Introdução: Helicobacter pylori é uma bactéria que infecta aproximadamente metade da população mundial e é considerada uma importante causa de câncer gástrico. A terapia de erradicação nem sempre é eficaz, pois pode ocorrer a resistência aos antimicrobianos. Objetivo: Determinar o perfil de sensibilidade de isolados de H. pylori frente aos antibióticos amoxicilina, claritromicina e ciprofloxacina na população do Rio Grande do Sul empregando distintas padronizações. Material e Métodos: Estudo transversal. Avaliaram-se 54 amostras de H. pylori obtidas através de cultivo de biópsias gástricas em Agar Belo Horizonte e incubação a 37°C em microaerofilia, durante cinco dias. A sensibilidade aos antibióticos foi determinada segundo as orientações das padronizações britânica (BSAC) e australiana (CDS Method) (quantitativas), além da francesa (CA-SFM) (qualitativa). Resultados e discussão: Sete (13%) isolados de H. pylori foram resistentes à claritromicina, um (1,9%) à amoxicilina e três (5,5%) à ciprofloxacina. Estes índices de resistência são considerados satisfatórios e demonstram que todos esses antibióticos podem ser utilizados na terapia empírica na população local, sobretudo a amoxicilina e a claritromicina como primeira linha de tratamento. As metodologias quantitativas BSAC e CDS Method revelaram concordância muito semelhante nos resultados de sensibilidade, sendo a interpretação mais facilitada na técnica CDS Method. A padronização CA-SFM parece ser mais atrativa sob o aspecto econômico, mas fornece resultados apenas qualitativos. Conclusão: Os antibióticos amoxicilina e claritromicina ainda são uma boa opção no tratamento anti-H. pylori na população do Rio Grande do Sul. / Introduction: Helicobacter pylori is a bacteria which infects nearly half the world population and it is considered an important cause of gastric cancer. The eradication therapy is not always effective because resistance to antimicrobials may occur. Objective: To determine the susceptibility profile of H. pylori isolates to the antibiotics amoxicillin, clarithromycin and ciprofloxacin in the population of Rio Grande do Sul using different standardizations. Material and Methods: Transversal study. Were evaluated 54 samples of H. pylori obtained by gastric biopsies which were cultured on Belo Horizonte agar and incubated at 37°C in a microaerophilic environment for five days. The antibiotics susceptibility was determined according to the guidelines of the British Society for Antimicrobial Chemotherapy (BSAC), the Australian (CDS Method) (quantitative) and the French (CA-SFM) (qualitative). Results and discussion: Seven (13%) H. pylori isolates were resistant to clarithromycin, one (1,9%) to amoxicillin and three (5,5%) to ciprofloxacin. These indices of resistance are considered satisfactory and show that all of these antibiotics can be used in the empirical therapy of the local population, especially amoxicillin and clarithromycin as a first line treatment. The quantitative methodologies BSAC and CDS Method revealed very similar agreement in the susceptibility results. Moreover, the CDS method had an easier interpretation technique. The CA-SFM standards seem to be more attractive on the economic aspect but they only provide qualitative results. Conclusion: The antibiotics amoxicillin and clarithromycin are still a good option for anti-H. pylori treatment in the population of Rio Grande do Sul.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Grupo DASA / Melioidose à uma doenÃa infecciosa grave causada por Burkholderia pseudomallei, um bacilo Gram-negativo encontrado no solo e na Ãgua. A doenÃa à endÃmica no sudeste asiÃtico e hiperendÃmica no norte da AustrÃlia, onde a letalidade permanece com uma taxa de 21%. No Brasil, à considerada uma doenÃa emergente desde marÃo de 2003. Nos Ãltimos oito anos, 12 casos ocorreram no Estado do Cearà e um notificado pelo Governo holandÃs, por se tratar de um turista que morreu de melioidose, apÃs visita ao CearÃ. Em razÃo da ocorrÃncia clÃnica de melioidose e do isolamento de B. pseudomallei no ambiente do Estado do CearÃ, este trabalho objetivou estudar as cepas clÃnicas e ambientais de B. pseudomallei isoladas no Estado no perÃodo de 2003 a 2011, visando a identificar as cepas por mÃtodos fenotÃpicos e moleculares, determinar o perfil de sensibilidade contra cinco agentes antimicrobianos (amoxicilina/clavulanato, ceftazidima, imipenem, doxicilina e sulfametoxazol/trimetoprim), realizar a genotipagem das cepas pela amplificaÃÃo aleatÃria de DNA polimÃrfico - Random Amplified Polymorphic DNA (RAPD), detectar o gene de virulÃncia Type Three Secretion System (TTSS), alÃm de avaliar os aspectos clÃnico-epidemiolÃgicos que caracterizaram a emergÃncia desta doenÃa no Brasil. Todas as 20 cepas (dez clÃnicas e dez ambientais) de B. pseudomallei foram precisamente identificadas tanto pela metodologia VITEK2 quanto pelo sequenciamento da regiÃo 16S do DNA, mostraram resultado negativo no teste de assimilaÃÃo de L-arabinose, e exibiram-se positivas para a detecÃÃo do gene de virulÃncia TTSS. As concentraÃÃes inibitÃrias mÃnimas (CIMs), obtidas por microdiluiÃÃo em caldo MÃeller-Hinton, demonstraram que todos os isolados (100%) foram sensÃveis ao imipenem, à doxicilina e ao sulfametoxazol- trimetoprim, no entanto, para amoxicilina/clavulanato e ceftazidima, a sensibilidade foi de 80 e 90%, respectivamente. A tÃcnica de RAPD evidenciou uma variabilidade genÃtica de 63% entre as cepas de B. pseudomallei oriundas do Estado do CearÃ, as quais foram agrupadas em trÃs clusters diferentes. Este trabalho decerto contribuirà para o conhecimento das caracterÃsticas fenotÃpicas e genotÃpicas das cepas de B. pseudomallei isoladas no Cearà e da atualizaÃÃo da vigilÃncia epidemiolÃgica dos casos de melioidose ocorridos no Estado, alÃm de contribuir para a conscientizaÃÃo dos ÃrgÃos de saÃde competentes para a inclusÃo do Cearà como zona endÃmica para esta enfermidade. / Melioidosis is a serious infectious disease caused by Burkholderia pseudomallei, a Gram negative rod, commonly found in soil and water. The disease is endemic in Southeastern Asia and hyperendemic in Northern Australia. Despite the initiation of empiric therapy, mortality remains at 21% in patients with melioidosis in Australia. In Brazil, it is considered an emerging disease, since April 2003, when it was first diagnosed in Ceara, Northeastern Brazil. In the last eight years, thirteen cases were reported, twelve local cases and one case reported by the Dutch government because of a tourist who died of melioidosis after a visit to Ceara. Considering the occurrence of melioidosis in CearÃ, this work aimed at studying these clinical and environmental strains of Burkholderia pseudomallei isolated from Cearà from 2003 to 2011, focusing on the bacterial and molecular identification; determining the susceptibility profile against five antimicrobial agents (amoxicillin/clavulanate, ceftazidime, imipenem, doxycycline and trimethoprim/sulfamethoxazole); genotyping through Random Amplified Polymorphic DNA (RAPD), detecting the virulence gene Type Three Secretion System (TTSS); and analyzing epidemiological and clinical aspects that characterized the emergence of this disease in Brazil. All 20 strains (10 clinical and 10 environment) from B. pseudomallei were accurately identified by both VITEK2  and sequencing of the 16S DNA, showed to be negative for the assimilation of L-arabinose and were positive results for the detection of the virulence gene TTSS. The minimum inhibitory concentrations (MICs) obtained through microdilution in MÃeller-Hinton broth, showed that all (100%) isolates were sensitive to imipenem, doxycycline and trimethoprim-sulfamethoxazole, however, the susceptibility rate to amoxicillin/clavulanate and ceftazidime was of 80 and 90%, respectively, with no differences between clinical and environmental strains. RAPD-PCR showed a genetic relatedness of 63% among the B. pseudomallei strains from the State of CearÃ, which were grouped in two different clusters. This work will contribute to the knowledge of phenotypic and genotypic characteristics of B. pseudomallei strains isolated in Cearà and the update of epidemiological surveillance of melioidosis cases in the state, also contribute to the awareness of agencies health authority for inclusion of Cearà State as an endemic area for this disease.

To investigate the bacterial composition of infected and non-infected dog bite wounds (DBW), a prospective study was performed on dogs with various grades of bite wounds presenting at the Onderstepoort Veterinary Academic Hospital, University of Pretoria, and a nearby animal shelter. Fifty dogs with bite wounds inflicted within the previous 72 hours were selected. This represented 104 wounds. Wounds were clinically graded according to severity. Swabs were collected from all wounds for bacterial culture and cytology. Infection was diagnosed if 2 of the following 3 criteria were met: macroscopic purulence, microscopic presence of phagocytosed bacteria, or pyrexia. Non-infected wounds were either classed as sterile (established by culture) or contaminated (culture positive but bacteria not phagocytosed on cytology). To determine the origin of the bacteria, swabs were collected from the skin near the wounds and gingiva of 15 bite victims. All swabs were cultured aerobically and anaerobically and all aerobic cultures were evaluated for antimicrobial susceptibility using the Kirby Bauer disk diffusion test. The victims were predominately male, uncastrated, small-breed dogs. Of the 104 wounds studied, 21 were judged to be infected and 83 non-infected. Infected wounds were significantly more likely to culture positive (Fisher's exact test: p = 0.02). Sixteen per cent of wounds did not culture bacteria, 67% grew aerobes only, 1% anaerobes only and 67% a mixture of aerobes and anaerobes. A total of 213 isolates were cultured representing a mean of 2 isolates per wound. Of the aerobe species cultured, 22%, 19% and 17% belonged to the genera of Pasteurella, Streptococcus and Staphylococcus respectively. The species of Pasteurella multocida (66%) and Staphylococcus intermedius (70%) were predominant. Pasteurella canis and pyogenic streptococci were common in infected wounds, whereas Bacillus spp., Actinomyces spp. and oral streptococci were usually found in contaminated wounds. Three anaerobic genera were cultured, namely, Prevotella, Clostridium and Peptostreptococcus, and were usually associated with wounds with dead space. This study also describes the first documented case of Capnocytophaga canimorsus in an infected dog bite wound. Notably clinical and cytological assessment was capable of establishing whether antimicrobials were required or not. Although no single antimicrobials was considered to be effective against all the bacteria, amoxycillin plus clavulanic acid, 1st and 3rd generation cephalosporins, ampicillin or amoxycillin and potentiated sulphonamides gave the best in vitro sensitivity results. / Dissertation (MMedVet(Surgery) Small Animal Surgery)--University of Pretoria, 2007. / Companion Animal Clinical Studies / unrestricted

Background. Severe periodontal disease is highly prevalent worldwide, affecting 20% of the population between the ages of 35 and 44 years. The etiological epidemiology in Peru is scarce, even though some studies describe a prevalence of 48.5% of periodontal disease in the general population. Periodontitis is one of the most prevalent oral diseases associated with site-specific changes in the oral microbiota and it has been associated with a socioeconomic state. This study aimed to determine the etiology and resistance profile of bacteria identified in a group of Peruvian patients with periodontal disease. Methods. Six subgingival plaque samples were collected from eight patients with severe periodontitis. Bacterial identification was carried out by an initial culture, PCR amplification, and subsequently DNA sequencing. We evaluated the antibiotic susceptibility by the disk diffusion method. Results. Variable diversity in oral microbiota was identified in each one of the eight patients. The bacterial genus most frequently found was Streptococcus spp. (15/48, 31.3%) followed by Rothia spp. (11/48, 22.9%), Actinomyces spp. (9/48, 18.8%), and Eikenella spp. (4/48, 8.3%). The most common species found was Rothia dentocariosa (8/48, 16.7%). The antimicrobial susceptibility assay varied according to the species tested; however, among all the isolates evaluated, Actinomyces naeslundii was resistant to penicillin and tetracycline; Eikenella corrodens was resistant to dicloxacillin; and Rothia dentocariosa was resistant to amoxicillin + clavulanic acid and metronidazole but also susceptible to trimethoprim-sulfamethoxazole. Conclusions. The most prevalent periodontal bacterium found in this study was Rothia dentocariosa. Specific antimicrobial therapy is required to improve the treatment outcomes of patients with periodontal disease and avoid antibiotic resistance. / Revisión por pares

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21298号 / 医博第4387号 / 新制||医||1030(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 中川 一路, 教授 木原 正博, 教授 小池 薫 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The growing threat of antimicrobial resistance is among the largest concerns in the world today. One method under development to combat this issue is the encapsulation of microbes in microfluidic droplets for single-cell testing. This method may be able to circumvent the need for a traditional positive cell culture which consumes the majority of the testing time using current diagnostic methods. This dissertation presents a method by which to deterministically encapsulate microbes using an artificial intelligence object detection algorithm and a Droplet-On-Demand microfluidic device. To accomplish this, the Droplet-On-Demand microfluidic device was first developed using a unique 3D-printing manufacturing method. An annular Channel-in-Channel droplet generator was developed which produced droplets within the hydrophobic 3D-printed polymeric microfluidic device. Supporting microfluidic unit operations were also developed including pumps, a 3-way flow-thru valve, and a detection window used for visualizing microfluidic particles. Control software was developed using python which controlled pneumatically-actuated membranes within the microfluidic device, the imaging system, and the object detection algorithm. 20-μm and 2-μm test particles were used as non-biological test particles while red blood cells and fluorescent E.coli baceria were used as biological test particles. All test particles were identified and encapsulated and show the flexibility of the system overall and the ability to identify a variety of particles of interest in microfluidic systems. Growth tests were conducted using E.coli bacteria encapsulated within microfluidic droplets with a fluorescent metabolic indicator. The fluorescence of droplets containing actively growing encapsulated bacteria was quantified using a unique first-principles model paired with an image processing protocol to provide relative concentration data to quantify the growth of the E.coli over time. These growth results indicated that bacterial growth in droplets could be detected and quickly quantified in 4 hours and thus provide practical results to clinicians on the susceptibility of bacteria to an antibiotic. This Droplet-On-Demand technology has the capability of providing clinically applicable data from the most basic and fundamental biological source, an individual cell; and that can be done with low concentrations and on any cell that can be visually identified.

The genus Gluconobacter consists of acetic acid bacteria which have the ability to generate acidic products from their substrates, particularly acetic acid from ethanol. For this reason, the gluconobacters live in acidic, sugary environments such as flowers, honey bees, fruits, cider, vinegar, wine and beer. The gluconobacters carry out a strictly respiratory type of metabolism using only oxygen as a terminal electron acceptor. They do not completely oxidize a substrate to carbon dioxide. Instead, they partially oxidize the substrate using membrane-bound dehydrogenases and excrete the product into the surrounding growth medium. It is these limited oxidations that make the gtuconobacters industrially useful. Although much is known about the physiology of the limited oxidations in the gluconobacters, little is known of their genetics, particularly, their plasmids. The overall purpose of this dissertation was to determine if Gluconobacter plasmids correlate with oxidative capability and/or antibiotic resistance. To achieve this goal, I first needed a way to screen strains of Gluconobacter for their ability to oxidize many different substrates. 'developed an assay that used an unusual artificial electron acceptor, tetranitroblue tetrazolium (TNBT) and then tested the ability of six strains to oxidize 13 chemical compounds. Although most strains were able to oxidize the 13 compounds tested, they accomplished this with varying extents of oxidation. These differences were noted even with strains representing the same species. / Ph. D.

Under vissa omständigheter kan bakterier kan ta sig in i blodbanan där de kan orsaka allvarliga infektioner (bakteriemi). Metoden som används för identifiering och resistensbestämning av bakterier i blododlingar kräver minst 16 timmars inkubation. Fram tills en resistensbestämning utförts kan empirisk antibiotikabehandling användas, men med ökande resistensutbredning blir det alltmer osäkert om denna behandling är verksam. Nyligen har en lappdiffusionsmetod för resistensbestämning direkt från blododling validerats, som kan läsas av efter 4, 6 och/eller 8 timmars inkubation. Det finns även publicerade arbeten där flödescytometriassisterad resistensbestämning används, men då krävs att bakterierna finns i tillräckligt hög koncentration, befinner sig i tillväxtfas och finns som renkultur. Syftet med examensarbetet var att optimera hantering av blododlingsflaskor så att bakterier från blododlingsflaskorna kunde isoleras med tillräcklig kvalitet och koncentration för att kunna utföra resistensbestämning med flödescytometri. Upprening av bakterierna utfördes med olika tvättbuffertar och sedan utfördes resistensbestämning med flödescytometri och  referensmetoden buljongspädning. Resultaten från uppreningen visade att sterilt vatten och Tween20 gynnade bakteriernas återhämtningsförmåga mest. Resistensbestämning utfördes med Klebsiella pneumoniae ATCC700603,  K. pneumoniae CCUG56233 och K. pneumoniae ATCC13882, som tvättats med sterilt vatten och Tween20. För CCUG56233 skiljde 1 spädningssteg i koncentrationsskalan mellan metoderna. ATCC-isolaten erhöll likartade MIC-värden (minimum inhibitory concentration) vid alla analyser men där fanns en skillnad på 2 spädningssteg mellan buljongspädning och analys med flödescytometri. Detta kan förklaras av skillnaden i inkubationstid mellan metoderna. Slutsatsen som kan dras är därför att resultaten från de två metoderna vid resistensbestämning är likartade och att sterilt vatten är mest lämpligt att använda vid upprening av bakterier. Fler undersökningar bör dock utföras.

O Actinobaculum suis é um dos principais micro-organismos relacionados a infecções de trato urinário em fêmeas suínas. As características de crescimento deste agente dificultam o isolamento bacteriano tradicional, o que pode tornar a sua prevalência subestimada. Este estudo teve por objetivos desenvolver a reação em cadeia pela polimerase (PCR) para detecção do A. suis, avaliar a sensibilidade e especificidade desta técnica e comparar seu desempenho com o isolamento bacteriano. Além disso, as cepas isoladas foram caracterizadas através do polimorfismo de comprimento de fragmentos amplificados (AFLP) e submetidas à determinação da concentração inibitória mínima para caracterização dos perfis de susceptibilidade antimicrobiana. Foram analisados 45 suabes prepuciais de machos e 192 urinas de fêmeas suínas provenientes de três granjas. Os resultados indicaram que a PCR desenvolvida foi específica para o A. suis e apresentou limiar de detecção entre 1,0 X 101 UF/mL e 1,0 X 102 UFC/mL. A frequência de A. suis encontrada através da PCR foi de 82,2% (37/45) nos suabes prepuciais e de 8,9% (17/192) nas urinas de fêmeas. No que se refere ao isolamento, nenhuma das amostras de urina foi positiva para o agente, enquanto 31,1% (14/45) dos suabes foram positivos. A partir das amostras positivas isoladas dos suabes prepuciais foram selecionadas 20 cepas de A. suis. Os perfis de susceptibilidade entre estas cepas foram semelhantes, no entanto diferiram dos isolados utilizados como controle e provenientes de uma fêmea com infecção urinária. A técnica de PCR foi mais eficiente que o isolamento na identificação de amostras positivas para A. suis. Através do AFLP com uma única enzima foi possível caracterizar todos os isolados e relacionar os dados obtidos com a origem das cepas e o perfil de resistência. Até o presente não há relatos na literatura de caracterização genotípica de A. suis através do AFLP ou detecção do agente através da PCR. / Actinobaculum suis is an important agent related to urinary infection in swine females. The growth characteristics of this agent hamper the traditional bacterial isolation, which can make their prevalence underestimated. The purpose of this study was to develop the polymerase chain reaction (PCR) for Actinobaculum suis detection, to evaluate the sensitivity and specificity of this technique and compare the results with bacterial isolation. Moreover, the isolates were characterized by amplified fragment length polymorphism (AFLP) and subjected to determination of minimum inhibitory concentration for characterization of the antimicrobial susceptibility profiles. Forty-five preputial swabs from boars and a hundred and ninety-two urine samples from sows of three herds were analyzed. The results indicate that the developed PCR was specific for A. suis, presenting a limit detection between 1.0 X 101 UFC/ml and 1.0 X 102 UFC/ml. A.suis frequency by PCR was 82.2% (37/45) in male preputial swabs and 8.9% (17/192) in females urine. Through traditional isolation, none of the urine samples were positive, while A.suis growing was observed in 31.1% (14/45) of the swabs. From the positive samples of the preputial swabs were selected 20 A.suis strains. The susceptibility profiles among these strains were similar, but differed from the female isolates used as control. The PCR technique was more effective than isolation for the A.suis detection. The AFLP with a single enzyme was able to characterize all isolates and relate the data obtained with the strains origin and resistance profile. Until present, there are no reports of genotypic characterization of A. suis strains through AFLP or agent detection by PCR.

A Bordetella avium é o agente etiológico da bordetelose aviária, uma doença altamente contagiosa que afeta o trato respiratório superior das aves. B. avium adere-se preferencialmente às células do epitélio ciliado traqueal, promovendo inflamação e deformação da mucosa respiratória. As infecções do trato respiratório das aves resultam em grandes prejuízos para toda indústria avícola, desta forma, o presente estudo teve como objetivo a caracterização genotípica e de sensibilidade a antimicrobianos de isolados de B. avium provenientes de perus com histórico de aerossaculite. Dentre os 300 animais examinados, isolou-se B. avium de 13 aves e foram selecionadas 20 cepas do agente para os estudos posteriores. Através do antibiograma realizado pela técnica de disco difusão observou-se um alto número de cepas resistentes aos antimicrobianos beta lactâmicos (amoxacilina, ampicilina, penicilina e ceftiofur), assim como para lincomicina, sulfonamidas e combinação sulfonamidas/trimetoprima (cotrimoxazol) e uma grande heterogeneidade resultando em 15 perfis distintos. Os antimicrobianos com maiores níveis de sensibilidade foram o florfenicol, seguidos pelas quinolonas, doxiciclina e pelas tetraciclinas. Todas as cepas foram caracterizadas através da PFGE e do AFLP, apresentando 15 pulsotipos e 16 perfis genotípicos respectivamente. Os métodos fenotípicos e genotípicos apresentaram capacidade discriminatória semelhante e revelaram uma grande diversidade dentre os isolados analisados. / Bordetella avium is the etiologic agent of avian bordetellosis, a highly contagious disease that affects the upper respiratory tract of birds. B. avium adheres preferentially to ciliated tracheal epithelial cells, promoting inflammation and deformation of the respiratory mucosa. Infections of the respiratory tract of birds resulting in large losses for the entire poultry industry in this way, this study aimed to characterize genotypic and antimicrobial susceptibility of isolates of B. avium from turkeys with a history of Airsacculitis. Among the 300 animals examined, B. avium was isolated from 13 turkeys and 20 strains were selected for further studies. Through the antibiogram performed by disk diffusion technique was observed a high number of strains resistant to beta-lactamic antibiotics (amoxicillin, ampicillin, penicillin and ceftiofur), as well as, lincomycin, sulfonamides and sulfonamide combination/ trimethoprim (cotrimoxazole) and a high level of heterogeneity resulting in 15 different profiles. The antimicrobials with higher levels of sensitivity were florfenicol, followed by quinolones, doxycycline and tetracycline. All strains were characterized through to PFGE and AFLP, presenting 15 pulsotypes and 16 genetic profiles, respectively. Phenotypic and genotypic methods showed similar discriminatory capacity and presented a high diversity among isolates examined.