A presente tese teve como objetivos analisar o padrão de resistência a antimicrobianos de bactérias isoladas de infecções endodônticas agudas por meio de uma revisão sistemática e meta-análise (capítulo 1); identificar e determinar a diversidade genotípica, a sensibilidade bacteriana e os fatores de virulência de anaeróbios facultativos isolados em casos de abscesso apical agudo (capítulo 2); e realizar, por meio de uma proposta de informativo, a difusão de informações geradas com orientações sobre o uso de agentes antibióticos em infecções endodônticas agudas para dentistas e pacientes (capítulo 3). A revisão sistemática foi realizada por meio de pesquisa em base de dados na internet e na literatura cinza até maio de 2015. As normas do PRISMA foram seguidas. Estudos clínicos em humanos que avaliaram o perfil de resistência a antimicrobianos de isolados de infecções endodônticas agudas primárias por meio de métodos laboratoriais foram incluídos. O efeito randômico da Meta-análise foi empregado, e o desfecho foi descrito reunindo as taxas de resistência para cada antibiótico testado. Os dados de sete estudos foram extraídos. A taxa de resistência para 15 diferentes agentes antibióticos foram avaliadas, variando entre 3,5% a 40%. Baixas taxas de resistência foram observadas para amoxicilina + clavulanato e amoxicilina, e altas taxas foram observadas para tetraciclina. No capítulo 2, amostras de canais radiculares foram coletadas de sete dentes com diagnóstico de abscesso apical agudo. Bactérias anaeróbias facultativas foram identificadas com o auxílio de métodos fenotípicos e MALDI-TOF MS. A sensibilidade antimicrobiana das cepas isoladas foi determinada para benzilpenicilina, eritromicina e clindamicina por meio da difusão em disco. Bactérias anaeróbias facultativas foram isoladas de 3 dentes. Streptococcus spp., Staphylococcus aureus, Enterococcus faecalis, e Actinomyces viscosus foram identificados. Streptococcus spp. e S. aureus foram sensíveis à benzilpenicilina. E. faecalis (24 cepas) isolados de um mesmo paciente tiveram a sensibilidade antimicrobiana determinada por meio da concentração inibitória mínima para benzilpenicilina, amoxicilina e amoxicilina + clavulanato utilizando-se o E-test. A diversidade genotípica e a presença de fatores de virulência (ace, asa, gelE, efaA, cylA, esp) nas cepas de E. faecalis foi analisada por meio do PFGE e PCR, respectivamente. A expressão da gelatinase e da hemolisina foi testada, e a produção de biofilme quantificada. E. faecalis foram sensíveis aos antibióticos beta-lactâmicos. O mesmo perfil cromossonal de DNA foi revelado para cepas de E. faecalis isoladas. Os genes gelE, ace e efaA foram detectados em 18 cepas. A expressão da gelatinase e a produção de biofilme foram observadas. Cepas de E. faecalis tiveram o mesmo perfil de DNA cromossomal, porém parecem apresentar diferentes perfis de virulência. No capítulo 3 foram apresentadas duas propostas de textos informativos. A primeira com o objetivo de orientar o dentista sobre o uso correto dos antibióticos sistêmicos em endodontia. A segunda visa informar os pacientes sobre a utilização dos antibióticos e esclarecer possíveis dúvidas. / The present thesis aimed to analyze the antimicrobial resistance profile of bacteria isolated from acute endodontic infections through a systematic review and meta-analysis (Chapter 1); to identify and determine the genotypic diversity, antimicrobial susceptibility and virulence factors of isolated facultative anaerobes in isolates from acute apical abscess (Chapter 2); and to provide information for dentist and patients generated from guidelines on the use of antibiotic agents in acute endodontic infections (Chapter 3). The electronic databases and the gray literature were searched up to May 2015 for systematic review. PRISMA guidelines were followed. The clinical studies in of humans that have evaluated the antimicrobial resistance of the isolates of primary acute endodontic infections through laboratorial methods were included. A random effect meta-analysis was employed, and the outcome was described as being the pooled resistance rates for each antimicrobial agent. The data from 7 studies were extracted. The resistance rates for 15 different antimicrobial agents were evaluated, ranging from 3.5% to 40.0%. Lower resistance rates were observed for amoxicillin+clavulanate and amoxicillin, and higher resistant rates were detected for tetracycline. In Chapter 2, root canal samples were collected from seven teeth. Facultative anaerobic bacteria were identified by phenotypic methods and MALDI-TOF MS. Antimicrobial susceptibility of strains was determined to benzylpenicillin, erythromycin and clindamycin by disk-diffusion. Facultative anaerobic bacteria were isolated from 3 teeth. Streptococcus spp., Staphylococcus aureus, Enterococcus faecalis, and Actinomyces viscosus were found. Streptococcus spp. and S. aureus were susceptible to benzylpenicillin. E. faecalis strains (n=24) isolated from the same patients had their susceptibility determined by minimum inhibitory concentration of benzylpenicillin, amoxicillin and amoxicillin + clavulanate using the E-test. The genotypic diversity and virulence factors (ace, asa, gelE, efaA, cylA, esp) were analyzed in E. faecalis strains by PFGE and PCR, respectively. Phenotypic expression of gelatinase and cytolysin were tested. Biofilm production was quantified. All the E. faecalis strains were susceptible to β-lactam antibiotics. The same chromosomal DNA fragmentation profile was revealed to E. faecalis strains isolated. The gelE, ace and efaA genes were detected in 18 E. faecalis strains. Gelatinase expression and biofilm production were observed. E. faecalis strains had the same chromosomal DNA profile, but showed virulence profiles different. In Chapter 3, two sugestion for information texts were presented. The first aims to guide dentists on the proper use of systemic antibiotics in endodontics. The second aims to inform patients about the use of antibiotics and clarify possible doubts.

Staphylococcus aureus e Streptococcus uberis são dois patógenos causadores mastite bovina que podem apresentar capacidade de produção de biofilme, o que pode resultar em infecções intramamárias crônicas, menor resposta à terapia, redução de produção de leite e maior risco de descarte das vacas infectadas. Os objetivos deste estudo foram avaliar a: 1) capacidade de formação de biofilme de S. aureus e S. uberis isolados de vacas com mastite clínica (MC) e subclínica (MSC); 2) sensibilidade in vitro e a multirresistência destes agentes a antimicrobianos selecionados (n=12); 3) associação entre a capacidade de formação de biofilme e resistência aos antimicrobianos de S. aureus e S. uberis. Um total de 197 cepas S. aureus e 128 S. uberis foram isoladas a partir de amostras de leite de vacas com MSC e MC, oriundas de 24 rebanhos. Os isolados de S. aureus e S. uberis foram avaliados quanto a capacidade de formação de biofilme pelo método??? e a sensibilidade in vitro aos antimicrobianos foi determinada pela técnica de disco difusão em ágar. A capacidade de formação de biofilme foi classificada em 4 categorias: forte, moderado, fraco e não formador de biofilme. Do total de cepas avaliadas, S. aureus (54,8%) e S. uberis (52,9%) apresentaram capacidade de formação de biofilme (forte, moderado ou fraco). Entre os isolados de S. aureus formadores de biofilme, a frequência de distribuição dos isolados foi de 19,3% na categoria forte, 18,8% moderado, e 16,7% na categoria fraco. Para os isolados de S. uberis, a frequência de distribuição entre as categorias de formação de biofilme foi 17,6% forte, 25,2% moderado, 17,6% fraco. Dentre as cepas de S. aureus isoladas de casos de MC, 55,8% foram classificados como forte formador de biofilme, enquanto 7,6% das cepas isoladas de MSC apresentaram capacidade de formação de biofilme. Todos os isolados de S. uberis (n=30; 100%) provenientes de MC apresentaram capacidade de formação de biofilme na categoria moderado. Quanto à sensibilidade aos antimicrobianos, os isolados de S. aureus apresentaram resistência à penicilina (92,9%), ampicilina (50,8%) e tetraciclina (18,3%); e os isolados de S. uberis apresentaram resistência à penicilina (86,5%), oxacilina (85,5%), tetraciclina (37,5%). Os isolados de S. aureus apresentaram maior chance de resistência aos antimicrobianos ampicilina, tetraciclina e ceftiofur que S. uberis. Em conclusão, S. aureus e S. uberis apresentam elevada capacidade de produção de biofilme, mas não houve interação entre a característica de multirresistência e formação de biofilme. Isolados de S. aureus e S. uberis foram altamente resistentes aos antimicrobianos das classes de beta-lactâmicos e tetraciclinas. / Staphylococcus aureus and Streptococcus uberis are both mastitis causing pathogens that can present ability to produce biofilm, which can result in chronic intramammary infection, reduced response to the therapy, reduction of milk yield, and greater risk of cows\' culling. The objectives of this study were to evaluate the: 1) biofilm-forming capacity of S. aureus and S. uberis isolated from clinical (CM) and subclinical mastitis (SCM); in vitro sensibility and multi-resistance of these agents to the antimicrobials; 3) association between the biofilm-forming capacity and antimicrobial resistance. A total of 197 S. aureus and 128 S. uberis were isolated from milk samples collected from cows with SCM and CM from 24 dairy herds. The biofilm-forming ability were classified in 4 categories: strong, moderate, weak, and non-biofilm producers. Of all isolates evaluated, S. aureus (54.8%) and S. uberis (52.9%) presented biofilm-forming ability (strong, moderate or weak). Among the biofilm-forming isolates, the frequency of distribution of S. aureus was 19.3% for the strong, 18.8% for the moderate, and 16.7% for the weak categories. For the S. uberis isolates, the frequency of distribution among the biofilm-forming categories was 17.6% strong, 25.2% moderate, and 17.6% weak. In relation to the mastitis presentation form, the strong biofilm-forming category had 55.8% of S. aureus isolates from CM cases; and among all biofilm-forming categories, the strong category was the one with the higher number of isolates of S. aureus (n=43; 19,2%). All S. uberis isolates (n=30; 100%) from CM presented moderate biofilm-forming ability. In relation to the antimicrobial susceptibility, the isolates of S. aureus were resistant to penicillin (92.9%), ampicillin (50.8%) and tetracycline (18.3%); and the isolates of S. uberis presented resistance to penicillin (86.5%), oxacillin (85.5%) and tetracycline (37.5%). The isolates of S. aureus and S. uberis, S. aureus had higher odds to be resistant to ampicillin, tetracycline and ceftiofur than S. uberis. In conclusion, S. aureus and S. uberis presented high ability of production of biofilm, but there was no interaction between multi-resistance and biofilm production ability. Isolates of S. aureus and S. uberis were highly resistant to antimicrobials of the class of beta lactams and tetracycline.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Enterococcus faecalis (E. faecalis), conhecidamente patógeno oportunista, tem sido frequentemente associado a infecções sistêmicas graves. É também encontrado na cavidade oral, com destaque em infecção endodôntica refratária. O objetivo deste estudo foi avaliar características moleculares de E. faecalis isolados de infecção endodôntica primária no Brasil e comparar com isolados orais e não orais de pacientes do Reino Unido e do Japão, assim como E. faecalis resistentes à vancomicina. O presente estudo também investigou o relacionamento entre E. faecalis de diferentes origens (oral e não oral) e de diferentes áreas geográficas para obter uma melhor compreensão do envolvimento dos diferentes reservatórios no surgimento e propagação de clones virulentos, aqueles que possuem genes que conferem infectividade e virulência, assim como resistência aos antibióticos. Para tal, foram estudados E. faecalis isolados em infecções endodônticas no Brasil (n = 20) e orais no Reino Unido (n = 10), e em infecções não orais no Japão (n = 9). Além disso, 20 E. faecalis isolados ambientais do Hospital Universitário de Gales (Cardiff, Reino Unido), classificados como Enterococcus resistentes à vancomicina (VRE) também foram examinados. A Concentração Inibitória Mínima (CIM) dos isolados do Brasil foi obtida pelo método de diluição em agar de acordo com as recomendações do Clinical and Laboratory Standards Institute (CLSI). Reação em cadeia da polimerase (inglês - PCR) foi a técnica empregada para detectar os genes de virulência e aqueles associados à resistência aos antibióticos, enquanto Reação de Amplificação Aleatória de DNA Polimórfico (inglês - RAPD-PCR) foi escolhida para a tipagem molecular. Dentre os genes de virulência examinados, o gene que codifica a gelatinase gelE foi o mais prevalente entre os isolados (77-100%). Entre isolados orais, foram detectados os genes agg de substâncias de agregação, esp de proteína de evasão imune, cylB de citolisina, genes de resistência à tetraciclina tetM e tetL e à eritromicina ermB com diferentes prevalências. Os isolados clínicos hospitalares do Japão apresentaram perfil genético similar aos isolados orais, mas com maior prevalência de ermB e cylB. Todas as amostras de VRE foram positivas para os genes gelE, esp, agg, vanA, ermB e tetM, 95% foram positivos para cylB e 17% positivo para tetL. Todas as amostras foram negativas para ermA, asa373, vanB, vanC1 e vanC2/3. RAPD-PCR revelou agrupamento de VRE em comparação com outros isolados. Neste estudo, os isolados de E. faecalis de infecções orais apresentaram genes de resistência à tetraciclina, um antimicrobiano frequentemente usado no tratamento local de infecções dentárias, abrindo um debate muito importante sobre o papel e a eficácia desta droga para infecções orais. Claramente, são necessários mais estudos nesta área principalmente em relação à expressão de fatores de virulência entre isolados endodônticos para melhor nortear as estratégias de tratamento. As pressões externas no microambiente dos canais radiculares podem ser responsáveis pela seleção de espécies mais resistentes e virulentas. Por fim, embora isolados orais apresentem genes de virulência fundamentais para a patogenicidade, estes foram detectados em menor incidência em comparação com os isolados não-orais e VRE. / Enterococcus faecalis is an opportunistic pathogen known to cause serious systemic infection. It is also encountered in the oral cavity and has been implicated in persistent root canal infection. The aim of this study was to evaluate a range of molecular characteristics of E. faecalis isolated from primary endondontic infections in Brazil and compare to isolates from oral and non-oral infections in patients from UK and Japan, as well as isolates of vancomycin resistant E. faecalis, VRE, from a hospital environment. The present study was undertaken to explore the relatedness of E. faecalis from different origins, oral and non oral, and from different geographic areas to gain a better understanding of the involvement of the different reservoirs in the emergence and spread of virulent clones, those that acquired a number of genes conferring infectivity and virulence and in addition antibiotic resistance. To do this, E. faecalis from oral infections in Brazilian (n=20) and UK patients (n=10), and non-oral infection in japanese patients (n=9) were studied. In addition, 20 environmental VRE isolates from the University Hospital of Wales (Cardiff, UK) were also examined. For braziliam isolates, antimicrobial susceptibility was ascertained by agar dilution, using the recommendations of the Clinical and Laboratory Standards Institute (CLSI). For all isolates, PCR with validated primers was used to detect genes associated with antibiotic resistance and virulence, whilst RAPD-PCR was used to fingerprint isolates. Of the virulence genes examined, gelatinase gene gelE was most prevalent amongst isolates (77-100%). In the case of oral isolates, the genes of aggregation substances agg, immune evasion protein esp, cytolysin cylB, tetracycline resistance tetM and tetL and erythromycin resistance ermB were detected with varying prevalence. Japanese hospital isolates had a similar genetic profile to oral isolates but with higher prevalence of ermB and cylB. All VRE strains were positive for gelE, esp, agg, vanA, ermB and tetM, 95% were positive for cylB and 17% positive for tetL. All isolates were negative for ermA, asa373 vanB, vanC1 and vanC2/3. RAPD-PCR revealed clustering of VRE compared with other isolates. In this study, isolates of E. faecalis from oral infections showed antibiotic resistance genes for tetracycline, an agent used in the local treatment of dental infection. This opens up a much-needed debate on the role and efficacy of this antibiotic for oral infections. Clearly, more research in this area is required particularly in relation to the possession and expression of virulence factors in the root canal environment, to better inform our management strategies. Environmental pressures in root canals may be responsible for the selection of more resistant species and the possession of virulence determinants. This knowledge is important in guiding procedures for controlling the increasing problem of antibiotic resistance amongst clinically relevant bacteria. Furthermore, whilst oral isolates had genes that could contribute to pathogenicity, these were detected at lower incidence compared with non-oral and VRE isolates.

Erysipelothrix rhusiopathiae é um importante patógeno em suinocultura e apesar do uso freqüente de vacinas contra o mesmo na maior parte das propriedades produtoras do país, a ocorrência de quadros clínicos da infecção tem sido amplamente observada e diagnosticada. Tendo em vista o ressurgimento deste agente como causa de prejuízos econômicos para a indústria suinícola nacional e seu potencial risco à saúde pública, este estudo teve como objetivo caracterizar 151 amostras de Erysipelothrix spp. isoladas de suínos nos útlimos 30 anos por meio de sorotipagem, determinação da susceptibilidade antimicrobiana, AFLP e PFGE. Dentre os 151 isolados, 139 foram classificados em 18 sorotipos diferentes (1a, 1b, 2a, 2b, 4, 5, 6, 7, 8, 10, 11, 12, 15, 17, 19, 21, 24 e 25), sendo que o sorotipo 2b foi o mais freqüente. Os perfis de susceptibilidade antimicrobiana foram muito semelhantes entre os isolados, o que impossibilitou a subtipagem dos isolados de Erysipelothrix spp. pelos testes de sensibilidade. Dentre os primers testados no AFLP, o HI-G foi o mais adequado à tipagem molecular de Erysipelothrix spp. Apesar do AFLP/HI-G e da PFGE apresentarem o mesmo índice discriminatório (0,98), a PFGE apresentou melhor relação com os dados epidemiológicos que o AFLP/HI-G, tendo em conta os agrupamentos por ela gerados. Independente da técnica molecular empregada, não foi observado a discriminação entre isolados recentes e históricos, bem como um padrão epidemiológico fixo de agrupamento dos mesmos. Contudo, o AFLP/HI-G pode ser uma alternativa interessante para diferenciar as espécies de Erysipelothrix, assim como a PFGE tem grande potencial para agrupar isolados deste gênero de acordo com os sorotipos. / Erysipelothrix rhusiopathiae is an important pathogen in swine production and even with the frequent use of vaccines against it in most of Brazilian pig farms, the occurrence of clinical manifestations of its infection has been widely observed and diagnosed. Given the resurgence of this agent as a cause of economic losses to the national pig industry and its potential risk to public health, this study aimed to characterize 151 samples of Erysipelothrix spp. isolated from swine in last 30 years by serotyping, determination of antimicrobial susceptibility, AFLP and PFGE. Among the 151 isolates, 139 were classified in 18 different serotypes (1a, 1b, 2a, 2b, 4, 5, 6, 7, 8, 10, 11, 12, 15, 17, 19, 21, 24 and 25) and serotype 2b was the most frequent. The susceptibility profiles were very similar among the isolates, which precluded the subtyping of Erysipelothrix spp. isolates by sensitivity tests. Among the primers tested in AFLP, the HI-G was the most suitable for molecular typing of Erysipelothrix spp. Despite AFLP/HI-G and PFGE provided the same discriminatory index (0.98), PFGE presented better relationship with epidemiological data than AFLP/HI-G, given the types of groups generated by it. Regardless of the molecular technique employed, there was no discrimination between recent and historical isolates as well as a fixed epidemiological pattern of grouping them. Nevertheless AFLP/HI-G could be an interesting alternative for Erysipelothrix species discrimination, even as PFGE has a good potential to diferenciate this genus according to serotypes.

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.

Erysipelothrix rhusiopathiae é um importante patógeno em suinocultura e apesar do uso freqüente de vacinas contra o mesmo na maior parte das propriedades produtoras do país, a ocorrência de quadros clínicos da infecção tem sido amplamente observada e diagnosticada. Tendo em vista o ressurgimento deste agente como causa de prejuízos econômicos para a indústria suinícola nacional e seu potencial risco à saúde pública, este estudo teve como objetivo caracterizar 151 amostras de Erysipelothrix spp. isoladas de suínos nos útlimos 30 anos por meio de sorotipagem, determinação da susceptibilidade antimicrobiana, AFLP e PFGE. Dentre os 151 isolados, 139 foram classificados em 18 sorotipos diferentes (1a, 1b, 2a, 2b, 4, 5, 6, 7, 8, 10, 11, 12, 15, 17, 19, 21, 24 e 25), sendo que o sorotipo 2b foi o mais freqüente. Os perfis de susceptibilidade antimicrobiana foram muito semelhantes entre os isolados, o que impossibilitou a subtipagem dos isolados de Erysipelothrix spp. pelos testes de sensibilidade. Dentre os primers testados no AFLP, o HI-G foi o mais adequado à tipagem molecular de Erysipelothrix spp. Apesar do AFLP/HI-G e da PFGE apresentarem o mesmo índice discriminatório (0,98), a PFGE apresentou melhor relação com os dados epidemiológicos que o AFLP/HI-G, tendo em conta os agrupamentos por ela gerados. Independente da técnica molecular empregada, não foi observado a discriminação entre isolados recentes e históricos, bem como um padrão epidemiológico fixo de agrupamento dos mesmos. Contudo, o AFLP/HI-G pode ser uma alternativa interessante para diferenciar as espécies de Erysipelothrix, assim como a PFGE tem grande potencial para agrupar isolados deste gênero de acordo com os sorotipos. / Erysipelothrix rhusiopathiae is an important pathogen in swine production and even with the frequent use of vaccines against it in most of Brazilian pig farms, the occurrence of clinical manifestations of its infection has been widely observed and diagnosed. Given the resurgence of this agent as a cause of economic losses to the national pig industry and its potential risk to public health, this study aimed to characterize 151 samples of Erysipelothrix spp. isolated from swine in last 30 years by serotyping, determination of antimicrobial susceptibility, AFLP and PFGE. Among the 151 isolates, 139 were classified in 18 different serotypes (1a, 1b, 2a, 2b, 4, 5, 6, 7, 8, 10, 11, 12, 15, 17, 19, 21, 24 and 25) and serotype 2b was the most frequent. The susceptibility profiles were very similar among the isolates, which precluded the subtyping of Erysipelothrix spp. isolates by sensitivity tests. Among the primers tested in AFLP, the HI-G was the most suitable for molecular typing of Erysipelothrix spp. Despite AFLP/HI-G and PFGE provided the same discriminatory index (0.98), PFGE presented better relationship with epidemiological data than AFLP/HI-G, given the types of groups generated by it. Regardless of the molecular technique employed, there was no discrimination between recent and historical isolates as well as a fixed epidemiological pattern of grouping them. Nevertheless AFLP/HI-G could be an interesting alternative for Erysipelothrix species discrimination, even as PFGE has a good potential to diferenciate this genus according to serotypes.

Background: Infections caused by carbapenem-resistant Enterobacteriaceae (CRE) such as Escherichia coli and Klebsiella pneumoniae are among the most urgent threats of the infectious disease realm. The incidence of these infections has been increasing over the years and due to very limited treatment options, mortality is estimated at about 50%. By 2050, mortality from antimicrobial resistant infections is expected to surpass cancer at 10 million deaths annually. Methods: We evaluated 18 contemporary antimicrobials against 122 carbapenem-resistant Enterobacteriaceae using a variety of antimicrobial susceptibility testing methods according to Clinical Laboratory Standards Institute guidelines. Time-kill studies were performed on clinical isolates with variable resistance to meropenem, amikacin, and polymyxin B. Phenotypic expression assays were performed on all isolates and whole genome sequencing was performed on 8 isolates to characterize molecular resistance mechanisms. Pharmacodynamic modeling of meropenem and polymyxin B was also conducted. Results: CRE were primarily K. pneumoniae, and Enterobacter spp. 60% expressed Klebsiella pneumoniae carbapenemase (KPC) only, 16% expressed Verona Integron-encoded Metallo-beta-lactamase (VIM) only, 5% expressed KPC and VIM, and 20% expressed other mechanisms of resistance. Antimicrobial susceptibility testing indicated the most active antimicrobials against CRE were ceftazidime/avibactam, imipenem/relebactam, amikacin, tigecycline, and the polymyxins. Etest® strips did not reliably measure polymyxin B resistance. The automated testing system, BD Phoenix™, consistently reported lower MICs than the gold standard broth microdilution. Time-kill studies showed regrowth at clinically achievable concentrations of meropenem alone (4, 16, and 64 mg/L), polymyxin B alone (0.25 and 1 mg/L), or amikacin alone (8 and 16 mg/L), but combinations of meropenem with either polymyxin B or amikacin were bactericidal and synergistic. Meropenem administered simultaneously or prior to polymyxin B exhibited superior activity to polymyxin B administered first. Conclusions: Novel carbapenemase-inhibitor combinations (ceftazidime/avibactam and imipenem/relebactam) exhibit the best activity against KPC-producing CRE. The polymyxins, amikacin, and tigecycline exhibit the best activity against VIM-producing CRE. Meropenem in combination with polymyxin B is bactericidal and synergistic when the meropenem MIC is ≤32 mg/L, and meropenem should never be administered after polymyxin B. Meropenem and amikacin is bactericidal and synergistic when the amikacin MIC is ≤16 mg/L. Etest® strips should not be used for characterizing polymyxin B or colistin activity. Clinicians should be aware that automated testing systems may produce biased susceptibility results relative to the gold standard method, broth microdilution, which may influence interpretation of in vitro results.

Rapid determination of drugs effective against bacterial strains is critically important to stopping further spread of an infection and reducing antibiotic resistance. Antimicrobial susceptibility testing (AST) is done to determine what type of antibiotic and what concentration will be effective in treating an infection. Current, growth-dependent, AST methods are reliant on the growth rate of the bacteria and can take several days to several weeks to get results. A piezoelectric plate sensor can be used to measure an instant change in the minute physiological stresses of the bacteria cells when they are exposed to an effective concentration of antibiotic. This work aims to investigate the feasibility of piezoelectric plate sensors used for instantaneous AST (iAST) results and develop a technological framework for scaling this technology to a clinical lab setting. Four Clinical and Laboratory Standards Institute (CLSI) quality control strains of bacteria were tested with a wide range of antibiotics from various drug classes using the piezoelectric sensor. Results were obtained within 30 minutes and compared to standard of care AST methods used in clinical labs, and CLSI prescribed ranges for each strain of bacteria. This thesis will also discuss a framework for developing more scalable sensors, and challenges associated with the different sensor designs.

Infectious diseases resulting from bacterial pathogens are the most common causes of patient morbidity and mortality worldwide. The rapid identification of the pathogens and their antibiotic resistances is crucial for proper clinical management. However, the standard culture-based diagnostic approach requires a minimum of two days from the initial specimen collection to result reporting. As a consequence, broad-spectrum antibiotics are often prescribed under the worst-case assumption without knowledge of the pathogens or their resistances. The current clinical practice results in improper treatment of the patient and causes the rapid emergence of multi-drug resistant pathogens. A rapid diagnostics system has therefore been developed which performs hybrid electrokinetic sample preparation and volume reduction, for single-cell antimicrobial susceptibility testing (AST). The system combines multiple electrokinetic forces for sample preparation, which reduces the sample volume for over 3 orders of magnitude and minimizes the matrix effects of physiological samples for enhanced sensitivity. The device is integrated with a single-cell AST system with microfluidic confinement and electrokinetic loading to phenotypically determine the bacterial antibiotic resistance at the single-cell level. The applicability of the system has been demonstrated for performing direct AST with urine and blood samples within one hour, enabling rapid infectious disease diagnostics in non-traditional healthcare settings.

Gonorrhea is caused by the human pathogen Neisseria gonorrhoeae. More than 106 million new cases of N. gonorrhoeae infections occur each year worldwide. There is no vaccine available against gonococcal infections and treatment of gonorrhea with antibiotics is the only way to eradicate infection. The high prevalence of antibiotic resistance (AMR) in this microorganism makes the effective treatment of gonococcal infections increasingly problematic. The emergence of AMR, especially to extended spectrum cephalosporins (i.e. cefixime and ceftriaxone) which are the last possibilities for single dose treatment options for gonococcal infections, is a serious concern. Gonorrhea may become an untreatable infection in the near future. Saskatchewan (SK) has one of the highest rates of gonorrhea in Canada. In order to better characterize the gonorrhea epidemic in SK, the objectives of the present research were to determine the prevalence and trends of AMR and emerging AMR mechanisms in N. gonorrhoeae isolates. AMR mechanisms were ascertained for the first time in SK in order to identify genetic causes of resistance. This was completed by determining and analyzing the DNA sequences of various genes - penA, mtrR, porB ponA, gyrA, parC mtrR, 23S rRNA alleles and erm –implicated in gonococcal AMR. The population dynamics of the N. gonorrhoeae isolates in SK was investigated by DNA based molecular methods to determine strain distribution, evolution of AMR phenotypes, and association between strain types (STs) and AMR genotypes and phenotypes. N. gonorrhoeae isolates (n=427) from Saskatchewan (2003-2011) were susceptible to antibiotics now recommended for treatment - cefixime, ceftriaxone and spectinomycin. Over 95% of the isolates tested were also susceptible to penicillin (96%) and ciprofloxacin (95.5%), antibiotics no longer recommended for treatment, and azithromycin (99.4%). Tetracycline resistance was also high (50.1%). N. gonorrhoeae isolates that were resistant to the antibiotics tested and also those isolates with MICs ≥0.003 mg/L to cefixime and ceftriaxone were analyzed (n=146) to determine their resistance mechanisms. This analysis revealed that reduced susceptibility to ceftriaxone and cefixime and resistance to penicillin is mediated by specific mutations in penicillin binding protein 2 (PBP2), in the promoter and dimerization domains of MtrR and porin protein (PorB). Novel mutations and combinations of mutations were noted. Ciprofloxacin resistant N. gonorrhoeae isolates carried double mutations in GyrA (S91F and D95G/N) and a S87R or S88P substitution in ParC. Isolates resistant to azithromycin had specific mutations in all the four alleles of 23S rRNA as well as in the DNA binding domain of MtrR. Most resistance was chromosomally mediated while plasmid-mediated resistance to penicillin (0.93% of penicillin resistant isolates) and tetracycline (3.3%) was low. DNA based strain typing methods such as porB-DNA sequencing, N. gonorrhoeae multi-antigen sequence typing (NG-MAST) and multilocus sequence typing (MLST) showed that the gonococcal population in SK differs appreciably from both other Canadian provinces and from strains reported internationally. MLST analysis, which ascertains the evolution of isolates over time, demonstrated that penicillin and tetracycline resistant isolates in SK evolved through spontaneous mutations in established lineages. Ciprofloxacin and azithromycin resistant N. gonorrhoeae isolates, on the other hand, were introduced into SK from outside the province. Significant associations between particular mutation pattern combinations in resistance determining genes and specific NG-MAST STs were identified e.g. NG-MAST ST 25 was associated with specific combined mutation patterns in PBP2, MtrR and PorB and antibiotic susceptibility; and, NG-MAST ST 3654 was associated with another PBP2/MtrR/PorB mutation pattern, chromosomal resistance to penicillin and tetracycline and elevated MICs to cefixime. This research shows the importance of regional antimicrobial susceptibility monitoring. In the context of SK, this means that local surveillance of gonococcal AMR may be used to develop policies for regional treatment guidelines which promote the prudent use of antimicrobials for treatment, including those antibiotics which may no longer be used in other regions due to higher AMR rates. Further, the significant association between particular AMR mutation pattern combinations and specific STs indicates that AMR might be predicted. These results should assist in the development of non-culture-based tests for the diagnosis of gonococcal AMR similar to nucleic acid amplification tests used to diagnose N. gonorrhoeae infections.