Orientador: Arnaldo Yoshiteru Kuaye / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-12T19:50:34Z (GMT). No. of bitstreams: 1 Rosado_MarciliaSantos_M.pdf: 15617760 bytes, checksum: 0a6732c38e48cb504e3b62de52b13c78 (MD5) Previous issue date: 2009 / Resumo: Dentre os microrganismos que apresentam capacidade de aderir e formar biofilmes em superfícies de processamento de alimentos, como equipamentos e utensílios, estão às bactérias do gênero Enterococcus, presentes em alimentos como leite e derivados. Sua presença em queijos tem sido muito estudada por sua capacidade de conferir características tecnológicas desejáveis ao produto. Este trabalho objetivou avaliar as características tecnológicas de Enterococcus faecium 946Ec, isolado de queijo de coalho, a possível formação de biofilme em superfície de aço inoxidável e seu controle por agentes sanitizantes. A cultura de E. faecium 946Ec foi caracterizada tecnologicamente, apresentando lenta produção de ácido, baixa atividade proteolítica e produção de diacetil; a curva de crescimento no caldo MRS apresentou contagem de 1,48 x 109 UFC.mL-1 após 24 horas. A avaliação da formação de biofilme foi realizada em cupons de aço inoxidável AISI 304, com rugosidade média de 0,366 mm, nos tempos de 0, 1,2, 4, 6,8 e 8 dias e temperaturas de 4,5, 10,5, 25, 39,5 e 45,5 °C, segu ndo delineamento composto central rotacional. O inóculo inicial utilizado foi de aproximadamente 1x104 UFC.cm-2. A análise de variância mostrou ajuste significativo (p<0,05) do modelo, permitindo a construção de um modelo matemático capaz de predizer a adesão em função do tempo e temperatura. A faixa ótima para a formação do biofilme foi observada nas combinações de 3 a 7,5 dias e 22 a 43 °C. A microscopia eletrônica de varredura (MEV) permitiu a visualização das topografias, das bactérias aderidas e produção e exopolissacarídeos. A contagem média a 25 °C por 4 dias foi de 4,08 x 105 UFC.cm-2 pela MEV, bem próxima a obtida por contagem padrão em placas de 8,93 x 105 UFC.cm-2. A ação de hipoclorito de sódio 100 mg.L-1 Cloro Residual Total (CRT) e ácido peracético 300 mg.L-1 Ácido Peracético (APA) sobre os biofilmes de E. faecium formados na superfície de 60 cupons de aço inoxidável, por 10 minutos, foram avaliados e os microrganismos foram detectados em 60 e 57 cupons, com reduções decimais de 3,00 e 4,02 ciclos log UFC.cm-2 respectivamente. Embora o ácido peracético tenha sido o mais eficiente, o microrganismo não foi eliminado nos tratamentos, o que demonstra a dificuldade de sanitização das superfícies após a formação do biofilme / Abstract: Among the microorganisms that exhibit ability to adhere and form biofilms on surfaces of food processing, such as equipment and utensils, are the bacteria of the genus Enterococcus, present as contaminants in foods such as milk and dairy products. Its presence in cheese has been widely studied for its capacity to provide the technological characteristics desirable product. The objective of this work was to evaluate the technological characteristics of Enterococcus faecium 946Ec, isolated from the artesanal Coalho cheeses, the possible formation of biofilms in surface of stainless steel and its control by sanitizers. The culture was characterized technologically, as a low acid producer, with low proteolytic activity and a diacetyl producer. The curve of growth of Enterococcus faecium 946Ec in MRS broth presented counts of 1,48x109 CFU.mL-1 after 24 hours. The evaluation of the biofilm formation was realized in coupons from stainless steel AISI 304, with roughness average of 0,366 mm, in times of 0, 1.2, 4, 6,8 and 8 days and in temperatures of 4,5; 10,5; 25; 39,5 and 45,5 °C, ac cording to a central composite design. The analysis of variance indicated the significant (p<0,05) adjust of the model, allowing the construction of a mathematical model capable of predicting the adhesion in function of time and temperature. The optimum interval for the formation of biofilms was observed in combinations of 3 to 7,5 days and 22 to 43 °C. The scanning electron microscopy allowed the vi sualization of the topography, the biofilm formation and exopolysaccharides production. The average counting at 25 °C for 4 days was 4,08 x 10 5 CFU.cm-2 using the SEM method, compared with results of the standard plate count of 8,93 x 105 CFU.cm-2. The action of sodium hypochlorite sanitizers 100 mg.L-1 Total Residual Chlorine (TRC) and peracetic acid 300 mg.L-1 Peracetic Ácid (PAA), during 10 minutes, was evaluated in 60 coupons with biofilm formation. The microorganism was detected in 57 and 60 coupons submitted to peracetic acid and sodium hypochlorite respectively, with decimal reductions of 3,00 and 4,02 cycles log CFU.cm-2 respectively. Although peracetic acid has been the most efficient, the microorganism has not been eliminated in the treatments, which demonstrates the difficulty of sanitization of the surfaces after the biofilm formation / Mestrado / Mestre em Tecnologia de Alimentos

A l’instar des D,D-transpeptidases de la famille des protéines de liaison à la pénicilline (PLP), les L,D-transpeptidases catalysent la formation des ponts interpeptidiques du peptidoglycane. Chez un mutant de Enterococcus faecium, la totalité du peptidoglycane est synthétisée par les L,D-transpeptidases entrainant une résistance à toutes les β-lactamines à l’exception des carbapénèmes. Le peptidoglycane de Mycobacterium tuberculosis étant majoritairement synthétisé par des L,D-transpeptidases, ces enzymes sont une cible potentielle pour le développement de nouveaux traitements de la tuberculose. Les objectifs de cette thèse sont de comprendre la spécificité des L,D­transpeptidases pour les carbapénèmes et d’identifier les sites de fixation des précurseurs du peptidoglycane à ces enzymes. Les résultats montrent que l’oxyanion formé lors de l’attaque du cycle β-lactame des carbapénèmes par la cystéine active est stabilisé dans le site actif des L,D­transpeptidases. Cette stabilisation combinée à l’absence d’hydrolyse de l’acylenzyme conduisent à l’inactivation rapide, totale et irréversible des L,D­transpeptidases par les carbapénèmes. La structure de l’acylenzyme indique que ces propriétés sont indépendantes de la nature de la chaine latérale des antibiotiques qui pourrait être optimisée. La localisation de l’accepteur d’acyle dans la Poche II de Ldtfm ainsi que des interactions supplémentaires avec les chaines glycanes du peptidoglycane montrent que d’autres sites que celui ciblé par les β­lactamines, mimes du donneur d’acyle, sont des cibles pour le développement de nouveaux antibiotiques qui pourraient agir en synergie avec les β­lactamines. / L,D-transpeptidases, as D,D-transpeptidases belonging to the penicillin-binding protein (PBP) family, catalyse the last cross-links step of peptidoglycan biosynthesis. The peptidoglycan of an Enterococcus faecium mutant is exclusively cross-linked by L,D-transpeptidases leading to resistance to all β-lactams except the carbapenems. Since peptidoglycan cross-links are predominantly synthesized by L,D-transpeptidases in Mycobacterium tuberculosis these enzymes are potential targets for chemotherapy of tuberculosis. The aims of the thesis are to identify the kinetic features that account for the specificity of L,D-transpeptidases for carbapenems and to characterise the binding sites for the peptidoglycan precursors in these enzymes. Our results show that the oxyanion resulting from nucleophilic attack of carbapebems by the catalytic cysteine is stabilized into the active site of L,D-transpeptidases. This stabilisation, combined to the absence of hydrolysis of the acylenzyme, leads to the rapid, total and irreversible inactivation of L,D-transpeptidases by carbapenems. Resolution of the acylenzyme structure shows that these kinetic features are independent from the carbapenem side chain that could be modified to optimize the antibiotics. The binding of the acyle acceptor has been identified in Pocket II of Ldtfm that is distinct from the binding site for β-lactams (Pocket I), which mimic the acyle donor. This site and additional peptidoglycan binding sites reveal additional targets for development of new antibiotics that might act in synergy with β-lactams.

Lower respiratory tract infectious diseases are a world-wide healthcare burden with bacterial pathogens accounting for a large portion of primary and secondary infections. The human respiratory tract is home to hundreds of species of microbes that comprise the human airway microbiome. These commensals play a crucial role in human health in part by providing colonization resistance against pathogens. In a previous study from the Surette lab it was shown that specific bacterial isolates from the respiratory microbiome inhibits the growth of pathogens aerobically. This included an isolate of Staphylococcus aureus which inhibited the growth of Enterococcus faecium. This activity was further characterized in this thesis and the underlying mechanism was explored through comparative genomics. As well, this observation provided proof-of-concept for a large-scale screen for additional isolates which inhibit pathogen growth. I hypothesized that the respiratory tract microbiota included many other bacteria capable of inhibiting the growth of respiratory tract pathogens in both aerobic and anaerobic environments, and that anaerobic conditions will identify new activities not detected aerobically. To examine and identify potential beneficial bacteria, I have screened ~5000 respiratory tract bacteria from the Surette lab’s airway isolate collection against four pathogens: Streptococcus pneumoniae, Staphylococcus aureus, Pseudomonas aeruginosa and Klebsiella pneumoniae. The respiratory tract commensals were pinned onto the pathogen-lawn and their interaction was expressed as zones of clearing or altered growth phenotypes of the pathogen. The results of the screen showed that anti-pathogen activity was a common feature of respiratory tract commensals. In particular, S. pneumoniae was inhibited by taxonomically diverse members of the microbiota representing three phyla (Proteobacteria, Firmicutes and Actinobacteria). Many of the facultative anaerobes that inhibited S. pneumoniae expressed their activity in anerobic conditions. / Thesis / Master of Science (MSc) / The human respiratory tract harbours commensal and pathogenic bacteria, and the latter cause most of the lower respiratory tract infections. The commensal bacteria help to train the immune system and impede the growth of pathogens through colonization resistance. A previous study by the Surette lab identified bacterial isolates from the respiratory tract that inhibit the growth of select pathogens, among them, a particular strain of Staphylococcus aureus. Based on the results of the earlier study, I hypothesized that the respiratory tract bacteria is a good source of commensals that can inhibit the growth of S. aureus and other respiratory pathogens, such as Streptococcus pneumoniae, Pseudomonas aeruginosa and Klebsiella pneumoniae. To find potential therapeutic bacteria, I screened ~5000 respiratory tract isolates from the Surette lab’s strain collection for the ability to impair growth of target pathogens. Additionally, I further characterized the activity of the previously identified S. aureus strain against various Lactobacillalles strains and used comparative genomics to identify potential biosynthetic genes required for biosynthesis of molecules with antibacterial activity within the genome of S. aureus. The research reported in this thesis demonstrates that many commensal bacteria that live within our airways have the ability to inhibit the growth of bacterial pathogens. This work may provide a new source of antibiotics against respiratory infections and new strategies to reduce susceptibility to infections in vulnerable populations.

Saturated steam (SS) and propylene oxide (PPO) fumigation are two common methods to improve microbiological quality and safety of tree nuts. Validation of these processes is needed to ensure adequate control of bacterial pathogens. Since pathogens cannot be studied in food processing environments, surrogates with resistance comparable to the pathogens needed to be identified. The objective was to investigate the suitability of Enterococcus faecium, Pediococcus acidilactici, and Staphylococcus carnosus as surrogate bacteria for Salmonella spp. on whole cashews and macadamia nuts, processed with SS or PPO. Whole cashews and macadamia nuts were co-inoculated with a cocktail of Salmonella enterica and one of the three potential surrogates. Nuts were dried to original aw, packaged in poly-woven bags (2.3 kg) and commercially processed using vacuum assisted steam at 80 ͦ C or PPO fumigation. Salmonella and the potential surrogates were enumerated by serial dilution, and plated onto TSA with overlay of XLT-4 (Salmonella) or media selective for the potential surrogates. Mean log reductions (CFU/g) of Salmonella and each potential surrogate were compared using a paired T-test. SS results: reduction of Salmonella (6.0 ± 0.14) was significantly larger than E. faecium (4.3± 0.12), or P. acidilactici (3.7± 0.14) on whole cashews. Salmonella (5.9 ± 0.18) was significantly larger than P. acidilactici (4.4± 0.18) on whole macadamia nuts. PPO results: reduction of Salmonella (7.3 ± 0.19) was significantly greater than E. faecium (6.4± 0.31), or P. acidilactici (6.3± 0.33) on whole macadamia nuts. Reduction of Salmonella was significantly greater than E. faecium and P. acidilactici reduction on cashews. P. acidilactici may be considered a surrogate for Salmonella reduction on whole macadamia nuts and whole cashews processed using SS at 80 ͦ C. E. faecium and P. acidilactici may be considered surrogates for Salmonella reduction on whole macadamia nuts and whole cashews processed using PPO. Reduction of St. carnosus exceeded that of Salmonella indicating it is not a suitable surrogate for Salmonella using either processing intervention. / Master of Science in Life Sciences / Tree nuts are produced and consumed worldwide, playing a role as a snack or ingredient in foods in several cultures. Traditionally, tree nuts have been believed to be microbiologically safe due to their composition and lack of water available to harbor pathogenic bacterial growth. However, recent years have proven to be problematic for many tree nuts and nut products, numerous recalls have occurred in the United States for verified contamination of <i>Salmonella</i>. Since <i>Salmonella</i> can be found in food systems worldwide, and is a leading cause of foodborne illness due to bacterial contamination of food, steps must be taken to improve the safety of nuts grown locally and imported products. With several processing options for tree nuts, two that are commonly practiced to reduce microorganisms include a fumigation of product using propylene oxide (PPO) and a thermal inactivation treatment through use of saturated steam. The comparison of <i>Salmonella</i> inactivation and non-pathogenic surrogate bacteria on cashews and macadamia nuts, being processed in these two manners, was investigated. Possible bacteria strains that were investigated for surrogacy were <i>Enterococcus faecium</i> NRRL B2354 (ATCC #8459), <i>Pediococcus acidilactici</i> (ATCC #8042), and <i>Staphylococcus carnosus</i> (ATCC #51365). Surrogates are bacteria that have similar inactivation characteristics to <i>Salmonella</i> when processed, that can be purposely introduced before processing to ensure inactivation of <i>Salmonella</i> and is harmless if consumption occurs. Studies continue to ensure safety of tree nuts as well as complying with pending and future regulations.

Les entérocoques font partie de la flore normale intestinale des animaux et des humains. Plusieurs études ont démontré que les entérocoques d’origine animale pouvaient représenter un réservoir de gènes de résistance aux antibiotiques pour la communauté humaine et animale. Les espèces Enterococcus faecalis et Enterococcus faecium sont importantes en santé publique; elles sont responsables d’environ 12% de toutes les infections nosocomiales aux États-Unis. Au Canada, les cas de colonisation et/ou d’infections à entérocoques résistants à la vancomycine ont plus que triplé de 2005 à 2009. Un total de 387 isolats E. faecalis et E. faecium aviaires, et 124 isolats E. faecalis porcins ont été identifiés et analysés pour leur susceptibilité aux antibiotiques. De hauts pourcentages de résistance envers les macrolides et les tétracyclines ont été observés tant chez les isolats aviaires que porcins. Deux profils phénotypiques prédominants ont été déterminés et analysés par PCR et séquençage pour la présence de gènes de résistance aux antibiotiques. Différentes combinaisons de gènes de résistance ont été identifiées dont erm(B) et tet(M) étant les plus prévalents. Des extractions plasmidiques et des analyses par hybridation ont permis de déterminer, pour la première fois, la colocalisation des gènes erm(B) et tet(M) sur un plasmide d’environ 9 kb chez des isolats E. faecalis porcins, et des gènes erm(B) et tet(O) sur un plasmide de faible poids moléculaire d’environ 11 kb chez des isolats E. faecalis aviaires. De plus, nous avons démontré, grâce à des essais conjugatifs, que ces plasmides pouvaient être transférés. Les résultats ont révélé que les entérocoques intestinaux aviaires et porcins, lesquels peuvent contaminer la viande à l’abattoir, pouvaient représenter un réservoir de gènes de résistance envers la quinupristine-dalfopristine, la bacitracine, la tétracycline et les macrolides. Afin d’évaluer l’utilisation d’un antisérum polyclonal SA dans l’interférence de la résistance à de fortes concentrations de bacitracine (gènes bcrRAB), lors d’un transfert conjugatif répondant aux phéromones, un isolat multirésistant E. faecalis aviaire a été sélectionné. Après induction avec des phéromones produites par la souche réceptrice E. faecalis JH2-2, l’agrégation de la souche donatrice E. faecalis 543 a été observée ainsi que des fréquences de transfert élevées en bouillon lors d’une courte période de conjugaison. Le transfert conjugatif des gènes asa1, traB et bcrRAB ainsi que leur colocalisation a été démontré chez le donneur et un transconjugant T543-1 sur un plasmide de 115 kb par électrophorèse à champs pulsé (PFGE) et hybridation. Une CMI de > 2 048 µg/ml envers la bacitracine a été obtenue tant chez le donneur que le transconjuguant tandis que la souche réceptrice JH2-2 démontrait une CMI de 32 µg/ml. Le séquençage des gènes asa1, codant pour la substance agrégative, et traB, une protéine régulant négativement la réponse aux phéromones, a révélé une association de cet élément génétique avec le plasmide pJM01. De plus, cette étude présente qu’un antisérum polyclonal SA peut interférer significativement dans le transfert horizontal d’un plasmide répondant aux phéromones codant pour de la résistance à de fortes doses de bacitracine d’une souche E. faecalis aviaire multirésistante. Des isolats cliniques E. faecium d’origine humaine et canine ont été analysés et comparés. Cette étude rapporte, pour la première fois, la caractérisation d’isolats cliniques E. faecium résistants à l’ampicilline (EFRA) d’origine canine associés à CC17 (ST17) au Canada. Ces isolats étaient résistants à la ciprofloxacine et à la lincomycine. Leur résistance envers la ciprofloxacine a été confirmée par la présence de substitutions dans la séquence en acides aminés des gènes de l’ADN gyrase (gyrA/gyrB) et de la topoisomérase IV (parC/parE). Des résistances élevées envers la gentamicine, la kanamycine et la streptomycine, et de la résistance envers les macrolides et les lincosamides a également été observées. La fréquence de résistance envers la tétracycline était élevée tandis que celle envers la vancomycine n’a pas été détectée. De plus, aucune résistance n’a été observée envers le linézolide et la quinupristine-dalfopristine. Les données ont démontré une absence complète des gènes esp (protéine de surface des entérocoques) et hyl (hyaluronidase) chez les isolats canins EFRA testés tandis qu’ils possédaient tous le gène acm (adhésine de liaison au collagène d’E. faecium). Aucune activité reliée à la formation de biofilm ou la présence d’éléments CRISPR (loci de courtes répétitions palindromiques à interespaces réguliers) n’a été identifiée chez les isolats canins EFRA. Les familles de plasmide rep6 and rep11 ont significativement été associées aux isolats d’origine canine. Les profils PFGE des isolats d’origine humaine et canine n'ont révélé aucune relation (≤ 80%). Ces résultats illustrent l'importance d'une utilisation judicieuse des antibiotiques en médecine vétérinaire afin d’éviter la dissémination zoonotique des isolats EFRA canins. Nous pensons que ces résultats contribueront à une meilleure compréhension des mécanismes de résistance aux antibiotiques et de leurs éléments mobiles ainsi qu’à de nouvelles stratégies afin de réduire le transfert horizontal de la résistance aux antibiotiques et des facteurs de virulence. / Enterococci are part of normal intestinal gut flora of animals and humans. Many studies have shown that enterococci from animal origin could represent an antimicrobial resistance genes reservoir for the human community. The two species Enterococcus faecalis and Enterococcus faecium are important in public health; they are responsible for approximately 12% of all nosocomial infections in the United States. In Canada, cases of colonization and/or infections to vancomycin resistant enterococci have more than tripled from 2005 to 2009. A total of 387 poultry E. faecalis and E. faecium isolates, and 124 porcine E. faecalis isolates were identified and analyzed for their antibiotic susceptibilities. High percentages of resistance to macrolides and tetracyclines were found in both avian and porcine isolates. Two predominant phenotypic profiles were determined and analyzed by PCR and sequencing for the presence of antimicrobial resistance genes. Various combinations of antibiotic resistance genes were detected; erm(B) and tet(M) were the most common genes. For the first time, plasmid extraction and hybridization revealed colocalization of erm(B) and tet(M) on a plasmid of ~9 kb in porcine E. faecalis isolates, and of erm(B) and tet(O) on a low-molecular-weight plasmid of ~11 kb in poultry E. faecalis isolates. Furthermore, we demonstrated, through mating experiments, these plasmids could be transferred. Results indicate that the intestinal enterococci of healthy pigs and poultry, which can contaminate meat at slaughter, could be a reservoir for quinupristin-dalfopristin, bacitracin, tetracycline, and macrolide resistance genes. To assess the use of a polyclonal antiserum AS on the contact interference of a high level bacitracin resistant (bcrRAB genes) pheromone-responsive plasmid, a multiresistant E. faecalis isolate of poultry origin was selected. After induction with pheromones produced by the recipient strain E. faecalis JH2-2, clumping of the donor E. faecalis strain 543 was demonstrated as well as high transfer frequencies in short time broth mating. Conjugative transfer of asa1, traB and bcrRAB genes and their co-localization were also demonstrated in the donor strain and a transconjugant T543-1 on a plasmid band of 115 kb by PFGE and Southern blotting. A MIC to bacitracin of > 2 048 µg/ml was obtained for both strains 543 and T543-1 whereas the recipient strain JH2-2 demonstrated a MIC of 32 µg/ml. Sequencing of the asa1 gene encoding for an AS, and traB for a pheromone shutdown protein, confirmed the association of this genetic element to the pheromone-responsive plasmid related to pJM01. More significantly, this study presents the evidence that a polyclonal antiserum AS can significantly interfere with the horizontal transfer of a pheromone-responsive plasmid encoding high-level bacitracin resistance of a poultry multidrug resistant E. faecalis strain. Clinical isolates of E. faecium of human and canine origin were analyzed and compared. This report describes for the first time the characterization of canine clinical ampicillin-resistant E. faecium (AREF) isolates related to CC17 (ST17) in Canada. These isolates were resistant to ciprofloxacin and lincomycin. Resistance to ciprofloxacin was confirmed by amino acid substitutions in DNA gyrase (gyrA/gyrB) and topoisomerase IV (parC/parE) genes. High-level gentamicin, -kanamycin and -streptomycin resistances and macrolides resistance were also observed. The frequency of tetracycline resistance was high whereas vancomycin resistance was not detected. Also, no resistance was observed to linezolid and quinupristin-dalfopristin antibiotics. Data demonstrated the complete absence of enterococcal surface protein (esp) and hyaluronidase (hyl) genes among the canine AREF isolates tested while all were acm (collagen adhesin from E. faecium) positive. However, most of them were shown to harbor efaAfm gene, encoding for a cell wall adhesin. No biofilm formation or clustered regularly interspaced short palindromic repeats (CRISPR) elements were identified in these canine AREF isolates. rep6 and rep11 families of plasmids were significantly associated with isolates from dogs. The PFGE patterns of human and dog isolates were considered unrelated (≤ 80%). These findings also support the importance of prudent use of antibiotics in veterinary medicine to avoid zoonotic spread of canine AREF isolates. We are confident that our results may help to better understand the mechanisms of antibiotic resistance and mobile element carrying them as well as new strategies to reduce the horizontal transfer of antibiotic resistance and virulence traits.

Les entérocoques font partie de la flore normale intestinale des animaux et des humains. Plusieurs études ont démontré que les entérocoques d’origine animale pouvaient représenter un réservoir de gènes de résistance aux antibiotiques pour la communauté humaine et animale. Les espèces Enterococcus faecalis et Enterococcus faecium sont importantes en santé publique; elles sont responsables d’environ 12% de toutes les infections nosocomiales aux États-Unis. Au Canada, les cas de colonisation et/ou d’infections à entérocoques résistants à la vancomycine ont plus que triplé de 2005 à 2009. Un total de 387 isolats E. faecalis et E. faecium aviaires, et 124 isolats E. faecalis porcins ont été identifiés et analysés pour leur susceptibilité aux antibiotiques. De hauts pourcentages de résistance envers les macrolides et les tétracyclines ont été observés tant chez les isolats aviaires que porcins. Deux profils phénotypiques prédominants ont été déterminés et analysés par PCR et séquençage pour la présence de gènes de résistance aux antibiotiques. Différentes combinaisons de gènes de résistance ont été identifiées dont erm(B) et tet(M) étant les plus prévalents. Des extractions plasmidiques et des analyses par hybridation ont permis de déterminer, pour la première fois, la colocalisation des gènes erm(B) et tet(M) sur un plasmide d’environ 9 kb chez des isolats E. faecalis porcins, et des gènes erm(B) et tet(O) sur un plasmide de faible poids moléculaire d’environ 11 kb chez des isolats E. faecalis aviaires. De plus, nous avons démontré, grâce à des essais conjugatifs, que ces plasmides pouvaient être transférés. Les résultats ont révélé que les entérocoques intestinaux aviaires et porcins, lesquels peuvent contaminer la viande à l’abattoir, pouvaient représenter un réservoir de gènes de résistance envers la quinupristine-dalfopristine, la bacitracine, la tétracycline et les macrolides. Afin d’évaluer l’utilisation d’un antisérum polyclonal SA dans l’interférence de la résistance à de fortes concentrations de bacitracine (gènes bcrRAB), lors d’un transfert conjugatif répondant aux phéromones, un isolat multirésistant E. faecalis aviaire a été sélectionné. Après induction avec des phéromones produites par la souche réceptrice E. faecalis JH2-2, l’agrégation de la souche donatrice E. faecalis 543 a été observée ainsi que des fréquences de transfert élevées en bouillon lors d’une courte période de conjugaison. Le transfert conjugatif des gènes asa1, traB et bcrRAB ainsi que leur colocalisation a été démontré chez le donneur et un transconjugant T543-1 sur un plasmide de 115 kb par électrophorèse à champs pulsé (PFGE) et hybridation. Une CMI de > 2 048 µg/ml envers la bacitracine a été obtenue tant chez le donneur que le transconjuguant tandis que la souche réceptrice JH2-2 démontrait une CMI de 32 µg/ml. Le séquençage des gènes asa1, codant pour la substance agrégative, et traB, une protéine régulant négativement la réponse aux phéromones, a révélé une association de cet élément génétique avec le plasmide pJM01. De plus, cette étude présente qu’un antisérum polyclonal SA peut interférer significativement dans le transfert horizontal d’un plasmide répondant aux phéromones codant pour de la résistance à de fortes doses de bacitracine d’une souche E. faecalis aviaire multirésistante. Des isolats cliniques E. faecium d’origine humaine et canine ont été analysés et comparés. Cette étude rapporte, pour la première fois, la caractérisation d’isolats cliniques E. faecium résistants à l’ampicilline (EFRA) d’origine canine associés à CC17 (ST17) au Canada. Ces isolats étaient résistants à la ciprofloxacine et à la lincomycine. Leur résistance envers la ciprofloxacine a été confirmée par la présence de substitutions dans la séquence en acides aminés des gènes de l’ADN gyrase (gyrA/gyrB) et de la topoisomérase IV (parC/parE). Des résistances élevées envers la gentamicine, la kanamycine et la streptomycine, et de la résistance envers les macrolides et les lincosamides a également été observées. La fréquence de résistance envers la tétracycline était élevée tandis que celle envers la vancomycine n’a pas été détectée. De plus, aucune résistance n’a été observée envers le linézolide et la quinupristine-dalfopristine. Les données ont démontré une absence complète des gènes esp (protéine de surface des entérocoques) et hyl (hyaluronidase) chez les isolats canins EFRA testés tandis qu’ils possédaient tous le gène acm (adhésine de liaison au collagène d’E. faecium). Aucune activité reliée à la formation de biofilm ou la présence d’éléments CRISPR (loci de courtes répétitions palindromiques à interespaces réguliers) n’a été identifiée chez les isolats canins EFRA. Les familles de plasmide rep6 and rep11 ont significativement été associées aux isolats d’origine canine. Les profils PFGE des isolats d’origine humaine et canine n'ont révélé aucune relation (≤ 80%). Ces résultats illustrent l'importance d'une utilisation judicieuse des antibiotiques en médecine vétérinaire afin d’éviter la dissémination zoonotique des isolats EFRA canins. Nous pensons que ces résultats contribueront à une meilleure compréhension des mécanismes de résistance aux antibiotiques et de leurs éléments mobiles ainsi qu’à de nouvelles stratégies afin de réduire le transfert horizontal de la résistance aux antibiotiques et des facteurs de virulence. / Enterococci are part of normal intestinal gut flora of animals and humans. Many studies have shown that enterococci from animal origin could represent an antimicrobial resistance genes reservoir for the human community. The two species Enterococcus faecalis and Enterococcus faecium are important in public health; they are responsible for approximately 12% of all nosocomial infections in the United States. In Canada, cases of colonization and/or infections to vancomycin resistant enterococci have more than tripled from 2005 to 2009. A total of 387 poultry E. faecalis and E. faecium isolates, and 124 porcine E. faecalis isolates were identified and analyzed for their antibiotic susceptibilities. High percentages of resistance to macrolides and tetracyclines were found in both avian and porcine isolates. Two predominant phenotypic profiles were determined and analyzed by PCR and sequencing for the presence of antimicrobial resistance genes. Various combinations of antibiotic resistance genes were detected; erm(B) and tet(M) were the most common genes. For the first time, plasmid extraction and hybridization revealed colocalization of erm(B) and tet(M) on a plasmid of ~9 kb in porcine E. faecalis isolates, and of erm(B) and tet(O) on a low-molecular-weight plasmid of ~11 kb in poultry E. faecalis isolates. Furthermore, we demonstrated, through mating experiments, these plasmids could be transferred. Results indicate that the intestinal enterococci of healthy pigs and poultry, which can contaminate meat at slaughter, could be a reservoir for quinupristin-dalfopristin, bacitracin, tetracycline, and macrolide resistance genes. To assess the use of a polyclonal antiserum AS on the contact interference of a high level bacitracin resistant (bcrRAB genes) pheromone-responsive plasmid, a multiresistant E. faecalis isolate of poultry origin was selected. After induction with pheromones produced by the recipient strain E. faecalis JH2-2, clumping of the donor E. faecalis strain 543 was demonstrated as well as high transfer frequencies in short time broth mating. Conjugative transfer of asa1, traB and bcrRAB genes and their co-localization were also demonstrated in the donor strain and a transconjugant T543-1 on a plasmid band of 115 kb by PFGE and Southern blotting. A MIC to bacitracin of > 2 048 µg/ml was obtained for both strains 543 and T543-1 whereas the recipient strain JH2-2 demonstrated a MIC of 32 µg/ml. Sequencing of the asa1 gene encoding for an AS, and traB for a pheromone shutdown protein, confirmed the association of this genetic element to the pheromone-responsive plasmid related to pJM01. More significantly, this study presents the evidence that a polyclonal antiserum AS can significantly interfere with the horizontal transfer of a pheromone-responsive plasmid encoding high-level bacitracin resistance of a poultry multidrug resistant E. faecalis strain. Clinical isolates of E. faecium of human and canine origin were analyzed and compared. This report describes for the first time the characterization of canine clinical ampicillin-resistant E. faecium (AREF) isolates related to CC17 (ST17) in Canada. These isolates were resistant to ciprofloxacin and lincomycin. Resistance to ciprofloxacin was confirmed by amino acid substitutions in DNA gyrase (gyrA/gyrB) and topoisomerase IV (parC/parE) genes. High-level gentamicin, -kanamycin and -streptomycin resistances and macrolides resistance were also observed. The frequency of tetracycline resistance was high whereas vancomycin resistance was not detected. Also, no resistance was observed to linezolid and quinupristin-dalfopristin antibiotics. Data demonstrated the complete absence of enterococcal surface protein (esp) and hyaluronidase (hyl) genes among the canine AREF isolates tested while all were acm (collagen adhesin from E. faecium) positive. However, most of them were shown to harbor efaAfm gene, encoding for a cell wall adhesin. No biofilm formation or clustered regularly interspaced short palindromic repeats (CRISPR) elements were identified in these canine AREF isolates. rep6 and rep11 families of plasmids were significantly associated with isolates from dogs. The PFGE patterns of human and dog isolates were considered unrelated (≤ 80%). These findings also support the importance of prudent use of antibiotics in veterinary medicine to avoid zoonotic spread of canine AREF isolates. We are confident that our results may help to better understand the mechanisms of antibiotic resistance and mobile element carrying them as well as new strategies to reduce the horizontal transfer of antibiotic resistance and virulence traits.

Introdução: Enterococcus faecium resistente à vancomicina (EFRV) surgiu como um importante patógeno multirresistente e de etiologia potencialmente letal nas infecções associadas aos cuidados de saúde em todo o mundo. Objetivo: O objetivo deste estudo de coorte retrospectivo foi avaliar os fatores associados à mortalidade em pacientes com bacteremia causadas por EFRV em um grande hospital de referência terciária no sul do Brasil. Métodos: Foram avaliados todos os casos documentados de bacteremia identificados entre maio de 2010 e julho de 2012. Regressão de Cox foi realizada para determinar se as características relacionadas ao hospedeiro ou o tratamento antimicrobiano estavam associadas com a mortalidade por qualquer causa em 30 dias. No total, 35 pacientes documentados com bacteremia por EFRV foram identificados durante o período de estudo. Resultados: A mediana do escore APACHE-II da população do estudo foi 26 (IQR 10). A mortalidade global em 30 dias foi de 65,7%%. Todos isolados de EFRV eram sensíveis à linezolida, daptomicina e quinopristina - dalfopristina. A linezolida foi o único agente antimicrobiano com atividade in vitro contra EFRV que foi administrada à coorte. Após a análise multivariada, o tratamento com linezolida (HR 0,08, 95% CI, 0,02-0,27) e a presença de insuficiência renal aguda no início da bacteremia (HR 4,01, 95% CI, 1,62-9,94) foram associadas de forma independente com o desfecho principal. Conclusão: Apresentação com insuficiência renal aguda e ausência de tratamento com um antibiótico eficaz representa um risco de mortalidade em pacientes com bacteremia por EFRV. / Background: Vancomycin-resistant Enterococcus faecium (VREF) has emerged as a relevant multidrug-resistant pathogen and potentially lethal etiology of health care-associated infections worldwide. Objective: The objective of this retrospective cohort study was to assess factors associated with mortality in patients with VREF bacteremia in a major tertiary referral hospital in southern Brazil. Methods: All documented cases of bacteremia identified between May 2010 and July 2012 were evaluated. Cox regression was performed to determine whether the characteristics related to the host or antimicrobial treatment were associated with the all-cause 30-day mortality. In total, 35 patients with documented VREF bacteremia were identified during the study period. Results: The median APACHE-II score of the study population was 26 (IQR 10). The overall 30-day mortality was 65.7%. All VREF isolates were sensitive to linezolid, daptomycin and quinopristin-dalfopristin. Linezolid was the only antimicrobial agent with in vitro activity against VREF that was administered to the cohort. After multivariate analysis, linezolid treatment (HR, 0.08; 95%CI, 0.02 – 0.27) and presence of acute kidney injury at the onset of bacteremia (HR, 4.01; 95%CI, 1.62 – 9.94) were independently associated with the main outcome. Conclusion: Presentation with acute kidney injury and lack of treatment with an effective antibiotic poses risk for mortality in patients with VREF bacteremia.

Introdução: Enterococcus faecium resistente à vancomicina (EFRV) surgiu como um importante patógeno multirresistente e de etiologia potencialmente letal nas infecções associadas aos cuidados de saúde em todo o mundo. Objetivo: O objetivo deste estudo de coorte retrospectivo foi avaliar os fatores associados à mortalidade em pacientes com bacteremia causadas por EFRV em um grande hospital de referência terciária no sul do Brasil. Métodos: Foram avaliados todos os casos documentados de bacteremia identificados entre maio de 2010 e julho de 2012. Regressão de Cox foi realizada para determinar se as características relacionadas ao hospedeiro ou o tratamento antimicrobiano estavam associadas com a mortalidade por qualquer causa em 30 dias. No total, 35 pacientes documentados com bacteremia por EFRV foram identificados durante o período de estudo. Resultados: A mediana do escore APACHE-II da população do estudo foi 26 (IQR 10). A mortalidade global em 30 dias foi de 65,7%%. Todos isolados de EFRV eram sensíveis à linezolida, daptomicina e quinopristina - dalfopristina. A linezolida foi o único agente antimicrobiano com atividade in vitro contra EFRV que foi administrada à coorte. Após a análise multivariada, o tratamento com linezolida (HR 0,08, 95% CI, 0,02-0,27) e a presença de insuficiência renal aguda no início da bacteremia (HR 4,01, 95% CI, 1,62-9,94) foram associadas de forma independente com o desfecho principal. Conclusão: Apresentação com insuficiência renal aguda e ausência de tratamento com um antibiótico eficaz representa um risco de mortalidade em pacientes com bacteremia por EFRV. / Background: Vancomycin-resistant Enterococcus faecium (VREF) has emerged as a relevant multidrug-resistant pathogen and potentially lethal etiology of health care-associated infections worldwide. Objective: The objective of this retrospective cohort study was to assess factors associated with mortality in patients with VREF bacteremia in a major tertiary referral hospital in southern Brazil. Methods: All documented cases of bacteremia identified between May 2010 and July 2012 were evaluated. Cox regression was performed to determine whether the characteristics related to the host or antimicrobial treatment were associated with the all-cause 30-day mortality. In total, 35 patients with documented VREF bacteremia were identified during the study period. Results: The median APACHE-II score of the study population was 26 (IQR 10). The overall 30-day mortality was 65.7%. All VREF isolates were sensitive to linezolid, daptomycin and quinopristin-dalfopristin. Linezolid was the only antimicrobial agent with in vitro activity against VREF that was administered to the cohort. After multivariate analysis, linezolid treatment (HR, 0.08; 95%CI, 0.02 – 0.27) and presence of acute kidney injury at the onset of bacteremia (HR, 4.01; 95%CI, 1.62 – 9.94) were independently associated with the main outcome. Conclusion: Presentation with acute kidney injury and lack of treatment with an effective antibiotic poses risk for mortality in patients with VREF bacteremia.

Introdução: Enterococcus faecium resistente à vancomicina (EFRV) surgiu como um importante patógeno multirresistente e de etiologia potencialmente letal nas infecções associadas aos cuidados de saúde em todo o mundo. Objetivo: O objetivo deste estudo de coorte retrospectivo foi avaliar os fatores associados à mortalidade em pacientes com bacteremia causadas por EFRV em um grande hospital de referência terciária no sul do Brasil. Métodos: Foram avaliados todos os casos documentados de bacteremia identificados entre maio de 2010 e julho de 2012. Regressão de Cox foi realizada para determinar se as características relacionadas ao hospedeiro ou o tratamento antimicrobiano estavam associadas com a mortalidade por qualquer causa em 30 dias. No total, 35 pacientes documentados com bacteremia por EFRV foram identificados durante o período de estudo. Resultados: A mediana do escore APACHE-II da população do estudo foi 26 (IQR 10). A mortalidade global em 30 dias foi de 65,7%%. Todos isolados de EFRV eram sensíveis à linezolida, daptomicina e quinopristina - dalfopristina. A linezolida foi o único agente antimicrobiano com atividade in vitro contra EFRV que foi administrada à coorte. Após a análise multivariada, o tratamento com linezolida (HR 0,08, 95% CI, 0,02-0,27) e a presença de insuficiência renal aguda no início da bacteremia (HR 4,01, 95% CI, 1,62-9,94) foram associadas de forma independente com o desfecho principal. Conclusão: Apresentação com insuficiência renal aguda e ausência de tratamento com um antibiótico eficaz representa um risco de mortalidade em pacientes com bacteremia por EFRV. / Background: Vancomycin-resistant Enterococcus faecium (VREF) has emerged as a relevant multidrug-resistant pathogen and potentially lethal etiology of health care-associated infections worldwide. Objective: The objective of this retrospective cohort study was to assess factors associated with mortality in patients with VREF bacteremia in a major tertiary referral hospital in southern Brazil. Methods: All documented cases of bacteremia identified between May 2010 and July 2012 were evaluated. Cox regression was performed to determine whether the characteristics related to the host or antimicrobial treatment were associated with the all-cause 30-day mortality. In total, 35 patients with documented VREF bacteremia were identified during the study period. Results: The median APACHE-II score of the study population was 26 (IQR 10). The overall 30-day mortality was 65.7%. All VREF isolates were sensitive to linezolid, daptomycin and quinopristin-dalfopristin. Linezolid was the only antimicrobial agent with in vitro activity against VREF that was administered to the cohort. After multivariate analysis, linezolid treatment (HR, 0.08; 95%CI, 0.02 – 0.27) and presence of acute kidney injury at the onset of bacteremia (HR, 4.01; 95%CI, 1.62 – 9.94) were independently associated with the main outcome. Conclusion: Presentation with acute kidney injury and lack of treatment with an effective antibiotic poses risk for mortality in patients with VREF bacteremia.

Pathogens in low-moisture foods are an emerging food safety concern due to increased survival and thermotolerance in matrices with low water activity. However, limited data is publicly available for the thermotolerance of Listeria monocytogenes, Salmonella spp., and Enterococcus faecium NRRL B-2354 (a Salmonella surrogate). The aims of this study were to identify differences in thermal inactivation rates between these organisms in three different low-moisture foods. Three model low-moisture foods (peanut butter, powder infant formula, and wheat flour) were inoculated with either E. faecium, a Salmonella spp. cocktail, or a L. monocytogenes cocktail using a dry inoculation method for a total of 9 treatments. Samples were heat treated in a hot water bath at predetermined temperatures, and bacterial survival was detected via direct plating on tryptic soy agar with 0.6% yeast extract. In peanut butter and most of the powder infant formula treatments, Salmonella spp. had significantly higher D-values than L. monocytogenes using comparable temperatures (p < 0.05). However, D-values between Salmonella spp. and L. monocytogenes were comparable in wheat flour and one of the treatment temperatures in powder infant formula (p > 0.05). For all but one of the treatments at the same temperature, E. faecium had significantly higher D-values than L. monocytogenes and Salmonella spp. in each food matrix (p < 0.05). The observed matrix effect on thermotolerance for each of the bacteria was reported in descending order as powder infant formula > peanut butter > wheat flour in the majority of the comparable D-values. While Salmonella continues to be the pathogen of concern in low-moisture foods due to survival and outbreaks, these results indicate L. monocytogenes can exhibit similar thermotolerances in relevant model low-moisture foods matrices.