Vancomicina é o antimicrobiano de escolha no tratamento de bacteremias causadas por estafilococos resistentes à meticilina. No entanto, estudos recentes têm reportado que a vancomicina apresenta atividade reduzida contra Staphylococcus aureus resistentes à meticilina com concentrações inibitórias mínimas (CIMs) próximas do limite do ponto de corte de suscetibilidade, conforme critérios do CLSI, indicando falha terapêutica. Entretanto, existem poucos estudos à respeito dos Staphylococcus sp. coagulase negativa resistentes à meticilina (SCoNRM). Ademais, para determinação da CIM, deveria ser utilizado o método de referência microdiluição em caldo (MDC), mas a maioria dos laboratórios clínicos utiliza a técnica de Etest ou sistemas automatizados. Alguns estudos com S. aureus demonstraram discrepâncias entre MDC e Etest, contudo não existem dados referentes aos SCoN. Os objetivos deste estudo foram avaliar a correlação entre as CIMs de vancomicina determinadas pelas técnicas de MDC e Etest em 130 SCoNRM isolados de hemocultura, bem como verificar a relação entre valores de CIM e falha terapêutica entre pacientes com bacteremia por SCoNRM tratados com este antimicrobiano. A maioria dos resultados de CIM por MDC (98,5%) foram ≤1,0 mg/mL, enquanto o Etest apresentou 72,3% de CIM ≥ 1,5 mg/mL. As CIMs de vancomicina obtidas por Etest foram, em geral, uma a duas diluições maiores do que as CIMs obtidas por MDC. Os resultados indicam que a técnica de Etest gera valores de CIM consistentemente maiores do que os obtidos por MDC nos SCoNRM. Apenas 37 (28,5%) dos 130 pacientes com hemocultura positiva para SCoNRM apresentaram dados clínicos compatíveis com bacteremia. A maioria dos pacientes com bacteremia comprovada (n=24) apresentaram CIMs de vancomicina ≥1,5 mg/mL, sendo que 13 pacientes (35,1%) obtiveram CIM < 1,5 mg/mL. Este estudo não observou relação estatisticamente significativa entre valores de CIM de vancomicina que pudessem ser associados com falha terapêutica em pacientes com bacteremia por SCoNRM. / Vancomycin is the first-line therapy for methicillin-resistant staphylococci bacteremia. However, recent studies have reported that vancomycin demonstrates reduced activity against methicillin-resistant Staphylococcus aureus bacteremia, with vancomycin MICs at the high end of the CLSI susceptibility range indicating treatment failure. There is, however, little data considering methicillin-resistant coagulase-negative staphylococci (MRCoNS) bacteremia. Besides, the reference method that should be used for MIC determination is the broth microdilution (BMD), but many clinical laboratories use the commercial Etest technique or automated systems. Some reports have showed a growing number of vancomycin MIC discrepancies between BMD and Etest method for S. aureus, but there are no studies about CoNS. The aims of this study were to evaluated the correlation between the vancomycin MIC determined by the Etest and the BMD method for a total of 130 MRCoNS bloodstream isolates as well as to examine the relationship between vancomycin MICs and failure among patients with MRCoNS bacteremia treated with vancomycin. The vast majority (98.5%) of MIC results by BMD were ≤1.0 mg/mL in contrast to MIC by Etest which majority (72.3%) was ≥1.5 mg/mL. The vancomycin MICs obtained by the Etest for the same isolates were, in general, one to twofold higher than those obtained by the BMD method. The results indicate that the Etest provides vancomycin MIC values consistently higher than those obtained by BMD method for MRCoNS. Only 37 (28.5%) out of the 130 patients with a positive MRCoNS bloodstream culture met the eligibility criteria to be considered bacteremic. The majority of these patients (n = 24, 64.9%) presented vancomycin MIC ≥ 1.5 mg/mL, in opposite to 13 patients (35.1%) with MIC < 1.5 mg/mL. This study did not observe any statistical significative relationship between vancomycin MIC and treatment failure.

Vancomicina é o antimicrobiano de escolha no tratamento de bacteremias causadas por estafilococos resistentes à meticilina. No entanto, estudos recentes têm reportado que a vancomicina apresenta atividade reduzida contra Staphylococcus aureus resistentes à meticilina com concentrações inibitórias mínimas (CIMs) próximas do limite do ponto de corte de suscetibilidade, conforme critérios do CLSI, indicando falha terapêutica. Entretanto, existem poucos estudos à respeito dos Staphylococcus sp. coagulase negativa resistentes à meticilina (SCoNRM). Ademais, para determinação da CIM, deveria ser utilizado o método de referência microdiluição em caldo (MDC), mas a maioria dos laboratórios clínicos utiliza a técnica de Etest ou sistemas automatizados. Alguns estudos com S. aureus demonstraram discrepâncias entre MDC e Etest, contudo não existem dados referentes aos SCoN. Os objetivos deste estudo foram avaliar a correlação entre as CIMs de vancomicina determinadas pelas técnicas de MDC e Etest em 130 SCoNRM isolados de hemocultura, bem como verificar a relação entre valores de CIM e falha terapêutica entre pacientes com bacteremia por SCoNRM tratados com este antimicrobiano. A maioria dos resultados de CIM por MDC (98,5%) foram ≤1,0 mg/mL, enquanto o Etest apresentou 72,3% de CIM ≥ 1,5 mg/mL. As CIMs de vancomicina obtidas por Etest foram, em geral, uma a duas diluições maiores do que as CIMs obtidas por MDC. Os resultados indicam que a técnica de Etest gera valores de CIM consistentemente maiores do que os obtidos por MDC nos SCoNRM. Apenas 37 (28,5%) dos 130 pacientes com hemocultura positiva para SCoNRM apresentaram dados clínicos compatíveis com bacteremia. A maioria dos pacientes com bacteremia comprovada (n=24) apresentaram CIMs de vancomicina ≥1,5 mg/mL, sendo que 13 pacientes (35,1%) obtiveram CIM < 1,5 mg/mL. Este estudo não observou relação estatisticamente significativa entre valores de CIM de vancomicina que pudessem ser associados com falha terapêutica em pacientes com bacteremia por SCoNRM. / Vancomycin is the first-line therapy for methicillin-resistant staphylococci bacteremia. However, recent studies have reported that vancomycin demonstrates reduced activity against methicillin-resistant Staphylococcus aureus bacteremia, with vancomycin MICs at the high end of the CLSI susceptibility range indicating treatment failure. There is, however, little data considering methicillin-resistant coagulase-negative staphylococci (MRCoNS) bacteremia. Besides, the reference method that should be used for MIC determination is the broth microdilution (BMD), but many clinical laboratories use the commercial Etest technique or automated systems. Some reports have showed a growing number of vancomycin MIC discrepancies between BMD and Etest method for S. aureus, but there are no studies about CoNS. The aims of this study were to evaluated the correlation between the vancomycin MIC determined by the Etest and the BMD method for a total of 130 MRCoNS bloodstream isolates as well as to examine the relationship between vancomycin MICs and failure among patients with MRCoNS bacteremia treated with vancomycin. The vast majority (98.5%) of MIC results by BMD were ≤1.0 mg/mL in contrast to MIC by Etest which majority (72.3%) was ≥1.5 mg/mL. The vancomycin MICs obtained by the Etest for the same isolates were, in general, one to twofold higher than those obtained by the BMD method. The results indicate that the Etest provides vancomycin MIC values consistently higher than those obtained by BMD method for MRCoNS. Only 37 (28.5%) out of the 130 patients with a positive MRCoNS bloodstream culture met the eligibility criteria to be considered bacteremic. The majority of these patients (n = 24, 64.9%) presented vancomycin MIC ≥ 1.5 mg/mL, in opposite to 13 patients (35.1%) with MIC < 1.5 mg/mL. This study did not observe any statistical significative relationship between vancomycin MIC and treatment failure.

Linezolida foi o primeiro fármaco da classe das oxazolidinonas a ser aprovado para o uso clínico. Esta nova oxazolidinona inibe a síntese protéica impedindo a formação do complexo de iniciação formado pelo mRNA, tRNA f-Met e a subunidade 50S do ribossomo bacteriano. Embora a resistência à linezolida possa ser mediada pelo produto do gene cfr ou por mutações nas proteínas ribossômicas L3, L4 e L22, o mecanismo de resistência mais comum envolve mutações no domínio V do gene rRNA 23S. Entre março de 2008 a dezembro de 2011, 38 cepas de estafilococos coagulase-negativos (SCNs) resistentes à linezolida (20 S. epidermidis, 14 S. haemolyticus, 3 S. hominis e 1 S. warneri) isoladas de hemoculturas e pontas de cateter de pacientes internados em dois hospitais terciários do Estado de São Paulo foram incluídas neste estudo para a determinação dos mecanismos de resistência e análise da estabilidade do fenótipo resistente. As cepas de SCNs apresentaram altos níveis de resistência à linezolida (CIMs de 16-128 &#181;g/ml) e foram multi-resistentes, permanecendo sensíveis à vancomicina e teicoplanina. A mutação G2576T foi identificada no domínio V do gene rRNA 23S em todas as cepas de SCNs, exceto em uma cepa de S. haemolyticus. O gene cfr e mutações nas proteínas L4 e L22 não foram detectados. Em relação à proteína L3, todas as cepas de S. epidermidis do hospital A, incluindo a cepa controle sensível à linezolida, apresentaram a substituição Leu101Val, sugerindo que essa mutação seja um marcador clonal dessa população sem envolvimento com a resistência à linezolida. A única cepa proveniente do hospital B (S. epidermidis) foi selvagem para essa proteína ribossômica. Somente uma cepa de S. haemoyticus teve uma mutação no gene rplC, resultando na alteração Val154Leu. Em S. hominis, a mutação Phe147Ile foi identificada em uma cepa, enquanto a associação de Gly139Arg e Met156Thr foi observada nas outras duas cepas dessa espécie. A identificação dessas mutações na proteína L3 de cepas de S. haemoyticus e S. hominis resistentes à linezolida reforça o papel desses sítios na aquisição da resistência ao fármaco em Staphylococcus spp. No entanto, a presença de G2576T no rRNA 23S torna difícil determinar exatamente qual o envolvimento das mutações na L3 com os elevados níveis de resistência à linezolida apresentados por essas cepas. Na ausência da pressão seletiva do antimicrobiano, após 130 passagens, a resistência à linezolida mediada pela mutação G2576T permaneceu estável nas cepas de SCNs deste estudo, as quais, de acordo com os perfis de restrição do domínio V gerados por NheI, tinham tanto alelos rRNA 23S selvagens como mutados. O sequenciamento individual do domínio V das diferentes cópias do gene rRNA 23S mostrou G2576T em todas as cópias amplificadas por PCR: 4/4 e 5/5 em S. epidermidis e 3/3 em S. haemolyticus (CIMs de 16-32 &#181;g/ml). A estabilidade da cópia rRNA 23S mutada foi observada mesmo em uma cepa S. epidermidis sensível à linezolida, a qual apresentou uma redução da CIM de 4 para 1 &#181;g/ml mantendo seu único alelo mutado ao longo do processo de reversão ao fenótipo sensível. A similaridade genética foi determinada por PFGE e mostrou uma disseminação clonal das diferentes espécies de SCNs resistentes à linezolida. A análise das cepas de S. epidermidis por MLST mostrou a ocorrência do clone ST-2 (CC2) nos dois hospitais. O aumento da pressão seletiva devido a exposições cada vez mais frequentes à linezolida, provavelmente, favoreceu a seleção e a disseminação de clones endêmicos de SCNs com a mutação G2576T na instituição A desde 2008. De forma diferente, o uso mais restrito do fármaco na instituição B poderia explicar a ocorrência isolada de uma única cepa resistente desde 2005. / Linezolid was the first agent of the oxazolidinone class to be introduced clinically. This oxazolidinone inhibits protein biosynthesis by preventing the formation of the initiation complex that consists of the mRNA, the f-Met tRNA and the 50S subunit of the ribosome. Although linezolid resistance has been mediated by the cfr-encoded product or by ribosomal proteins (L3, L4 and L22), the most common mechanism of resistance involves mutations in the central loop of domain V of the 23S rRNA gene. From March 2008 to December 2011, 38 coagulase-negative staphylococci (CNS) strains (20 S. epidermidis, 14 S. haemolyticus, 3 S. hominis e 1 S. warneri) exhibiting resistance to linezolid were isolated from blood and catheter cultures from patients in two tertiary care hospitals in the State of São Paulo and were included in this study for the ascertainment of the resistance mechanisms to this antimicrobial agent and for the analysis of the stability of this resistance. The strains exhibited high-level resistance to linezolid (MICs 16-128 &#181;g/ml) and all were multidrug resistant, remaining susceptible to vancomycin and teicoplanin. The G2576T mutation in domain V region of 23S rRNA was identified in all isolates, except in a linezolid-resistant S. haemolyticus strain. The cfr gene and mutations in ribosomal proteins L4 and L22 were not detected. Regarding L3 protein analysis, all S. epidermidis strains of hospital A, including the linezolid-susceptible control strain, showed the L3 Leu101Val mutation, suggesting that this alteration is probably not involved in linezolid resistance. The one strain from hospital B (S. epidermidis) was wild-type for this ribosomal protein. Only one S. haemolyticus strain had a mutation in the L3 protein, Val154Leu. Two S. hominis strains showed Gly139Arg/Met156Thr mutations whereas one strain had Phe147Ile in L3 protein. The identification of these mutations in L3 protein of the linezolid-resistant S. haemolyticus and S. hominis strains strengthens the role of these sites in the acquisition of linezolid resistance in Staphylococcus spp. However, the presence of G2576T in the 23S rRNA gene makes difficult to determine exactly the role of L3 mutations in conferring elevated linezolid MIC values showed by these clinical strains. In the absence of antibiotic pressure, after 130 passages, linezolid resistance was stable in the clinical strains of this study, which did not have all copies of the 23S rRNA gene mutated, according to the restriction of the domain V fragment with NheI enzyme. Sequencing of the individual copies of the 23S rRNA gene in the serially passaged strains showed G2576T in all amplified copies by PCR: 4/4 and 5/5 in S. epidermidis and 3/3 in S. haemolyticus strains (MIC of 16-32 &#181;g/ml). The stability of the mutant rRNA copy was also observed in the linezolid-susceptible S. epidermidis strain (MIC of 4 &#181;g/ml). After the passages in antibiotic-free medium, the linezolid MIC of this strain fell to 1 &#181;g/ml and the G2576T mutation persisted in one 23S rRNA gene copy. The clonal relatedness of the strains was determined by PFGE and revealed a clonal dissemination of different CNS species. Regarding MLST analysis, all S. epidermidis strains belonged to the sequence type ST2 (CC2). Most likely, the increased selective pressure has contributed to the selection of endemic linezolid-resistant CNS clones showing the G2576T mutation that have been disseminated in the institution A since 2008. Differently, the restricted use of linezolid in the institution B could explain the occurrence of a single resistant strain since 2005.

Made available in DSpace on 2017-07-10T17:48:02Z (GMT). No. of bitstreams: 1 Gilberto_Henrique_Simoes.pdf: 609344 bytes, checksum: c392f5329a574e72a4a5033766bd505a (MD5) Previous issue date: 2014-04-15 / The complexity and diversity of the dairy production chain in the country are due to the different types of dairy production systems and their specific characteristics, among them, the spatial and cultural differences found across the country, making it difficult to standardize the production, planning, technicization and especially the microbiological quality of the raw material in the dairy chain. In order to illustrate the dairy production systems, the managements, critical points of contamination, isolation and identification of the main factors involved in the contamination in Marechal Cândido Rondon - PR, some experiments were conducted. Initially it was performed a selection among 735 dairy production systems for semi - structured guides (production characteristics and general management) and cluster analyzes, which formed five distinct and homogeneous groups of dairy production systems. Only 10% of properties were selected to represent each group, totalizing 73 properties. Subsequently it was analyzed the milk cooling tanks for the somatic cell count (SCC) and total the bacterial count (TBC). After the counts, only 35 properties were selected for the research because they showed contaminations rates above current limits for SCC and TBC according to the MAPA regulation (IN 62). After the formation of the systems, collections related to the research were made, which consisted of the semi-structured questionnaire guide (hygienic-sanitary management), milk collection of the tank and swabs of critical points of contamination in milk production (hand of the milker, milking machine and cooling tank). The experiment was proceeded with microbiological analysis and quantification of Staphylococcus sp, hand of the milker, milking machine, tank and milk 9.6 x103, 2.2 x104, 1.4 x104 and 3.8 x103 CFU/mL respectively, and quantification, isolation and identification of proteolytic and lipolytic bacterias, in which the agents of highest incidence were Escherichia coli, Escherichia fergusoni, Yersinia enterocolitica and Klebsiella oxytoca both with 8.6% frequency in the different dairy production systems studied / A complexidade e diversidade da cadeia produtiva leiteira existente no país devem-se aos diferentes tipos de sistemas produtivos leiteiros e as características próprias que os envolvem, entre elas as diferenças espaciais e culturais encontradas em todo o território nacional, dificultando assim uma padronização na produção, planejamento, tecnificação e principalmente da qualidade de produção microbiológica da matéria-prima na cadeia leiteira. Afim de caracterizar os sistemas de produção leiteiros, os manejos empregados, pontos críticos de contaminação e de isolamento e identificação dos principais agentes envolvidos nas contaminações da microrregião de Marechal Cândido Rondon PR, estudos foram realizados. Inicialmente efetuou-se seleção dentre 735 sistemas de produção leiteiros, nestes foram aplicados guias semiestruturados para levantamento de características de produção e manejo produtivo. A partir destes resultados foram empregadas técnicas de formação de clusters. Foram definidos cinco grupos homogêneos e distintos. Em cada grupo, 10% dos casos foram selecionados, totalizando 73 sistemas produtivos leiteiros. Posteriormente analisou-se o leite dos tanques de resfriamento para contagem de células somáticas (CCS) e contagem bacteriana total (CBT). Após as contagens, 35 propriedades foram selecionadas paras as pesquisas, pois obtiveram contaminações acima dos limites vigentes de CCS e CBT de acordo com IN 62 do MAPA. Após a formação dos sistemas sucedeu-se coletas referentes às pesquisas, que consistiram de questionário semi-estruturado, contemplando variáveis relativas ao manejo higiênico-sanitário, bem como, coleta de leite do tanque e swabs de pontos críticos de contaminação na produção leiteira, tais como: mão do ordenhador, ordenhadeira e tanque de resfriamento. Procedeu-se com análises microbiológicas e a quantificação de Staphylococcus sp da mão-do-ordenhador, ordenhadeira, tanque e leite 9,6x103, 2,2x104, 1,4x104 e 3,8x103 UFC/mL respectivamente; e quantificação, isolamento e identificação de bactérias proteolíticas e lipolíticas, nos quais os agentes de maior incidência foram a Escherichia coli, Escherichia fergusoni, Klebsiella oxytoca e Yersinia enterocolitica ambos com 8,6% de frequência nos diferentes sistemas de produção leiteiros estudados

Coagulase negative staphylococci (CoNS) are the most prevalent bacterial contaminants of platelet concentrates (PCs), and have been implicated in severe and fatal transfusion reactions. Of this group, Staphylococcus epidermidis is most frequently identified. The preliminary objective of this thesis was to confirm that S. epidermidis could form biofilms under platelet storage conditions. This was achieved using a modified crystal violet staining assay to detect plastic-adherent bacterial cells and examination of attachment processes by scanning electron microscopy. A collection of CoNS isolated from PCs obtained from reportedly healthy donors was then assessed for biofilm-forming potential at the genetic and phenotypic level. Despite the presumable commensal origin of these isolates, a high proportion of S. epidermidis strains displayed a biofilm positive phenotype. The threat of S. epidermidis biofilm formation during platelet storage identified herein signifies that any alterations made to platelet storage protocols should be evaluated with consideration of this risk. The advent of platelet additive solutions (PASs) as an alternative to plasma for PC storage provides a relevant example, since little is known about the effect of PAS on contaminant bacteria, and vice versa. Growth and biofilm formation by S. epidermidis and the Gram-negative bacterium Serratia liquefaciens were measured in PAS- or plasma-PCs over 5 days, simulating standard platelet storage conditions, after initial inoculation with low, clinically relevant bacterial concentrations. Assays for platelet quality were performed simultaneously. Only S. liquefaciens exhibited a slower doubling time in plasma-PCs than in PAS-PCs. Biofilm formation by both species was reduced during storage in PAS-PCs, increasing bacteria availability for detection. Although S. liquefaciens adversely affected platelet quality in both media, S. epidermidis contamination did not. Ultimately, culture-based detection remains the earliest indicator of bacterial presence in PAS-PCs. Lastly, since formation of platelet-bacteria aggregates is largely based on receptor-ligand interactions, it was postulated that biofilm formation by contaminant bacteria could be abrogated by receptor shielding. Methoxypoly(ethylene glycol) was applied to covalently modify the platelet surface using a process termed ‘PEGylation’. It is herein demonstrated that PEGylation of PCs inoculated with S. epidermidis results in significantly reduced bacterial binding and biofilm formation during platelet storage.

Coagulase negative staphylococci (CoNS) are the most prevalent bacterial contaminants of platelet concentrates (PCs), and have been implicated in severe and fatal transfusion reactions. Of this group, Staphylococcus epidermidis is most frequently identified. The preliminary objective of this thesis was to confirm that S. epidermidis could form biofilms under platelet storage conditions. This was achieved using a modified crystal violet staining assay to detect plastic-adherent bacterial cells and examination of attachment processes by scanning electron microscopy. A collection of CoNS isolated from PCs obtained from reportedly healthy donors was then assessed for biofilm-forming potential at the genetic and phenotypic level. Despite the presumable commensal origin of these isolates, a high proportion of S. epidermidis strains displayed a biofilm positive phenotype. The threat of S. epidermidis biofilm formation during platelet storage identified herein signifies that any alterations made to platelet storage protocols should be evaluated with consideration of this risk. The advent of platelet additive solutions (PASs) as an alternative to plasma for PC storage provides a relevant example, since little is known about the effect of PAS on contaminant bacteria, and vice versa. Growth and biofilm formation by S. epidermidis and the Gram-negative bacterium Serratia liquefaciens were measured in PAS- or plasma-PCs over 5 days, simulating standard platelet storage conditions, after initial inoculation with low, clinically relevant bacterial concentrations. Assays for platelet quality were performed simultaneously. Only S. liquefaciens exhibited a slower doubling time in plasma-PCs than in PAS-PCs. Biofilm formation by both species was reduced during storage in PAS-PCs, increasing bacteria availability for detection. Although S. liquefaciens adversely affected platelet quality in both media, S. epidermidis contamination did not. Ultimately, culture-based detection remains the earliest indicator of bacterial presence in PAS-PCs. Lastly, since formation of platelet-bacteria aggregates is largely based on receptor-ligand interactions, it was postulated that biofilm formation by contaminant bacteria could be abrogated by receptor shielding. Methoxypoly(ethylene glycol) was applied to covalently modify the platelet surface using a process termed ‘PEGylation’. It is herein demonstrated that PEGylation of PCs inoculated with S. epidermidis results in significantly reduced bacterial binding and biofilm formation during platelet storage.

Σκοπός της παρούσας ερευνητικής εργασίας ήταν η επιδημιολογική μελέτη των λοιμώξεων από πηκτάση-αρνητικούς σταφυλοκόκκους (CNS) σε ασθενείς με προσθετικά υλικά, όπως ενδαγγειακούς καθετήρες και η σύγκριση με στελέχη που προκαλούν βακτηριαιμία. Συνολικά μελετήθηκαν 168 Staphylococcus epidermidis και 58 S. haemolyticus από βακτηριαιμίες (BSIs, 100 στελέχη) και εντοπισμένες λοιμώξεις σχετιζόμενες με την εφαρμογή προσθετικών υλικών (PDAIs, 126 στελέχη) από ασθενείς του Πανεπιστημιακού Γενικού Νοσοκομείου Πατρών (ΠΓΝΠ) και του Νοσοκομείου Παίδων Πεντέλης (ΝΠΠ). Η πλειοψηφία των στελεχών (89.8%) ήταν ανθεκτικά στην methicillin (MR-CNS) και πολυανθεκτικά (90.7%). Βιομεμβράνη συνέθεταν τα 106/226 στελέχη, ενώ 208 παρήγαγαν β-λακταμάση. Τα γονίδια σύνθεσης προσκολλητινών aap, fnbA και bap βρέθηκαν σε συχνότητα 40.3%, 35.8% και 20.4% αντίστοιχα. Οι S. epidermidis έφεραν τα γονίδια atlE και fbe σε ποσοστά 88.1% και 81%, αντίστοιχα. Από τα γονίδια σύνθεσης τοξινών, συχνότερο ήταν το γονίδιο της τοξίνης τοξικής καταπληξίας tst (8.4%) ενώ τα γονίδια που κωδικοποιούν τις εντεροτοξίνες sea, sec βρέθηκαν μόνο σε μικρό ποσοστό στελεχών S. epidermidis και S. haemolyticus (5.3% και 3.1% του συνολικού πληθυσμού αντίστοιχα). Κανένα στέλεχος δεν έφερε τα γονίδια σύνθεσης των εντεροτοξινών seb και sed. Ο πληθυσμός των στελεχών S. epidermidis έδειξε μεγάλη γενετική ποικιλομορφία, με 67 PFGE τύπους, μεταξύ των οποίων δύο κύριοι τύποι (a, b) με 50 και 36 στελέχη αντίστοιχα. Έλεγχος με MLST ανέδειξε τρεις κύριους κλώνους (ST2, ST5 και ST16) που ανήκαν στο ίδιο κλωνικό σύμπλεγμα (Clonal Complex 2). Τα στελέχη του PFGE τύπου a παρουσίασαν υψηλότερα ποσοστά αντοχής στα αντιμικροβιακά clindamycin, ciprofloxacin, fusidic acid, SXT και στις αμινογλυκοσίδες, ενώ τα στελέχη του τύπου b έφεραν συχνότερα το γονίδιο aap (p=0.049). Τα στελέχη S. haemolyticus παρουσίασαν μικρότερη γενετική ποικιλομορφία, με έναν κύριο PFGE τύπο (h), που περιελάμβανε 44/58 στελέχη (75.9% του συνολικού πληθυσμού). Τα στελέχη CNS από BSIs ήταν συχνότερα ανθεκτικά στην methicillin (p<0.001) και στα υπόλοιπα αντιμικροβιακά (p<0.05), ενώ υπερείχαν και στην παραγωγή biofilm (p=0.003). Αντιθέτως, οι CNS από PDAIs έφεραν συχνότερα τα γονίδια των προσκολλητινών aap (p=0.006) και bap (p=0.045). Σε ένα δεύτερο σκέλος της παρούσας ερευνητικής εργασίας μελετήθηκε ένας πληθυσμός S. lugdunensis από το ΠΓΝΠ (37 στελέχη) και το ΝΠΠ (1 στέλεχος). Ο S. lugdunensis κατέχει ιδιαίτερη θέση μεταξύ των CNS, καθώς μπορεί να μιμηθεί την παθογόνο δράση του S. aureus και να προκαλέσει σοβαρές λοιμώξεις. Είκοσι δύο S. lugdunensis απομονώθηκαν από ασθενείς με λοιμώξεις δέρματος και μαλακών μορίων (Skin and Soft Tissue Infections: SSTIs), εννέα από εν τω βάθει λοιμώξεις (Deep Sited Infections: DSIs), συμπεριλαμβανομένων τριών στελεχών από ασθενείς με βακτηριαιμία, και επτά στελέχη από λοιμώξεις σχετιζόμενες με προσθετικά υλικά, κυρίως ενδαγγειακούς καθετήρες, (PDAIs). Όλα τα στελέχη ήταν ευαίσθητα στην methicillin (MS-CNS), στις αμινογλυκοσίδες (kanamycin, gentamicin), καθώς και στα: ciprofloxacin, rifampicin, teicoplanin, vancomycin, linezolid και daptomycin, ενώ μόνο τέσσερα στελέχη ήταν πολυανθεκτικά. Οι S. lugdunensis της συλλογής μας έδειξαν μικρή γενετική ποικιλομορφία. Τα 38 στελέχη ταξινομήθηκαν σε επτά κλώνους, με δύο κύριους PFGE τύπους (C και D), οι οποίοι περιελάμβαναν 22 και εννέα στελέχη αντίστοιχα. Τα 26 από τα 38 στελέχη έφεραν το οπερόνιο ica, ενώ συνολικά 14 ήταν biofilm-θετικά. Δεν παρατηρήθηκε συσχέτιση της παρουσίας του ica με κάποιο κλώνο, αλλά ούτε και με την παραγωγή βιομεμβράνης. Οι S. lugdunensis από PDAIs ήταν συχνότερα biofilm-θετικοί σε σχέση με τα στελέχη από SSTIs και DSIs, ενώ ο κύριος κλώνος C παρήγαγε biofilm σε μεγαλύτερο ποσοστό από τον D, δεύτερο σε συχνότητα κλώνο. Το γονίδιο fbl ανιχνεύθηκε σε όλα τα στελέχη S. lugdunensis που εξετάστηκαν, επιβεβαιώνοντας την φαινοτυπική ταυτοποίηση σε επίπεδο είδους. Ο επόμενος κατά σειρά συχνότητας παράγοντας παθογένειας που ανιχνεύθηκε ήταν το γονίδιο atlL, το οποίο βρέθηκε σε 36 από τα 38 στελέχη (94.7%). Ακολουθούν οι παράγοντες vwbl και slush, που βρέθηκαν σε 31 (81.6%) και 15 (39.5%) S. lugdunensis, αντίστοιχα. Τα στελέχη από εν τω βάθει λοιμώξεις (DSIs) έφεραν σε μεγαλύτερο ποσοστό τα γονίδια vwbl και slush σε σχέση με αυτά από PDAIs και SSTIs . Ο κλώνος C υπερείχε στην παρουσία του ermC, ενώ τα στελέχη που ανήκαν στον κλώνο D έφεραν σε μεγαλύτερο ποσοστό τα γονίδια vwbl και slush. / Coagulase-negative staphylococci (CNS), especially Staphylococcus epidermidis and S. haemolyticus, have emerged as opportunistic pathogens in patients with low immune response or indwelling medical devices. In the present study, bloodstream (BSIs) and prosthetic-device associated infections (PDAIs) CNS isolates were compared in terms of biofilm formation, antimicrobial resistance, clonal distribution, adhesin and toxin genes carriage. A collection of 226 CNS (168 S. epidermidis and 58 S. haemolyticus) recovered from BSIs (100) and PDAIs (126) of different patients in the Patras tertiary-care University General Hospital (UGHP) and Pentelis Paediatric Hospital in Athens (PPHA), was tested for biofilm formation, antimicrobial susceptibility, mecA, ica operon, adhesin (aap, bap, fnbA, atlE, fbe) and toxin (tst, sea, seb, sec, sed) genes carriage. All CNS were classified into pulsotypes by PFGE, whereas S. epidermidis strains were assigned to sequence types by MLST. In total, 106 isolates (46.9%) produced biofilm, whereas 150 (66.4%) carried ica operon. Most isolates carried mecA and were multidrug resistant (90.7%). The adhesin encoding genes aap, fnbA and bap were identified in 40.3%, 35.8% and 20.4% of the total population, respectively. Genes encoding AtlE and Fbe were found in 88.1% and 81% of S. epidermidis isolates, respectively. CNS recovered from BSIs prevailed in biofilm formation (P=0.003), resistance to antimicrobials and mecA carriage (P<0.001) as compared to isolates derived from PDAIs. CNS from PDAIs carried more frequently aap and bap genes (P=0.006 and P=0.045, respectively). No statistically significant difference in toxin genes carriage was identified (P>0.05). Even though PFGE showed genetic diversity, especially among S. epidermidis, analysis of representative strains from the main PFGE types by MLST, revealed three major clones (ST2, ST5, ST16). A clonal relationship was found concerning antimicrobial susceptibility, ica and aap gene carriage, reinforcing the aspect of clonal expansion in hospital settings. Pathogenesis of BSIs is associated with biofilm formation and high-level antimicrobial resistance, whereas PDAIs are related to the adhesion capability of CNS. In the second part of this study we analyzed a collection of S. lugdunensis isolates recovered from different inpatients hospitalized in UGHP (37 isolates) and PPHA (one isolate) during a six-year period (2008-2013). S. lugdunensis has emerged as a significant human pathogen with distinct clinical and microbiological characteristics. A collection of 38 S. lugdunensis was tested for biofilm formation, antimicrobial susceptibility, clonal distribution, virulence factors (ica operon, fbl, atlL, vwbl, slush) and antibiotic resistance genes (mecA, ermC) carriage. The majority (22) was isolated from skin and soft tissue infections (SSTIs), nine from deep-sited infections (DSIs), including three bacteraemias and seven from PDAIs. All isolates were oxacillin-susceptible, mecA-negative and fbl-positive. The higher resistance rate was detected for ampicillin (50%), followed by erythromycin and clindamycin (18.4%). Fourteen isolates (36.8%) produced biofilm, 26 carried ica operon, but no relation between ica carriage and biofilm formation was identified. Biofilm formation was more frequent in isolates recovered from PDAIs. Thirty six strains (94.7%) carried atlL, 31 (81.6%) vwbl, whereas, slush was detected in fifteen (39.5%). PFGE revealed low level of genetic diversity: strains were classified into seven pulsotypes, with two major clones C and D including 22 and nine strains, respectively. Type C strains, recovered from all infection sites, prevailed in biofilm formation and ermC carriage, whereas, type D strains, associated with SSTIs and DSIs, carried more frequently vwbl, slush or both genes. Despite susceptibility to antimicrobials, clonal expansion and carriage of virulence factors combined with biofilm-producing ability render this species an important pathogen that should not be ignored.

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]

Coagulase negative staphylococci (CoNS) are the most prevalent bacterial contaminants of platelet concentrates (PCs), and have been implicated in severe and fatal transfusion reactions. Of this group, Staphylococcus epidermidis is most frequently identified. The preliminary objective of this thesis was to confirm that S. epidermidis could form biofilms under platelet storage conditions. This was achieved using a modified crystal violet staining assay to detect plastic-adherent bacterial cells and examination of attachment processes by scanning electron microscopy. A collection of CoNS isolated from PCs obtained from reportedly healthy donors was then assessed for biofilm-forming potential at the genetic and phenotypic level. Despite the presumable commensal origin of these isolates, a high proportion of S. epidermidis strains displayed a biofilm positive phenotype. The threat of S. epidermidis biofilm formation during platelet storage identified herein signifies that any alterations made to platelet storage protocols should be evaluated with consideration of this risk. The advent of platelet additive solutions (PASs) as an alternative to plasma for PC storage provides a relevant example, since little is known about the effect of PAS on contaminant bacteria, and vice versa. Growth and biofilm formation by S. epidermidis and the Gram-negative bacterium Serratia liquefaciens were measured in PAS- or plasma-PCs over 5 days, simulating standard platelet storage conditions, after initial inoculation with low, clinically relevant bacterial concentrations. Assays for platelet quality were performed simultaneously. Only S. liquefaciens exhibited a slower doubling time in plasma-PCs than in PAS-PCs. Biofilm formation by both species was reduced during storage in PAS-PCs, increasing bacteria availability for detection. Although S. liquefaciens adversely affected platelet quality in both media, S. epidermidis contamination did not. Ultimately, culture-based detection remains the earliest indicator of bacterial presence in PAS-PCs. Lastly, since formation of platelet-bacteria aggregates is largely based on receptor-ligand interactions, it was postulated that biofilm formation by contaminant bacteria could be abrogated by receptor shielding. Methoxypoly(ethylene glycol) was applied to covalently modify the platelet surface using a process termed ‘PEGylation’. It is herein demonstrated that PEGylation of PCs inoculated with S. epidermidis results in significantly reduced bacterial binding and biofilm formation during platelet storage.

Estafilococos têm sido relatados como os agentes mais prevalentes de mastite bovina. Este gru-po bacteriano pode transportar múltiplos elementos de resistência provenientes inclusive de outras populações bacterianas, tornando-se um grande problema de saúde pública, uma vez que também está envolvido em vários processos de doença em humanos, incluindo infecções da pele e dos tecidos moles, septicemia, osteomielite e pneumonia. A caracterização do perfil de resis-tência dos estafilococos aos antimicrobianos é importante para controlar a sua disseminação. O presente trabalho avaliou a distribuição de multirresistência entre mais de 3.500 isolados de um estudo transversal repetido, realizado entre 2010 e 2011 nas principais regiões produtoras de leite do Brasil. As bactérias foram classificadas de acordo com métodos fenotípicos e os padrões de resistência antimicrobiana foram determinados pelo teste de difusão em disco ao invés de testes moleculares e ensaios quantitativos. Para avaliar os principais fatores relacionados à vari-ável resposta – proporção de bactérias resistentes a três ou mais classes de antimicrobianos, ou multirresistência – diversas variáveis explicativas foram acessadas por meio de um questionário epidemiológico. Um modelo misto foi construído com um componente aleatório multinível, a saber, a variação de multirresistência entre as bacias leiteiras (segundo nível) e a variação dass propriedades em cada bacia (primeiro nível) durante os quatro momentos de amostragem, ou seja, a variação entre-sujeitos e intra-sujeitos, respectivamente. A avaliação dos perfis de resis-tência revelou que as penicilinas, seguidas por tetraciclina e sulfonamida, foram os antimicrobi-anos com menor eficácia em estafilococos (n = 3009). Estafilococos coagulase negativa pare-cem ter semelhanças com S. aureus e alguns outros estafilococos coagulase positiva testados, demonstrando um padrão de grupo com moderada resistência a múltiplas drogas. Ilustrativa-mente, alguns estreptococos (n = 480) também foram submetidos a testes de suscetibilidade a antimicrobianos, os quais mostraram moderada a alta resistência à tetraciclina, gentamicina e clindamicina. O modelo misto indicou que o tratamento de mastite feito de imediato, a aplicação de terapia antimicrobiana pelo próprio produtor ao invés de um veterinário, e a interação entre este último fator e o sistema de produção intensivo aumentou a probabilidade de resistência múltipla em nível de rebanho. O coeficiente de correlação intraclasse (ICC) foi baixo e mostrou que a maior parte da variação de resistência múltipla é explicada pelas características em nível de hospedeiro, seguido por fatores em nível de rebanho (ICC = 0,126). Esses fatores são exem-plos importantes de como o uso leigo de antimicrobianos em vacas leiteiras tem grande poten-cial para seleção, expansão e manutenção de populações de bactérias resistentes a múltiplas drogas em ambientes de produção animal, em especial nos mais intensivos. / Staphylococci have already been reported as the most prevalent mastitis agents. Such bacterial species can carry multidrug-resistant elements coming from other bacteria species, and so on are becoming a great public health concern, once it is also involved in several human disease. The characterization of their antimicrobial resistance profile is important to better control their dissemination. The present work evaluated the distribution of multidrug-resistance among more than 3500 isolates from a repeated cross-sectional study performed from 2010 to 2011 in the main dairy regions of Brazil. The bacteria were classified according to phenotypic methods and the antimicrobial resistance patterns were determined by disk diffusion. To evalu-ate the main factors related to the response variable – the proportion of bacteria resistant to three or more antimicrobial classes, or multidrug resistance – several explanatory variables were accessed by means of an epidemiological questionnaire. A multivariable mixed model was created to access the strength of association between several putative risk factors and re-sistance to multiple drugs. Resistance profiles evaluation revealed that penicillins, followed by tetracycline and sulfonamide, were the antimicrobials with lower effectiveness in staphylococci (n = 3009). Coagulase negative staphylococci seemed to have similarities with coagulase posi-tive tested, performing moderate resistance pattern to multiple drugs. Some streptococci (n = 480) were also submitted to antimicrobial susceptibility tests, and showed moderate to high resistance to tetracycline, gentamycin and clindamycin. The mixed model indicated that mastitis treatment made immediately rather than cautiously; applicaton of antimicrobial therapy by the producer itself rather than a veterinarian practitioner; and the interaction between the produc-er as applicator and the intensive, modern production system increased the likelihood of multi-ple-resistance at herd level. The low intraclass correlation coefficient at region level showed that resistance variance is most explained at herd level, and it characteristics, rather than re-gion level. Probably the host level factors not evaluated in this study explains more than the herd level factors, but, considering the coverage of our study, these factors give us insights of how cattle antimicrobial consumption can affect the maintenance and expansion of multidrug resistant bacteria populations in animal production environments.