O glicerol é gerado em grandes quantidades durante a produção de biodiesel e tem se tornado um substrato potencialmente atrativo para a produção microbiana de produtos de valor agregado como 1,3-propanodiol (1,3-PD). O presente trabalho teve como objetivo estudar a produção de 1,3-PD por células imobilizadas de K. pneumoniae BLh-1, utilizando glicerol residual como fonte de carbono. Primeiramente, foi estudado o efeito da concentração celular e do diâmetro das esferas no sistema de imobilização. Os resultados mostraram que a melhor combinação foi 100 mg de biomassa por mL de suporte e o diâmetro de 3,40 mm, pois apresentaram a maior produtividade. Em experimentos realizados para comparar a produção de 1,3-PD com células imobilizadas e em suspensão, a produção de 1,3-PD foi similar nos dois experimentos. Mas o uso de células imobilizadas levou à obtenção de maior produtividade de 1,3-PD, devido à alta densidade celular utilizada, uma das vantagens da utilização desta técnica. Foram realizados experimentos de reutilização das células imobilizadas, para avaliar a estabilidade de produção de 1,3-PD durante cinco bateladas, nos experimentos com intervalo de tempo entre as bateladas à produção de 1,3-PD foi mais estáveis em relação ao uso das esferas sem intervalo entre as bateladas e no final da quinta batelada as esferas de alginato de cálcio estavam com sua estrutura danificadas. Para os experimentos em biorreatores melhorias na imobilização foram realizadas, para aumentar a durabilidade e a resitência das esferas. Nos experimentos em biorreatores a produção de 1,3-PD foi aproximadamente de 25 g L-1 em batelada, os biorreatores contínuos atingiram o regime estacionário, após 40 h de cultivo. Este resultado foi influenciado pela queda do pH do meio de cultivo, que dificulta a produção de 1,3-PD. O controle de pH, é essencial na produção de 1,3-PD por K. pneumoniae BLh1, mas este desestabiliza a estrutura das esferas de alginato de cálcio. Para trabalhos futuros haverá a necessidade de buscar novas alternativas de suportes de imobilização, para que se possa controlar o pH dos experimentos, pois a imobilização celular mostrou ser uma alternativa biotecnológica viável na produção de 1,3-PD. / Glycerol is generated in large amounts during the production of biodiesel and it is becoming a potentially attractive susbstrate for microbial production of value-added products such as 1,3-propanediol (1,3-PD). This research aimed at studying the production of 1,3-PD using immobilized cells of K. pneumoniae BLh-1 on residual glycerol as the carbon source. First, we studied the effect of cell concentration and the diameter of the beads in the immobilization system. The results showed that the best combination is 100 mg biomass by mL of support and diameter of 3.40 mm, as presented in increased productivity. In the experiments to compare the production of 1,3-PD with immobilized cells and suspension cells the production of 1,3-PD was similar in both experiments. But the use of immobilized cells led to obtaining greater productivity 1,3-PD, due to the high cell density used, one of the advantages of using this technique. Experiments were conducted reuse of immobilized cells, to assess the stability of production of 1,3-PD for five batches, in the experiments with interval between batches to the production of 1,3-PD was more stable in relation the use of beads without interval between batches and at the end of the fifth batch of the calcium alginate beads were with its damaged structure. In experiments in bioreactors the production of 1,3-PD was approximately of 25 g L-1 in batch, continuous bioreactors reached steady state after 40 h of cultivation. The control of pH is essential in the production of 1,3-PD K. pneumoniae BLh-1, but this destabilizes the structure of the spheres of calcium alginate. Future studies will be necessary to seek new alternatives supports of the immobilization, for can control the pH of the experiments, because the immobilization cellular proved to be a viable alternative biotechnological production of 1,3-PD.

Made available in DSpace on 2018-08-23T21:50:43Z (GMT). No. of bitstreams: 1 tese_12423_Dissertação Ingrid VERSÃO FINAL.pdf: 2910032 bytes, checksum: f74a79e0492f8dbd74374a927a556dd4 (MD5) Previous issue date: 2018-05-24 / Infecções causadas por Klebsiella pneumoniae produtora de carbapenemase KPC (Kp-KPC) constituem uma grande ameaça para a prática clínica, visto que são associadas a elevadas taxas de mortalidade e possuem escassas opções terapêuticas disponíveis. Atualmente estão sendo detectadas cepas dessa bactéria com resistência a todos os agentes antimicrobianos conhecidos, corroborando a urgente necessidade por novos tratamentos eficazes. O presente estudo teve como objetivo avaliar os efeitos de fendiona e seus derivados, Cu-fendiona e Ag-fendiona, sozinhos e combinados aos carbapenêmicos meropenem (MPM) e imipenem (IMP) em diferentes amostras de Kp-KPC, in vitro e em modelo de Galleria mellonella. Para tal foram investigados: (i) os valores de concentração inibitória mínima (CIM) e de concentração bactericida mínima (CBM) dos compostos; (ii) o efeito da combinação dos compostos com MPM e IMP por meio de checkerboard e curva tempo-morte; e (iii) o efeito de combinações sinérgicas em modelo in vivo de G. mellonella. Os resultados obtidos pela determinação da CIM e da CBM demonstraram boa atividade antimicrobiana pelos três compostos contra todas as 46 amostras. Os valores médios da CIM de fendiona, Cu-fendiona e Ag-fendiona foram 42,06, 9,88, e 10,10 μg/ml, respectivamente. Por meio do checkerboard foram testadas combinações dos compostos com MPM e IMP sobre 9 amostras clonalmente não-relacionadas, e não foi observada a presença de efeitos indiferentes ou antagônicos em qualquer uma das seis combinações testadas. Maiores taxas de sinergismo foram observadas nas três combinações contendo MPM, sendo esse efeito detectado em 66,67%, 77,78%, e 33,33% das amostras quando em associação a fendiona, Cu-fendiona e Ag-fendiona, respectivamente. Pelo método de curva tempo-morte foram testadas as combinações de MPM com Cu-fendiona e Ag-fendiona sobre duas amostras clonalmente não-relacionadas. Foi demonstrado que as combinações contendo concentrações de ½ xCIM de cada agente produziram efeito sinérgico, sendo verificado entre nove e 12 horas de teste. Observou-se que a combinação de MPM com o composto Ag-fendiona foi capaz de erradicar o inóculo (106 UFC/mL) aplicado no teste. Os níveis de toxicidade aguda de fendiona e seus derivados foram avaliados em modelo de G. mellonella, sendo os resultados considerados satisfatórios no que se refere à utilização desses compostos sozinhos ou combinados. A eficácia das combinações foi também avaliada in vivo em modelo de infecção de G. mellonella sobre as mesmas duas amostras empregadas na curva tempo-morte. Foi verificada uma superioridade estaticamente significativa do tratamento com as combinações em relação à administração dos agentes sozinhos. Assim, os resultados obtidos no nosso estudo demonstram o potencial de fendiona, Cu-fendiona e Ag-fendiona como candidatos a fármacos sozinhos ou combinados a antimicrobianos carbapenêmicos.

Drug-resistant bacteria, especially those belonging to the Enterobacteriaceae family, have become increasingly problematic in the nosocomial setting. However, a solution may be to exploit bacteria’s ability to produce inhibitory proteins, like bacteriocins, to suppress competitors and synergistically pair these proteins with antibiotics. Our lab has discovered a potentially novel plasmid-mediated antimicrobial protein produced by as specific strain of Klebsiella pneumoniae. To verify the genetic elements of this plasmid necessary to produce the antimicrobial, a gene interruption plasmid library was generated by transposon mutagenesis using the EZ-TN5TM system. These transposon plasmids were then electroporated into competent E. coli. The resulting E. coli were then plated and screened on agar containing kanamycin to ensure successful plasmid uptake and were now able to secrete the antimicrobial protein. The transposon’s unique sequence allowed primer- binding sites, which were used to sequence the plasmid. Four different sequences were analyzed by NCBI BLAST comparisons and matched with high similarity to: 1) a predicted colicin; 2) an uncharacterized Klebsiella protein, 3) a TraM recognition domain containing protein. The K. pneumoniae antimicrobial protein has been shown, when spotted on lawns of Citrobacter freundii, Enterobacter cloacae and Enterobacter aerogenes to inhibit their growth. It has been additionally shown to inhibit the growth of closely related strains including Klebsiella pnuemoniae strain 9997 when spotted on a lawn. When the protein was synergistically paired with subinhibitory levels of common antibiotics, there was an increase in the effectiveness of the antibiotic, it was paired with. The optical density, MTT, and CFUs demonstrate that when the K. pneumoniae protein is paired with Streptomycin or Kanamycin, growth is inhibited greater than the antibiotic alone. These results demonstrate the importance of studying polymicrobial interactions as a means to combat drug resistance and discover novel antimicrobial derived proteins for new therapeutics.

Made available in DSpace on 2015-11-11T12:04:10Z (GMT). No. of bitstreams: 2 22.pdf: 3102930 bytes, checksum: 2f67ffbd3b5474aacabccf998ea6f22e (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2014 / Fundação Oswaldo Cruz. Centro de Pesquisas Aggeu Magalhães. Recife, PE, Brasil / Este estudo visou a caracterização molecular de mecanismos de virulência e resistência aos antimicrobianos em isolados de K. pneumoniae MDR provenientes de um hospital universitário em Recife-PE. Seis isolados de K. pneumoniae produtores de carbapenemase foram obtidos de pacientes hospitalizados na UTI de um hospital universitário do Recife. Os isolados apresentaram resistência a todos os antimicrobianos beta-lactâmicos e quinolonas testados, mas sensibilidade a amicacina, polimixina B e tigeciclina, por meio de microdiluição em caldo. A tipagem molecular por PFGE revelou que os isolados são intimamente relacionados, apresentando três subclones distintos. Dois STs foram detectados, o ST340 e o ST11, ambos pertencentes ao CC258. Os genes blaKPC-2 e blaSHV-11 foram detectados em todos os isolados, seguido do gene blaCTX-M-15 em quatro dos seis isolados e por fim os genes blaCTX-M-2, qnrB19, aac(6')-31 em dois dos seis isolados. Os genes blaKPC-2 e blaCTX-M-15 estavam presentes em um mesmo plasmídeo de aproximadamente 133 Kb pertencente ao IncI-gama em quatro isolados. Nos demais isolados os genes blaKPC-2 e blaCTX-M-2 eram carreados também por um plasmídeo de, aproximadamente, 133 Kb, entretanto, não foi possível tipar o mesmo com as metodologia utilizada. O gene qnrB19 foi detectado sendo carreado por um plasmídeo de 15 Kb pertencente ao IncY. Todos os isolados apresentaram integron de classe 1, associado com a resistência aos aminoglicosídeos / Mutações na região QRDR de GyrA (Ser83Ile) e ParC (Ser80Ile) foram detectadas em todos os isolados analisados, sendo esse o principal mecanismo de resistência as quinolonas detectadas ao longo do estudo. Adicionalmente, a permeabilidade de membrana externa foi analisada, verificando-se a ausência da Ompk35 em todos os isolados e da OmpK36 em uma das amostras analisadas. A investigação dos genes de virulência revelou a presença de antígenos capsulares do tipo K2 entre os isolados. Genes codificadores das fímbrias do tipo I e III foram detectados, assim como genes envolvidos na síntese de LPS e operon da urease. A presenca de micro-organismos multirresistentes e virulentos em unidades hospitalares reforça a necessidade de medidas para a rápida contenção de possíveis infecções hospitalares causadas por esses patógenos

Klebsiella pneumoniae is one of the top eight pathogens in hospitals, causing around 10% of hospital-acquired infections (nosocomial infections). It often produces extended-spectrum β-lactamase enzymes (ESBLs). This has led to numerous outbreaks, especially in intensive care, neonatal and surgical wards, associated with increases in morbidity and mortality. In order to reduce the number of infections caused by multi-resistant K. pneumoniae and improve standards of infection control within hospitals, there is extensive use of biocides as disinfectants and antiseptics. However this raises concerns that intensive exposure of hospital pathogens to biocides may result in the emergence of resistance not just to themselves but also to antibiotics. The reduced susceptibility to biocides and their relationship with resistance to antibiotics was assessed in this thesis. The susceptibility of 64 isolates of K. pneumoniae to five biocides preparations, Chlorhexidine (CHX), Benzalkonium chloride (BZK), Trigene, MediHex-4 (MH-4), Mediscrub (MS) and 17 antibiotics, were tested. The isolates of K. pneumoniae were collected from Royal Infirmary Hospital in Edinburgh (RIE) between 2006 and 2008 from different sites of infection. Antimicrobial susceptibility was tested by the agar double dilution method (DDM) and disc diffusion methods following the British Society for Antimicrobial Chemotherapy (BSAC) guidelines. A few isolates of K. pneumoniae showed insusceptibility to cephalosporins, colistin, rifampicin, trimethoprim and penicillin but not to carbapenems. Biocide susceptibility testing showed that 57, 55 and 61 strains had reduced susceptibility to Chlorhexidine, Trigene and Benzalkonium chloride, respectively, but not to MediHex-4 and Mediscrub. The effect of efflux pumps were determined by carbonyl cyanide m-chlorophenylhydrazone (CCCP) (10mg/L), which decreased the MICs of Chlorhexidine and Medihex-4 by 2 – 128 fold but had no impact on the MICs of Benzalkonium chloride, Trigene and Mediscrub. Six isolates of K. pneumoniae were chosen for their varying sensitivity to Chlorhexidine (CHX), and were tested for their minimum bactericidal concentration (MBC) to biocides. The high MBCs of Mediscrub and Trigene, over 500-fold greater than the minimum inhibitory concentration (MICs), indicates that these compounds are mainly bacteriostatic. Conversely, the MBCs of Chlorhexidine and MediHex-4, which contains chlorhexidine, were less than 10-times the MIC value indicating they are effective in killing the organism. However, this thesis showed how the killing capability of Chlorhexidine was hindered by the presence of organic matter, which compromised its effect. The relationship between reduced susceptibility to biocides and the carriage of antiseptic resistance genes, cepA, qacΔE1 and qacE was determined by polymerase chain reaction. The antiseptic resistance genes cepA, qacΔE1 and qacE were found in 56, 34 and 1 isolates respectively, and the levels of gene expression were detected by the reverse transcription polymerase chain reaction (RT-PCR). These results have shown that there was a close link between carriage of efflux pump genes, cepA, qacΔE1 and qacE genes and reduced susceptibility to biocides. Most strains showed decreased susceptibility to Chlorhexidine, Trigene and Benzalkonium chloride and this correlated with the carriage of the cepA, qacΔE1 and qacE genes encoding efflux. There was no correlation between the reduced susceptibility to biocides and antibiotic resistance in clinical isolates of K. pneumoniae.

Klebsiella pneumoniae is a common cause of nosocomial and community-acquired infections, and the increasing incidence and prevalence of antibiotic resistant strains is proving to be particularly problematic to clinicians. K. pneumoniae is capable of employing a multitude of mechanisms by which to confer resistance to most available antibiotics. The carbapenem antibiotics are usually reserved for the treatment of complicated or multidrug resistant (MDR) K. pneumoniae infections. The recent emergence of not only MDR but also pan-drug resistant (PDR) K. pneumoniae strains has signified that it is now more important than ever to understand the mechanisms by which these strains confer resistance so that we may find ways to combat or hinder this progression. This project aimed to investigate the regulation of the transcriptional activator RamA, its ability to confer a MDR phenotype, and the mechanisms employed by K. pneumoniae to confer levels of carbapenem resistance sufficient to result in therapy failure. The analysis of a panel of K. pneumoniae strains, containing both RamA expressers and non-expressers, demonstrated that the overexpression of RamA was sufficient to confer an MDR phenotype. Two compounds, chlorpromazine (CPZ) and tigecycline, were shown to act as inducers of ramA, romA and acrA transcription. CPZ exhibited synergy with the antibiotics chloramphenicol, norfloxacin and tetracycline, all of which are known substrates of the AcrAB efflux pump. The current lack of novel classes of antimicrobials in development indicate a potential for a compound, such as CPZ, to be developed and exploited for clinical use. The ability of both CPZ and tigecycline to cause mutations within ramR however, indicate that both compounds may have the ability to select for efflux mutants as a result of their ability to upregulate ramA, which in turn causes the upregulation of the AcrAB efflux pump. The regulation of RamA by the upstream gene ramR, which encodes a TetR family protein was investigated in K. pneumoniae isolates. Sequencing of the ramR genes revealed that strains exhibiting an MDR phenotype commonly contained mutations within their gene sequences. The complementation of a wildtype ramR into a strain containing a 32 amino acid deletion within its ramR, was shown to increase susceptibility to various antibiotics of different classes, and additionally downregulate the expression of ramA, romA and acrA. CPZ, ciprofloxacin and tigecycline K. pneumoniae mutants were shown to exhibit increased MICs to a broad spectrum of antibiotics with respect to their parent strains, and possess mutations within their ramR genes. Complementation of the wildtype ramR resulted in partial reversion to the parental phenotypes, indicating another mechanism must also be involved in conferring the MDR phenotypes. These studies indicated that RamR plays an important role as a negative regulator of RamA, but also that it is not the sole regulator. The development of reduced susceptibility to the carbapenems was investigated in two clinical strains of K. pneumoniae, K1 and K2, isolated from the urine of a single patient at different stages of antibiotic therapy. The strains were shown to exhibit similar resistance phenotypes with the exception of their susceptibilities to the carbapenems. PCR and phenotypic analyses revealed that neither strain contained any carbapenemases or AmpC enzymes, but both contained OXA-1, SHV-1 TEM-1 and CTX-M-15. Analysis of their OMP profiles indicated that both strains lacked OmpK35, and K2 additionally lacked OmpK36. Mutation studies showed that the phenotype and OMP profile exhibited by K2 could be achieved in K1 via single step mutations using ertapenem, imipenem or meropenem. Susceptibility testing of CTXM- 15 clinical strains showed that strains containing CTX-M-15 showed reduced activity against ertapenem in the presence of clavulanic acid. These studies indicated a potential role for CTX-M-15 in conferring reduced susceptibility to the carbapenems when found in conjunction with altered permeability and active efflux. The mechanisms of antibiotic resistance employed by K. pneumoniae are numerous and complex. This work highlights several of these mechanisms and, more importantly, how they can work in synergy with one another to devastating consequences.

No description available.

No description available.

The cyanide-degrading bacterium Klebsiella oxytoca SYSU-011 was isolated from the waste water of a metal-plating plant. In this study, we found out that K. oxytoca was capable of utilizing tetracyanonickelate {K2[Ni(CN)4]}(TCN) as its sole nitrogen source. This organism could degrade TCN both aerobically (D.O.¡×100¢H) and anaerobically (D.O.¡×0¢H).The addition of ammonia (5 mM) in the growth medium would inhibit TCN-degrading. The TCN-degrading by-product, a greenish precipitate, was found in the spent medium and was identified as nickel cyanide [Ni(CN)2] by FT-IR spectroscopic studies. Ammonia was demonstrated as a product of the TCN-degrading process by K. oxytoca resting cells. The addition of glucose could greatly enhance the TCN-degradation. Nitrogenase was found to be the cyanide degrading enzyme in this organism. The activity of nitrogenase was inhibited by ammonia but could be induced by the addition of TCN or KCN.

Univ., Diss.--München, 1998.