranging functionality and perform various roles in bacterial activity and attract a particular interest to investigate their virulence and survival influences in the course of host infection. The initial part of this study was to find out whether the lipoproteins of S. aureus have similar genetic characteristics among all strains. PCR and Quantitative Real-Time PCR experiments were performed to analyse the genetic and the expression levels for some lipoprotein genes. The majority of PCR results showed high similarity in lipoprotein genetic structure among the examined strains. Phylogenetic trees from concatenated lipoprotein genes alignment were generated to represent the lipoprotein genes distribution of S. aureus strains. To identify and characterise proteomic of S. aureus lipoproteins a comprehensive quantitative proteome profiling of S. aureus lipoproteins using gel-free /in-solution trypsin digestion system followed by LC-MS/MS quantification identified 38 lipoproteins that represent two-thirds of the S. aureus MRSA252 lipoprotein. In addition, S. aureus-mediated infections with live C. elegans were performed on solid assays to investigate the host-pathogen relationships. S. aureus MRSA252 exhibited a high level of nematocidal activity with average time for half of the worms to die of ~ 2 d and infected C. elegans showed visible signs of illness. To evaluate lipoprotein transcripts expression level and microbe/host-specific pathogenic factors RNA of both S. aureus and C. elegans were characterised after isolation from the infected C. elegans and subjected to RNA Sequencing, the large-scale data has provided useful information on pathogen and host activities during infection. RNA sequencing analysis showed different types of regulations and interactions of lipoprotein transcripts during host exposures to indicate 3 transcripts significantly were up-regulated and 11 down-regulated. RNA sequencing analysis showed that 62 lipoprotein transcripts were expressed during C. elegans infection model. Proteomic analysis using the application of gel-free proteomic technique identified 38 lipoproteins that were expressed in the non-infection condition representing approx. two-thirds of the S. aureus MRSA252 lipoproteins. The results suggest that some lipoproteins were involved in pathogenesis of C. elegans but their function were not clear. More research is needed for explore the roles of lipoproteins in pathogenesis and the interactions of S. aureus with the host immune responses.

A conjugation system in Staphylococcus aureus has been investigated and shown to be determined, at least in part, by genes carried on plasmids. Conjugation required cell-to-cell contact but not calcium ions. The frequency of conjugation depended on the recipient used and on the incubation conditions. Two conjugative plasmids were mapped by restriction enzyme analysis but experiments to clone the conjugation-determining region were unsuccessful although separate regions specifying gentamicin resistance, ethidium bromide resistance and cadmium resistance were cloned. The gentamicin resistance determinant was probably part of Tn4001. Deletion of various sized pieces of DNA from one of the plasmids resulted in reduction of its ability to specify conjugation but no specific part of this plasmid could be implicated in the process. Further experiments led to the conclusion that this particular plasmid (p8325-4) is probably not self-transmissible but transferred by a phage-mediated system. Strains of Staphylococcus aureus produced a pheromone-like substance that elicited a clumping response in Streptococcus faecalis but no evidence was found for the involvement of staphylococcal conjugative plasmids in this. The conjugative plasmid, p8325-2, mobilized a small plasmid (pT181) but not a chromosomal gene. Insertion of transposon Tn551 was used to produce mutants of the conjugative plasmid p8325-2. Some twenty-six mutants were studied and the position of Tn551 in them mapped. There were preferred regions of insertion for Tn551 and twenty out of the twenty-six mutants had altered ability to conjugate. One showed a significantly higher frequency of conjugation and the other nineteen, all with substantially lower frequencies of conjugation, were mapped to two well-separated regions of the plasmid. Similarity between the locations of these putative regions and those reported for some other conjugative plasmids from staphylococci is striking and suggests a common origin.

Staphylococcus aureus was discovered in 1880 by the Scottish surgeon Sir Alexander Ogston. Its ability to utilise a panoply of secreted and cell associated virulence factors, combined with an ability to rapidly adapt to antibiotic selective pressure, has led it to become one of the most important Gram positive pathogens of our time. It plays a significant role in chronic diseases such as eczema, as well as the acute and potentially life threatening conditions toxic shock syndrome (TSS), meningitis, pneumonia and staphylococcal scalded skin syndrome (SSSS) to name just a few. Its presence in hospitals is endemic in many countries, where methicillin resistant S. aureus (MRSA) strains have earned the 'superbug' moniker. Using a large, well-defined, clinical strain collection in combination with isogenic lab strains, this study endeavored to characterise the genetic factors associated with S. aureus toxicity, and identify novel means of controlling toxin expression. We found that: • A broad range of toxicities exists among S. aureus strains, that the beta- and delta-haemolysins are involved, and that hospital acquired-MRSA (HA-MRSA) strains are less toxic than MSSA or community acquired-MRSA (CA-MRSA). • NaCl and the emollient Oilatum downregulate toxin expression. • The mecA gene, conferring methicillin resistance, mediates mutation rates. • The large type II SCCmec element of HA-MRSA confers a significant metabolic burden which is mitigated by the down regulation of toxins. • The smaller type IV SCCmec element of CA-MRSA does not confer a significant metabolic burden, enabling CA-MRSA to utilise their full arsenal of virulence factors.

Cell division is a fundamental biological process that occurs in all kingdoms of life. Our understanding of cell division in bacteria stems from studies in the rod-shaped model organisms: Gram-negative Escherichia coli and Gram-positive Bacillus subtilis. The molecular underpinnings of cell division regulation in non-rod-shaped bacteria remain to be studied in detail. Rod-shaped bacteria possess many positive and negative regulatory proteins that are essential to the proper placement of the division septa and ultimately the production of two identical daughter cells, many of which are absent in cocci. Given that essential cell division proteins are attractive antibacterial drug targets, it is imperative for us to identify key cell division factors especially in pathogens, to help counter the emergence of multi-drug resistance. In Staphylococcus aureus, a spherical Gram-positive opportunistic pathogen that causes a range of diseases from minor skin infections to life-threatening sepsis, we have identified the role of an essential protein, GpsB, in the regulation of cell division. We discovered that GpsB preferentially localizes to cell division sites and that overproduction of GpsB results in cell enlargement typical of FtsZ inhibition, while depletion of GpsB results in cell lysis and nucleoid-less minicell formation. The identification of GpsB’s interaction partners will allow us to understand the molecular mechanism by which GpsB regulates cell division.