La compréhension de la fonction des protéines et des systèmes biologiques passe par une connaissance fine des mécanismes moléculaires sous-jacents. La cristallographie et la résonance magnétique nucléaire permettent d'appréhender ces mécanismes au niveau atomique en fournissant des informations sur la structure et sur la dynamique des macromolécules biologiques. Nous nous sommes ainsi intéressés à deux protéines, la lipase lip2 de la levure Yarrowia lipolytica et la protéine membranaire OmpA de la bactérie Klebsiella pneumoniae. Nous avons recherché des conditions d'expression de la protéine lip2 marquée uniformément ou spécifiquement sur une boucle (appelée " lid ") afin d'en étudier la dynamique. Des conditions de marquage uniforme à l'azote 15 de lip2 recombinante dans Yarrowia lipolytica ont été mises au point, mais le marquage acide aminé spécifique n'a pu être réalisé à cause de phénomènes de dilution isotopique trop importants dans cette levure. Nous avons résolu par cristallographie aux rayons X la structure du domaine C-terminal de la protéine OmpA et étudié sa dynamique en solution par RMN (techniques de relaxation 15N). Nous avons caractérisé la dynamique de son domaine N-terminal membranaire reconstitué en liposomes par RMN du solide : en utilisant la rotation à l'angle magique à 60kHz et à la détection 1H sur un spectromètre 1 GHz, nous avons pu attribuer une majorité des résonances du tonneau ? et établir un profil de paramètre d'ordre des vecteurs NH. Des expériences de protéolyse ménagée ont révélé par ailleurs un site de coupure unique à la trypsine au sein de la boucle extracellulaire L3. Enfin, une première caractérisation de la protéine complète exprimée dans la membrane externe d'Escherichia coli a été entreprise par RMN du solide sur membranes externes natives. / Understanding the function of proteins and biological systems requires an accurate knowledge of the underlying molecular mechanisms. Crystallography and nuclear magnetic resonance provide a detailed description of these mechanisms, with an atomic resolution, by providing data on both structures and motions. We investigated two proteins, the lip2 lipase from the yeast Yarrowia lipolytica and the membrane protein OmpA from the bacteria Klebsiella pneumoniae. We tried to produce lip2 with uniform and amino-acid specific stable isotope labelling on its functional loop (the lid) for NMR experiments. The homologous recombinant expression in Yarrowia lipolytica turned out to be the most efficient for uniform labelling but failed for specific labelling due to extensive isotope scrambling. We solved the structure of OmpA C-terminal domain by X-ray crystallography, and analyzed its dynamics in solution by NMR (15N relaxation techniques). We characterized its transmembrane N-terminal domain in proteoliposomes by solid state NMR: using state of the art ultra-fast MAS (60 kHz), 1H detection and a 1 GHz spectrometer, we could assign most ?-barrel resonances and establish a NH order parameter profile. In a complementary approach, we used proteolysis to reveal a unique trypsin cleavage site on the extracellular loop 3. Finally, a first characterization of the full-length protein expressed in the outer membrane of Escherichia coli was initiated by solid state NMR on intact outer membranes.

Background. Carbapenem-resistant Enterobacteriaceae, including Escherichia coli and Klebsiella pneumoniae, are newly emerging pathogens of public health importance. Currently no nationally representative or mandatory surveillance or reporting system exists to examine trends of these important pathogens. Objective. The purpose of the current study was to estimate trends in overall microbial burden and carbapenem resistance in E. coli and K. pneumoniae and to understand the extent to which hospitals which report to voluntary surveillance systems represent all hospitals in the United States. Design. We conducted a descriptive study to compare the hospitals participating in voluntary reporting systems of the University HealthSystem Consortium and the National Healthcare Safety Network with the Healthcare Utilization Project’s Nationwide Inpatient Sample, a nationally representative sample of hospital discharges. Methods. Descriptive analyses examined hospital characteristics (region, bed size, hospital control, teaching status, case mix index) and patient characteristics (age, sex, race/ethnicity, admission source, admission type, discharge status, primary payer) of participant hospitals versus all US hospitals. ICD-9-CM codes identified discharges coded for E. coli and K. pneumoniae diagnoses; linear regression was used to evaluate trends in overall microbial burden of E. coli and K. pneumoniae in all US Hospitals and US Academic Centers. Trends in E. coli and K. pneumoniae resistance to carbapenem were also evaluated in hospitals participating in voluntary surveillance systems (n=13). Results. Between 2002 and 2007, slight increasing trends in burden of both E. coli and K. pneumoniae were observed (E. coli: slope = 0.0537; K. pneumoniae slope = 0.0168). Hospitals participating in voluntary surveillance systems are larger and care for fewer elderly patients than all US hospitals. Conclusions. These results suggest that hospitals that participate in voluntary surveillance systems like the National Healthcare Safety Network and the University HealthSystem Consortium may underrepresent trends in smaller hospitals, as well as those that treat elderly patients. Increasing overall burden of infection due to these isolates only reinforces the importance carbapenem resistance in E. coli and K. pneumoniae. This important public health threat may warrant the creation of a national, mandatory reporting system for these and other antimicrobial resistant organisms.

Thesis (MSc (Biomedical Technology))--Cape Peninsula University of Technology, 2017. / Background: It is well established that Klebsiella pneumoniae (K. pneumoniae) is an opportunistic pathogenic organism that has been frequently identified as the cause of nosocomial and community acquired infections. Furthermore, studies have shown that over the last few decades strains of the genus Klebsiella have systematically developed resistance to numerous antibiotics. Aims and Methods: The primary aim of this study was to investigate the prevalence of K. pneumoniae in nosocomial and community isolates in the Western Cape province of South Africa. Various identification techniques such as the polymerase chain reaction (PCR) using the API 20 E, the VITEK®2 system, primers specific for the 16S-23S rDNA ITS region and the Matrix-assisted laser desorption/ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) were compared for the identification of this pathogen. The VITEK 2 system was used to detect antibiotic resistant profiles of the K. pneumoniae isolates and to identify the extended spectrum beta-lactamase (ESBL) phenotypic among these isolates. The PCR was used to detect Beta-lactam genes viz. CTX-M (blaCTX-M), TEM (blaTEM) and SHV (blaSHV) respectively in both the genome and plasmid DNA of K. pneumoniae using gene specific primers. Results: In total 57 agar plate bacterial cultures or glycerol stock bacterial cultures were obtained during 2011. Of the 57 isolates, the API 20 E test identified 47 (82.5%) of the isolates (n = 57) as K. pneumoniae while 10 isolates (17.5%) were identified as Raoultella species. The VITEK 2 method and PCR identified all 57 isolates as K. pneumoniae (100%). Of the isolates, 82.5% (47/57) were positively identified as Klebsiella species, 14% (8/57) were identified as Klebsiella variicola and 3.5% (2/57) were shown as no reliable identification (NRI) when using the MALDI-TOF MS. Examination of the 57 isolates using primers specific for the CTX-M (blaCTX-M), TEM (blaTEM) and SHV (blaSHV) respectively showed the following: PCR amplicons for the TEM gene were produced successfully for 46 (81%) of the 57 isolates included in this project, while 11 (19%) of the samples did not yield any TEM amplicons; PCR amplicons for the blaSHV gene were obtained successfully for 56 (98%) of the 57 DNA samples, while 1 sample (2%) did not yield any SHV amplicons; and PCR amplicons for the blaCTX-M gene were produced successfully by 89% (n = 51) of the DNA samples included in this project, while 11% (n = 6) did not yield any CTX-M amplicon. Extended-spectrum beta-lactamase phenotypes had been confirmed in 84% (n = 48) K. pneumoniae isolates while nine isolates were found to be non-ESBL. Resistance rates for these 48 isolates were high and showed resistance patterns of: Amoxicillin/Ampicillin, Amoxycillin/Clavulanate, Ceftriaxone/Cefotaxime, Cefuroxime/Cefprozil and Ceftazidime (100%, n = 48); Piperacillin/Tazobactam and Cefoxitin (98%, 47/48); Cefepime (96%, 46/48); Aztreonam (94%; 45/48); Tobramycin (81%, 39/48); Gentamycin and Ciprofloxacin (77%, 37/48); Trimethoprim/Sulfamethoxazole (67%, 32/48); and Tigecycline (25% 12/48). Conclusion: For the analysis by all four methods employed, a total agreement of 68.4% was obtained, indicating the positive identification of K. pneumoniae in 39 of the 57 samples analysed. An average agreement of 28.1% was then obtained for the comparison of results generated for three of the methods utilised, while a 3.5% average agreement was obtained for at least two methods. Furthermore, all four methods agreed that 82.5% of the isolates were Klebsiella species while three methods agreed that 17.5% of the isolates were Klebsiella species. Based on the results obtained in the current study, PCR and VITEK 2 were the methods of choice for the identification of K. pneumoniae. The current study also showed, that ESBL-K. pneumoniae strains are present in the Western Cape province, South Africa; with high resistance profiles to numerous antibiotics including the Cephalosporins.

This work will look at two factors that add to the diversity of carbapenem resistant bacteria. First, it focuses on the diversity of carbapenemase resistance plasmids. 446 plasmids were characterized by size, gene content and replicon groups. We identified that on average, over 30% of the encoded proteins on each plasmid have an unknown function. Plasmid sizes ranged from 1.6kb to 500kb, with an average of around 100kb and median of 80kb. Additionally, six replicon groups account for 80% of all the carbapenemase resistance plasmids. We also highlight the lack of data available for carbapenemase carrying plasmids from bacterial genera other than Escherichia and Klebsiella, and plasmids that carry the New Delhi metallo-β- lactamase or the Verona-integron encoded metallo-β-lactamase. Second, we characterized the β-lactamase diversity of a single carbapenemase resistant Klebsiella pneumoniae. This isolate encodes six distinct β-lactamases, all of which are functional, and three of which are redundant. Additionally, we determined that the CTX-M-15 cephalosporinase imparts a greater fitness when grown in aztreonam (a monobactam) than ceftazidime (a cephalosporin). Finally, we show that individually, these β-lactamases do not account for the elevated levels of resistance seen in the parent strain, indicating that the passive resistance mechanisms (i.e. efflux pumps, altered membrane porins) may play a larger role than originally thought.

Submitted by Alessandra Portugal (alessandradf@ioc.fiocruz.br) on 2013-09-24T14:50:16Z No. of bitstreams: 1 Thiago Pavoni.pdf: 1955163 bytes, checksum: b15140ec10d9f1473101f075a2a57a86 (MD5) / Made available in DSpace on 2013-09-24T14:50:16Z (GMT). No. of bitstreams: 1 Thiago Pavoni.pdf: 1955163 bytes, checksum: b15140ec10d9f1473101f075a2a57a86 (MD5) Previous issue date: 2011 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / Drogas antimicrobianas e bactérias resistentes aos antimicrobianos estão disseminadas em grandes quantidades no ambiente, como resultado do aumento e freqüente uso indiscriminado dos antibióticos. Bactérias e seus genes de resistência têm sido detectados em diferentes ambientes, tais como esgoto hospitalar, esgoto doméstico e águas de rios contaminados. O esgoto hospitalar é um importante poluente, representando riscos para a saúde pública se chegar aos sistemas de distribuição. Ambientes fortemente seletivos, como os hospitais, permitem a geração bactérias resistentes, as quais podem ser lançadas no esgoto hospitalar. O presente trabalho tem como objetivo investigar a presença bactérias resistentes aos antimicrobianos em efluentes de uma estação de tratamento de esgoto hospitalar no Rio de Janeiro, avaliando o potencial do sistema de tratamento para a eliminação de micro-organismos. A estação de tratamento de esgoto fica localizada na região metropolitana. O sistema de lodo ativado por aeração prolongada é constituído por três partes básicas: o tanque de aeração, o decantador e o tanque de cloração. Vinte e quatro amostras de esgoto foram coletadas no período de Julho a Dezembro de 2008. Oito amostras (1000 mL) foram coletadas a partir de diferentes pontos: afluente, efluente do tanque decantador e efluente clorado. Micro-organismos indicadores também foram investigados. Os isolados bacterianos foram identificados a partir de provas bioquímicas convencionais. A sensibilidade aos antimicrobianos das bactérias isoladas foi determinada através do método fenotípico de difusão em ágar, de acordo com as orientações do Clinical and Laboratory Standards Institute (CLSI). A identificação da produção fenotípica de beta-lactamases de espectro estendido e de carbapenemases entre os isolados também seguiram as recomendações do CLSI. Ensaios de PCR foram processados para a identificação dos genes blaKPC, blaTEM, blaSHV e blaCTX-M. A genotipagem das amostras bacterianas foi realizada por eletroforese em gel de campo pulsado. Concentrações significativas de coliformes totais e fecais foram detectadas nos efluentes hospitalares. Um total de 226 isolados foi identificado, entre os quais 213 (94%) pertenciam à família Enterobacteriaceae. Outros grupos de micro-organismos, como Pseudomonas aeruginosa, Acinetobacter baumannii e Aeromonas spp., foram também observados. A maioria das cepas era sensível ao imipenem e ao meropenem; e resistente à cefalotina, à cefotaxima e ao sulfametoxazol-trimetoprim. O fenótipo de ESBL foi caracterizado em 97 (43%) isolados. Os produtores de ESBL mais comuns foram: Klebsiella pneumoniae, Enterobacter cloacae e Escherichia coli. Micro-organismos patogênicos e altas taxas de resistência ainda puderam ser observados nos efluentes clorados. Os genes blaTEM, blaSHV e blaCTX-M foram detectados em 82%, 48% e 67% dos isolados do efluente hospitalar, respectivamente. Em muitos isolados, a ocorrência de mais de um tipo de ESBL foi observada, sendo a associação dos tipos TEM e CTX-M a mais frequente. O gene blaKPC foi detectado em dois isolados do efluente. Foi possível observar isolados clínicos e do esgoto geneticamente relacionados. Concluímos que, apesar do tratamento, o esgoto hospitalar pode ser considerado um veículo ambiental de disseminação de bactérias multirresistentes. A ocorrência destes micro-organismos nos efluentes é preocupante e tem impacto sobre a saúde pública. Medidas urgentes são necessárias para enfrentar este problema. Vale ressaltar que, em muitos países em desenvolvimento, os efluentes hospitalares não recebem tratamento adequado. / Antimicrobial drugs and antimicrobial - resistant bacteria are discharged in large quantities in the environment as a result of increasing ly frequent and indiscriminate use of antibiotics . Antimicrobial - resistant bacteri a and antimicrobial - resistant genes have been detected in different environments, such as domestic sewage, hospital sewage and sewage - contaminated river waters. Hospital sewage is an important pollutant , representing risks to public health if it reaches th e distribution system. The occurrence of strongly selective environments, such as hospitals, leads to an incre ase of multiresistant bacteria, which can be released in hospital sewage. The aim of this study was to investigate the antimicrobial - resistant bac teria isolated from a hospital sewage treatment plant in Rio de Janeiro city, evaluating the treatment plant’s potential to remove these microorganisms. The sewage treatment plant serve a hospital located in the metropolitan area of the Rio de Janeiro city (RJ), Brazil. The extended aeration activated sludge plant is divided into three parts, an aeration tank, a clarifier tank and a chlorine contact tank. During the study, twenty - four sewage samples were collected in the period from July to December 2008. E ight samples (1000 ml) were collected on each day from the following: influent; clarifier tank effluent; and chlorine contact tank effluent. Total and faecal coliforms concentrations were also determined . Isolates were identified using established biochemi cal procedures. The antimicrobial susceptibilities of bacterial isolates were determined using the agar diffusion method according to Clinical and Laboratory Standards Institute (CLSI) guidelines. Isolates were screened for the KPC - and ESBL - producing phen otype according to the CLSI. PCR experiments were used for the molecular detection of bla KPC , bla TEM , bla SHV and bla CTX - M genes. The genetic relat ionships of isolates were determined by PFGE. High concentrations of total and faecal coliforms were detected in the influent , clarifier tank and chlorine contact tank effluent. A total of 226 isolates were identified, among which 213 (94%) were Enterobacteriaceae . In addition , Pseudomonas aeruginosa , Acinetobacter baumannii and Aeromonas spp . in hospital effluent were observed . The majority of the strai ns were susceptible to imipenem and meropenem and resistant to cefalothin, cefotaxime and trimethoprim - sulphametoxazole. ESBL phenotype was characterized in 97 (43%) isolates. The most common ESBL - producing isolates were: Klebsiella pneumoniae , Enterobacter cloacae , and Escherichia coli . Pathogenic microorganisms and higher antimicrobial resistance rates were detected in chlorine contact tank effluent. The bla TEM , bla SHV and bla CTX - M genes were detected in 82%, 48% a nd 67% of isolates respectively. Many of the isolates harboured other β - lactam resistance enzymes and the a ssociation of types TEM and CTX - M was more frequent. The bla KPC was detected in isolates from effluents. PFGE analysis revealed clonal types among cl inical isolates and isolates from effluents. Despite the treatment of the wastewater, hospital effluent may be considered as a potential environmental vehicle of multiresi s tant microorganisms. The occurrence of multiresistant bacteria isolates in hospital effluents is worrisome and has a real impact on public health. Urgent measures are necessary in order to counteract this problem. It should be noted that effluents from hospitals in developing countries do not receive adequate treatment

The treatment options for multidrug resistant bacteria are dwindling and it is an important issue of research in medicine to solve. One of the more problematic bacterial species is Klebsiella pneumoniae, it can cause infections with high morbidity that are difficult to treat. Common antibiotics for treatment of these infections are carbapenems but K. pneumoniae can produce enzymes called carbapenemases that can hydrolyze carbapenems and most other beta-lactam antibiotics. In this study carbapenemase genes were introduced chromosomally to a previously susceptible K. pneumoniae strain using λ-Red recombineering. Further constructs were made with non-functional porins to examine how they affect combination treatment with carbapenems. Antibiotic combination therapy was evaluated against constructed carbapenemase- (KPC-2, NDM-1 and OXA-48) producing K. pneumoniae strains. Screening was done using time-lapse microscopy (oCelloScope), and combinations with better effect than treatment with a single antibiotic were chosen for time-kill assays. The results shows that a triple combination of colistin, meropenem and the beta-lactamase inhibitor avibactam gives an improved effect, up to twice the effect compared to monotherapy and up to 1.8 times increased effect compared to double combination. The synergistic effect was greater when adding colistin to treat the strains with non-functional porins, indicating that colistin can increase the permeability for other antibiotics into the cell. This is an interesting finding that need to be researched further.

Klebsiella pneumoniae is an opportunistic pathogen responsible for lobar pneumoniae, liver abscess, and septicemia. Clinical isolates are found to be extended spectrum beta lactamase positive with differential expression of the two classical porins, OmpK35 and OmpK36. Porin loss is associated with increased minimum inhibitory concentrations of beta lactam, cephalosporin, and carbapenem antibiotics that target the peptidoglycan. However, little is known about how porin loss affects other aspects of the cell envelope. The focus of this study was to characterize clinical isolates exhibiting differential porin expression and determine if the cumulative changes altered the resistance to phagocytosis by macrophages. The results support the hypothesis that porin loss significantly impacts the overall cell envelope composition, which in turn alters interactions with macrophages.

Submitted by Cássio da Cruz Nogueira (cassionogueirakk@gmail.com) on 2017-09-04T12:06:25Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_CaracterizacaoMolecularKlebsiella.pdf: 3383189 bytes, checksum: 1b8dc4391b448cb697a3dd6a7612a585 (MD5) / Approved for entry into archive by Irvana Coutinho (irvana@ufpa.br) on 2017-09-04T17:57:15Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_CaracterizacaoMolecularKlebsiella.pdf: 3383189 bytes, checksum: 1b8dc4391b448cb697a3dd6a7612a585 (MD5) / Made available in DSpace on 2017-09-04T17:57:15Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_CaracterizacaoMolecularKlebsiella.pdf: 3383189 bytes, checksum: 1b8dc4391b448cb697a3dd6a7612a585 (MD5) Previous issue date: 2016-12-13 / A resistência aos antimicrobianos em bactérias da família Enterobacteriaceae está aumentando de forma alarmante no mundo todo. As K. pneumoniae constituem um importante grupo de patógenos humanos, causadores de infecções hospitalares e comunitárias. Nestas bactérias, a produção de beta-lactamases é um dos principais mecanismos de resistência aos antimicrobianos e responsável pela falha da terapia antimicrobiana. Este trabalho teve como objetivo realizar a caracterização molecular de espécimes de K. pneumoniae produtoras de ESBL e KPC quanto a resistência aos antimicrobianos em pacientes hospitalizados em Belém-PA. Foram analisadas 124 espécimes de K. pneumoniae oriundas de um hospital público de Belém-Pará. Foram realizadas nesses espécimes testes de suscetibilidade a antimicrobianos, testes fenotípicos para detecção de betalactamases de espectro ampliado (ESBL) e K. pneumoniae produtora de carbapenemase (KPC). Posteriormente foram realizadas reações de cadeia de polimerase (PCR) e o sequenciamento de DNA para identificar os genes determinantes de resistência aos antimicrobianos. Foi observado que 83% dos isolados apresentaram o gene bla CTX-M, 85,5% o bla SHV, 83% o bla TEM e 5% o gene bla KPC. Quanto aos genes que codificam ESBL o gene bla CTX-M-71 foi isolado com maior freqüência e foi identificado em 60% dos isolados analisados. Os outros genes que codificam ESBL foram bla SHV-38 (5%), bla SHV-100 (5%) and bla SHV-12 (3,5%). O gene bla KPC-2 foi detectado em 100% dos isolados. Estas enterobactérias apresentaram fenótipos de multidroga resistência com elevados níveis para os quinolonas e aminoglicosideos. Foram observadas associações entre os genótipos e à resistência aos antibióticos. A presença de micro-organismos multirresistentes em unidades hospitalares reforça a necessidade de medidas para a rápida contenção de possíveis infecções causadas por esses patógenos. / The antimicrobial resistance in Enterobacteriaceae is increasing worldwide. The K. pneumoniae constitute an important group of human patogen, causing of hospital and communitarian infections. In these bacteria, the production of extended spectrum beta-lactamases (ESBL) is one of the main mechanisms of resistance the antimicrobials, responsible for the imperfection of the therapy against infections for gram-negative bacilli. This work aimed to do the molecular characterization of the K. pneumoniae producing ESBL and KPC about antimicrobial resistence in pacients from Belém-PA. A total of 124 K. pneumoniae isolates were collected from public hospital from Belém-PA and susceptibility test was performed to detect its susceptibility patterns antibiotics. Phenotypic tests for extended-spectrum beta-lactamases (ESBLs) and carbapenemase-producing Klebsiella pneumoniae (KPC) producing strains were performed to detect the resistance phenotype of the isolates. Then PCR amplification and sequencing analysis were performed for the drug resistance determinants genes. The results showed that 83% strains harbored bla CTX-M gene, 85,5% carried bla SHV , 83% carried bla TEM and 5% carried bla KPC. The most frequent gene ESBL detected was bla CTX-M-71, which was observed in 60% of isolates. Other ESBL genes were bla SHV-38 (5% of isolates), bla SHV-100 (5% of isolates) and bla SHV-12 (3,5% of isolates). O gene bla KPC-2 was detected in 100% of isolates.These enterobacterias showed multidrug resistance phenotypes with high levels for quinolones and aminoglycosides. Associations between genotypes and antibiotic resistance were observed.The presence of multidrug resistant micro-organisms in hospitals, reinforces the need for measures for rapid containment of possibles infections caused by these pathogens.

Extended spectrum β-lactamases or ESBLs, which are derived from non-ESBL precursors by point mutation of β-lactamase genes (bla), are spreading rapidly all over the world and have caused considerable problems in the treatment of infections caused by bacteria which harbour them. The mechanism of this resistance is not fully understood and a better understanding of these mechanisms might significantly impact on choosing proper diagnostic and treatment strategies. Previous work on SHV β-lactamase gene, blaSHV, has shown that only Klebsiella pneumoniae strains which contain plasmid-borne blaSHV are able to mutate to phenotypically ESBL-positive strains and there was also evidence of an increase in blaSHV copy number. Therefore, it was hypothesised that although specific point mutation is essential for acquisition of ESBL activity, it is not yet enough, and blaSHV copy number amplification is also essential for an ESBL-positive phenotype, with homologous recombination being the likely mechanism of blaSHV copy number expansion. In this study, we investigated the mutation rate of non-ESBL expressing K. pneumoniae isolates to an ESBL-positive status by using the MSS-maximum likelihood method. Our data showed that blaSHV mutation rate of a non-ESBL expressing isolate is lower than the mutation rate of the other single base changes on the chromosome, even with a plasmid-borne blaSHV gene. On the other hand, mutation rate from a low MIC ESBL-positive (≤ 8 µg/mL for cefotaxime) to high MIC ESBL-positive (≥16 µg/mL for cefotaxime) is very high. This is because only gene copy number increase is needed which is probably mediated by homologous recombination that typically takes place at a much higher frequencies than point mutations. Using a subinhibitory concentration of novobiocin, as a homologous recombination inhibitor, revealed that this is the case.

Submitted by Marilene Donadel (marilene.donadel@unioeste.br) on 2018-10-04T18:06:39Z No. of bitstreams: 1 Mabel_Arantes_2018.pdf: 2059864 bytes, checksum: 831822ad4d3c7ce18c2fda6d1319af80 (MD5) / Made available in DSpace on 2018-10-04T18:06:39Z (GMT). No. of bitstreams: 1 Mabel_Arantes_2018.pdf: 2059864 bytes, checksum: 831822ad4d3c7ce18c2fda6d1319af80 (MD5) Previous issue date: 2018-09-06 / The biological production of hydrogen (bioH2) through anaerobic fermentation of residual carbon sources is an interesting way to combine the environmental adequation of agro-industrial wastewaters to the generation of value-added products. Wastewaters with fermentable carbon sources have high potential to produce bioH2, being possible using different inoculum and biological reactors. It is the case found in brewery wastewater, generated in large amount at Brazil and around the world, and there are only few studies about it. In this context, this work studied the production of bioH2 by an anaerobic reactor operated in sequencing batch with immobilized biomass, AnSBBR, from brewery wastewater, using two inocula: mixed culture from the natural fermentation of synthetic wastewater based on sucrose and pure culture of Klebsiella pneumoniae, immobilized in expanded clay. Were evaluated the effects of influent concentration (2,8 a 7,2 gTRS L-1), volumetric organic load (2,6 a 12,6 gTRS (L day)-1) and cycle time (12 h e 24 h), in assays taking from 9 to 16 cycles. The inocula gotten through natural fermentation showed potential to produce bioH2 in preliminary experiments (shaken bottles, 0,5 L) but, in the tests on the reactor the production was low, instable and brief (less than 7 days). The pure inocula K. pneumoniae showed a good adaptability to the residue, in activity through the whole experiment. The raise in the volumetric organic load (VOLc) up to 12,6 gTRS (L day)-1 and the reduction of the cycle from 24 h to 12 h had a positive effect in the production and, under those conditions, the best values for the evaluation parameters ahead were obtained: volumetric productivity average of 0,9 LH2 (L day)-1, molar flow rate up to 10,8 mmolH2 h-1, molar yield average de 3,9 molH2 KgTRS-1, with H2 content in biogas between 18 and 42% and TRS and COD removal efficiency of 89% and 23%, respectively, with a predominance of acetic and butyric pathways. Those results indicate a promising production of H2 from brewery wastewater using K. pneumoniae as inocula in AnSBBR reactor, contributing to the development of the bioH2 technology by the proposal of a new inocula to achieve a higher use of this wastewater potential. / A produção biológica de hidrogênio (bioH2) por meio de fermentação anaeróbia de fontes de carbono residuais é uma forma interessante de aliar a adequação ambiental de efluentes agroindustriais à geração de produto de valor agregado. Águas residuárias que possuam fontes de carbono fermentescíveis apresentam alto potencial para a produção de bioH2, o que é possível com diferentes inóculos e reatores biológicos. É o caso da água residuária de cervejaria, gerada em grandes quantidades no Brasil e no mundo e para a qual poucos estudos foram realizados até o momento. Neste contexto, o presente trabalho avaliou a produção de biohidrogênio em reator anaeróbio operado em bateladas sequenciais com biomassa imobilizada, AnSBBR, a partir de água residuária de cervejaria, empregando dois inóculos: cultura mista proveniente da fermentação natural de meio sintético à base de sacarose e cultura pura de Klebsiella pneumoniae, suportados em argila expandida. Foram avaliados os efeitos da concentração afluente (2,8 a 7,2 gART L-1), da carga orgânica volumétrica aplicada (2,6 a 12,6 gART (L dia)-1) e do tempo de ciclo (12 h e 24 h) em ensaios com duração entre 9 e 16 ciclos. O inóculo obtido por fermentação natural apresentou potencial para produção de bioH2 em ensaios preliminares (frascos agitados, 0,5 L) mas na avaliação em reator a produção foi baixa, instável e breve (período inferior a 7 dias). Já o inóculo puro, K. pneumoniae apresentou boa adaptação ao resíduo e atividade durante todo o período avaliado. O aumento da carga orgânica volumétrica aplicada (COVc) até 12,6 gART (L dia)-1 e a redução do tempo ciclo de 24 h para 12 h tiveram um efeito positivo sobre a produção e nestas condições foram obtidos os melhores valores para os parâmetros de avaliação: produtividade volumétrica de H2 média de 0,9 LH2 (L dia)-1, vazão molar de H2 máxima de 10,8 mmolH2 h-1, rendimento de H2 médio de 3,9 molH2 KgART-1, com teor de H2 no biogás entre 18 e 42% e eficiência média de remoção de ART e de DQO de 89% e 23%, respectivamente, com predomínio das rotas metabólicas acética e butírica. Estes resultados indicam uma produção promissora de H2 a partir de água residuária de cervejaria empregando K. pneumoniae como inóculo em reator AnSBBR, contribuindo para o desenvolvimento da tecnologia do bioH2 ao propor um novo inóculo para o aproveitamento do potencial de uso desta água residuária.