Reprodutibilidade teste-reteste e validade concorrente de manovacuômetro digital

A manovacuometria é um teste simples, rápido e não invasivo que mensura as pressões respiratórias máximas (PRM). Diretrizes recomendam o uso do manovacuômetro digital devido à sua alta precisão. O objetivo deste estudo foi avaliar a reprodutibilidade teste-reteste e a validade concorrente de um manovacuômetro digital na mensuração das pressões inspiratórias e expiratórias máximas (PImáx e PEmáx) e da pressão inspiratória nasal durante o fungar (SNIP). Foram avaliados 30 indivíduos saudáveis (20-30 anos) utilizando os manovacuômetros digitais UFMG e MicroRPM(r) (Micro Medical, UK). Para avaliar a reprodutibilidade, foi utilizado o Coeficiente de Correlação Intraclasse (CCI) e teste t de student para amostras dependentes. Para análise da validade foram utilizados: a correlação de Pearson, o teste t de student para amostras dependentes, a análise de regressão linear e o método Bland-Altman. O nível de significância considerado foi de 5% (p;0,05). A correlação entre os valores observados nos dois instrumentos foi de alta magnitude para todas as variáveis (0,82 a 0,85); não houve diferença significativa entre os valores médios obtidos nos dois instrumentos (p>;0,05); foi observada forte associação entre as medidas das PRM obtidas pelos dois métodos e a análise de Bland-Altman não demonstrou superestimação ou subestimação sistemática das PRM e do SNIP. Em conclusão, os resultados sugerem que o manovacuômetro UFMG é confiável e válido para avaliação das PRM e SNIP em indivíduos saudáveis.The manovacuometer is a simple, quick and non-invasive test which measures the maximal respiratory pressures (MRS). Guidelines recommend the use of a digital manovacuometer due to its high accuracy. The purpose of this study was to assess the test-retest reliability and concurrent validity of a digital manovacuometer in measuring the maximal inspiratory and expiratory pressures (MIP/MEP) and nasal inspiratory pressure while sniffing (SNIP). A total of 30 healthy subjects were assessed (20-30 years old) using the UFMG and MicroRPM(r) (Micro Medical, UK) digital manovacuometers. To assess reliability, Intraclass Correlation Coefficient (ICC) and Student's t test it was used for dependent samples. For the validity assessment, the following were used: Pearson correlation, Student's t test for dependent samples, linear regression and the Bland-Altman method. The level of significance was set at 5% (p;0.05); the correlation between observed values from the two instruments was of high magnitude for all variables (0.82 to 0.85); no significant difference was found between the values obtained for both instruments (p>;0.05); a strong association was observed between measures of MIP and MEP obtained by the two methods and Bland-Altman analysis showed no systematic overestimation or underestimation of maximal respiratory pressures and SNIP. In conclusion, the results suggest that the UFMG manovacuometer is a reliable and valid instrument for assessing MIP, MEP and SNIP in healthy subjects.La manovacuometría es una prueba sencilla, rápida y no invasiva que mide las presiones respiratorias máximas (PRM). Directrices recomiendan el uso del manuvacuómetro digital debido a su alta precisión. El objetivo de este estudio fue evaluar la reproducibilidad test-retest y la validez concurrente de un manuvacuómetro digital para medir las presiones inspiratoria y espiratoria máximas (PImáx y PEmáx) y de la presión inspiratoria nasal durante la aspiración (SNIP). Se evaluaron 30 sujetos sanos (20-30 años) por medio de los manovacuómetros digitales UFMG y MicroRPM(r) (Micro Medical, UK). Para evaluar la reproducibilidad, se utilizó el coeficiente de correlación intraclase (CCI) y el test t de student para muestras dependientes. Para el análisis de la validez se utilizaron: la correlación de Pearson, el test t de student para muestras dependientes, el análisis de regresión lineal y el método Bland-Altman. El nivel de significación considerado fue del 5% (p;0,05). La correlación entre los valores observados en los dos instrumentos fue de alta magnitud para todas las variables (0,82 a 0,85); no hubo diferencia significativa entre los valores medios obtenidos en los dos instrumentos (p>;0,05); Se observó una fuerte asociación entre las medidas de las PRM obtenidas por los dos métodos y el análisis de Bland-Altman no demostró sobreestimación o subestimación sistemática de las PRM y del SNIP. En conclusión, los resultados sugieren que el manovacuómetro UFMG es fiable y válido para la evaluación de las PRM y SNIP en sujetos sanos

Exploring the CRISPR-Cas9 potential to revert beta-lactam resistance in clinically relevant gram-negative bacteria

Submitted by Thaysa Tagliaferri ([email protected]) on 2020-02-20T00:33:42Z No. of bitstreams: 1 Thesis_Tagliaferri_Corrections.pdf: 8489164 bytes, checksum: 5deccf1a9c5007f4c684202ce33b16ad (MD5)Approved for entry into archive by Camila Silva ([email protected]) on 2020-02-21T18:16:10Z (GMT) No. of bitstreams: 1 Thesis_Tagliaferri_Corrections.pdf: 8489164 bytes, checksum: 5deccf1a9c5007f4c684202ce33b16ad (MD5)Made available in DSpace on 2020-03-19T21:46:58Z (GMT). No. of bitstreams: 1 Thesis_Tagliaferri_Corrections.pdf: 8489164 bytes, checksum: 5deccf1a9c5007f4c684202ce33b16ad (MD5) Previous issue date: 2020-01-15CNPq - Conselho Nacional de Desenvolvimento Científico e TecnológicoFAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas GeraisCAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível SuperiorThe antimicrobial resistance (AMR) crisis urgently requires countermeasures for reducing the dissemination of plasmid-borne resistance genes. Of particular concern are opportunistic pathogens of Enterobacteriaceae family. One innovative approach to tackle AMR is the use of CRISPR-Cas9 system which has recently been employed for plasmid curing in model strains of Escherichia coli. During this thesis, this system was further exploited by targeting the blaTEM-1 resistance gene located on a high copy plasmid (i.e. > 100 copies/cell) and by directly tackling the blaTEM-1-and blaKPC genes from clinical isolates, both prevalent in multidrug-resistant bacteria. It is known that clinical Enterobacteriaceae strains possess multiple resistance mechanisms which could impair the efficiency of the system. In addition, the resistance levels are directly dependent of the plasmid copy number. Hence, the aim of this thesis was to explore further the CRISPR-Cas9 technology in challenging conditions, especially when facing high copy plasmids and when targeting clinical bacteria. This thesis aims at contributing to clarify all the possible scenarios obtained with the application of the CRISPR-Cas9 system in resistance reduction in order to establish this tool as an alternative method to counteract AMR. By using independent techniques as fluorescence-activated cell sorting (FACS), qPCR, and fluorescence microscopy, low levels of plasmid maintenance were detected in the cells upon CRISPR-Cas9 insertion. However, even though plasmid integrity could be detected, sequence alterations in the blaTEM-1 gene were observed, resulting in a dysfunction of the gene product and, therefore, in an antibiotic sensitive strain. In a clinical isolate of E. coli, plasmid clearance and re-sensitization to five beta-lactams were achieved. Reusability of antibiotics could be confirmed by infecting larvae of Galleria mellonella with CRISPR-Cas9-treated E. coli, as opposed to infection with the 16 unmodified clinical isolate. The drug sensitivity levels could also be increased in a clinical isolate of Enterobacter cloacae and to a lesser extent in Klebsiella variicola, both of which harboured also the blaCTX-M gene. When changing target to the blaKPC gene, resistance reduction to the intermediate level of imipenem was achieved in 63% of the retrieved clones of Klebsiella oxytoca clinical isolate. Interestingly, both still resistant and intermediate sensitive clones had the plasmid copy number and the blaKPC gene expression reduced. Moreover, fitness levels were also significantly decreased when either intermediate or still resistant clones were compared to the CRISPR-Cas9 untreated control. However here, CRISPR-Cas9 did not contribute to increase larvae survival after challenge with different clones of K. oxytoca. Finally, a functional CRISPR-Cas9 delivery system via bacteriophages was developed, expanding the possibilities of applicability of the technique. To conclude, our data demonstrated that the targeted strain was not able to sufficiently evade the CRISPR-Cas9-based manipulation and maintain resistance phenotype by means of plasmid amplification. Despite the versatile challenges imposed by clinical isolates, the interference with the resistance gene led after all from minor to clear resistance reductions. Overexpression of efflux pumps or alterations in porins in the clinical isolates, if having occurred, did not prevent resistance reduction. Moreover, all possible CRISPR-Cas9-based outcomes of targeting a resistance gene, i.e. plasmid clearance; resistance gene disruption; or reduction of plasmid copy number impacted the horizontal gene transfer of resistant plasmids. In light of the fact that antimicrobial resistance has spread worldwide with serious impact on human lives, the findings of this study provides relevant details regarding the possible outcomes when using the CRISPR-Cas9 as an alternative tool to reduce resistance in clinically relevant pathogens.A crise de resistência bacteriana a antimicrobianos (AMR) torna necessária o desenvolvimento de medidas efetivas para a redução da disseminação de genes de resistência transmitidos por plasmídeos, cenário em que os patógenos oportunistas da família Enterobacteriaceae apresentam um papel relevante. O sistema CRISPR-Cas9 representa uma abordagem inovadora e efetiva para combater a AMR, o qual foi recentemente empregado para a total eliminação de plasmídeos em linhagens laboratoriais de Escherichia coli. Nesta tese, este sistema foi amplamente explorado, tendo como alvo o gene de resistência blaTEM-1 tanto localizado em um plasmídeo de alto número de cópias (> 100 cópias / célula) quanto presente em isolados clínicos. Além disso, o gene blaKPC, prevalente em amostras multirresistentes, também foi utilizado como alvo para o CRISPR-Cas9, em isolados clínicos bacterianos. Sabe-se que as linhagens clínicas da família Enterobacteriaceae possuem múltiplos mecanismos de resistência que podem prejudicar a eficiência do sistema. Além disso, os níveis de resistência são diretamente dependentes do número de cópias do plasmídeo. Assim, o objetivo desta tese foi explorar ainda mais a tecnologia CRISPR-Cas9 em condições desafiadoras, isto é, na presença de plasmídeos de alto número de cópias e para reverter resistência em isolados clínicos. Com isso, o presente estudo visa contribuir para o esclarecimento de todos os possíveis cenários obtidos com a aplicação do sistema CRISPR-Cas9 na redução da resistência, a fim de consolidá-la como um método alternativo para neutralizar a AMR. Utilizando técnicas independentes como citômetro de fluxo (FACS), qPCR e microscopia de fluorescência, foram verificados níveis baixos de manutenção de plasmídeos nas células após a inserção do CRISPR-Cas9. No entanto, mesmo detectando a integridade do plasmídeo, a sequência do gene blaTEM-1 se encontrava alterada, resultando em uma 18 disfunção da proteína transcrita e, portanto, em um clone sensível a antibióticos. No isolado clínico de E. coli, CRISPR-Cas9 foi responsável pela completa remoção do plasmídeo, juntamente com a re-sensibilização bacteriana a cinco beta-lactâmicos. Antibióticos puderam ser reutilizados com eficácia para tratar larvas da espécie Galleria mellonella infectadas com clones de isolados clínicos de E. coli previamente tratados com CRISPR-Cas9. De forma contrária, baixos níveis de sobrevivência foram obtidos em larvas infectadas com o isolado clínico não tratado pelo CRISPR-Cas9. Os níveis de sensibilidade a antimicrobianos também aumentaram em isolados clínicos de Enterobacter cloacae e, em menor grau, em Klebsiella variicola, ambos contendo também o gene blaCTX-M. Ao alterar o alvo para o gene blaKPC, foi possível obter uma redução da resistência a imipenem para o nível intermediário em 63% dos clones de Klebsiella oxytoca. Curiosamente, em ambos os clones resistentes e com sensibilidade intermediária, uma redução no número de cópias do plasmídeo, bem como na expressão gênica do blaKPC foi obtida. Além disso, os níveis de fitness bacteriano também foram significativamente reduzidos quando clones com sensibilidade intermediária ou ainda resistentes foram comparados ao controle, este não tratado pelo CRISPR-Cas9. Apesar disso, nenhum impacto in vivo pôde ser alcançado quando larvas de G. mellonella infectadas com diferentes clones de K. oxytoca foram tratadas com imipenem. Finalmente, um sistema funcional de entrega do CRISPR-Cas9 por meio de bacteriófagos foi desenvolvido, ampliando as possibilidades de aplicabilidade da técnica. Para concluir, nossos dados demonstraram que as linhagens tratadas pelo CRISPR-Cas9 não foram capazes de evadir eficientemente do sistema e manter o fenótipo de resistência por meio de amplificação do plasmídeo. Apesar dos desafios versáteis impostos pelos isolados clínicos, a interferência por meio do CRISPR-Cas9 nos genes de resistência levou tanto a aumentos marginais quanto significativos na sensibilidade a 19 antimicrobianos. A superexpressão de bombas de efluxo ou alterações nas porinas nos isolados clínicos, caso tenham ocorrido, não impediram a redução da resistência. Além disso, todos os possíveis resultados obtidos pelo CRISPR-Cas9 quando usado para reduzir AMR, isto é, eliminação total do plasmídeo; ruptura do gene de resistência; ou redução do número de cópias do plasmídeo, impactam diretamente na transferência horizontal de genes. Tendo em vista o fato de que a resistência antimicrobiana se espalhou globalmente, gerando graves impactos na vida humana, os resultados deste estudo fornecem detalhes relevantes sobre o uso do CRISPR-Cas9 como uma ferramenta alternativa para reduzir a resistência em bactérias clinicamente relevantes

Exploring the CRISPR-Cas9 potential to revert beta-lactam resistance in clinically relevant gram-negative bacteria

The antimicrobial resistance (AMR) crisis urgently requires countermeasures for reducing the dissemination of plasmid-borne resistance genes. Of particular concern are opportunistic pathogens of Enterobacteriaceae family. One innovative approach to tackle AMR is the use of CRISPR-Cas9 system which has recently been employed for plasmid curing in model strains of Escherichia coli. During this thesis, this system was further exploited by targeting the blaTEM-1 resistance gene located on a high copy plasmid (i.e. > 100 copies/cell) and by directly tackling the blaTEM-1-and blaKPC genes from clinical isolates, both prevalent in multidrug-resistant bacteria. It is known that clinical Enterobacteriaceae strains possess multiple resistance mechanisms which could impair the efficiency of the system. In addition, the resistance levels are directly dependent of the plasmid copy number. Hence, the aim of this thesis was to explore further the CRISPR-Cas9 technology in challenging conditions, especially when facing high copy plasmids and when targeting clinical bacteria. This thesis aims at contributing to clarify all the possible scenarios obtained with the application of the CRISPR-Cas9 system in resistance reduction in order to establish this tool as an alternative method to counteract AMR. By using independent techniques as fluorescence-activated cell sorting (FACS), qPCR, and fluorescence microscopy, low levels of plasmid maintenance were detected in the cells upon CRISPR-Cas9 insertion. However, even though plasmid integrity could be detected, sequence alterations in the blaTEM-1 gene were observed, resulting in a dysfunction of the gene product and, therefore, in an antibiotic sensitive strain. In a clinical isolate of E. coli, plasmid clearance and re-sensitization to five beta-lactams were achieved. Reusability of antibiotics could be confirmed by infecting larvae of Galleria mellonella with CRISPR-Cas9-treated E. coli, as opposed to infection with the unmodified clinical isolate. The drug sensitivity levels could also be increased in a clinical isolate of Enterobacter cloacae and to a lesser extent in Klebsiella variicola, both of which harboured also the blaCTX-M gene. When changing target to the blaKPC gene, resistance reduction to the intermediate level of imipenem was achieved in 63% of the retrieved clones of Klebsiella oxytoca clinical isolate. Interestingly, both still resistant and intermediate sensitive clones had the plasmid copy number and the blaKPC gene expression reduced. Moreover, fitness levels were also significantly decreased when either intermediate or still resistant clones were compared to the CRISPR-Cas9 untreated control. However here, CRISPR-Cas9 did not contribute to increase larvae survival after challenge with different clones of K. oxytoca. Finally, a functional CRISPR-Cas9 delivery system via bacteriophages was developed, expanding the possibilities of applicability of the technique. To conclude, our data demonstrated that the targeted strain was not able to sufficiently evade the CRISPR-Cas9-based manipulation and maintain resistance phenotype by means of plasmid amplification. Despite the versatile challenges imposed by clinical isolates, the interference with the resistance gene led after all from minor to clear resistance reductions. Overexpression of efflux pumps or alterations in porins in the clinical isolates, if having occurred, did not prevent resistance reduction. Moreover, all possible CRISPR-Cas9-based outcomes of targeting a resistance gene, i.e. plasmid clearance; resistance gene disruption; or reduction of plasmid copy number impacted the horizontal gene transfer of resistant plasmids. In light of the fact that antimicrobial resistance has spread worldwide with serious impact on human lives, the findings of this study provides relevant details regarding the possible outcomes when using the CRISPR-Cas9 as an alternative tool to reduce resistance in clinically relevant pathogens

Potential of the endogenous and artificially inserted CRISPR-Cas system for controlling virulence and antimicrobial resistance of food pathogens

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated sequences (Cas) are prokaryotic defenses against viruses and foreign nucleic acids found in many bacterial and archaeal genomes. The discovery that CRISPR spacers are often identical to sequence fragments of mobile genetic elements was a major breakthrough that eventually led to the elucidation of CRISPR-Cas as an adaptive immunity system. The ability of the CRISPR-Cas systems to perform sequence-specific DNA cleavage evidenced its potential for gene deletion, insertion, or regulation. This allows the food microbiota to be easily genetically modulated, including virulence or resistance gene editing from pathogens which could lead to more safe and high-quality products. This review provides insights into the CRISPR-Cas systems, followed by the understanding of the synergistic or antagonistic relationship of resistance and virulence determinants in foodborne pathogens in connection to their intrinsic CRISPR system. By employing specific examples and recently reported studies this review also widens the discussion of the CRISPR-Cas use for controlling food pathogens by editing genes associated with virulence modulation and reversal of antimicrobial resistance

Newly isolated Drexlerviridae phage LAPAZ is physically robust and fosters eradication of Klebsiella pneumoniae in combination with meropenem

Due to the spread of multidrug resistance there is a renewed interest in using bacteriophages (briefly: phages) for controlling bacterial pathogens. The objective of this study was the characterization of a newly isolated phage (i.e. phage LAPAZ, vB_KpnD-LAPAZ), its antimicrobial activity against multidrug resistant Klebsiella pneumoniae and potential synergistic interactions with antibiotics. LAPAZ belongs to the family Drexlerviridae (genus: Webervirus) and lysed 30 % of tested strains, whereby four distinct capsular types can be infected. The genome consists of 51,689 bp and encodes 84 ORFs. The latent period is 30 min with an average burst size of 27 PFU/cell. Long-term storage experiments show that LAPAZ is significantly more stable in wastewater compared to laboratory media. A phage titre of 90 % persists up to 30 min at 50 ˚C and entire phage loss was seen only at temperatures > 66 ˚C. Besides stability against UV-C, antibacterial activity in liquid culture medium was consistent at pH values ranging from 4 to 10. Unlike exposure to phage or antibiotic alone, synergistic interactions and a complete bacterial eradication was achieved when combining LAPAZ with meropenem. In addition, synergism with the co-presence of ciprofloxacin was observed and phage resistance emergence could be delayed. Without co-addition of the antibiotic, phage resistant mutants readily emerged and showed a mixed pattern of drug sensitivity alterations. Around 88 % became less sensitive towards ceftazidime, meropenem and gentamicin. Conversely, around 44 % showed decreased resistance levels against ciprofloxacin. Whole genome analysis of a phage-resistant mutant with a 16-fold increased sensitivity towards ciprofloxacin revealed one de novo frameshift mutation leading to a gene fusion affecting two transport proteins belonging to the major facilitator-superfamily (MFS). Apparently, this mutation compromises ciprofloxacin efflux efficiency and further studies are warranted to understand how the non-mutated protein might be involved in phage-host adsorption

Test-retest reliability and concurrent validity of a digital manovacuometer

The manovacuometer is a simple, quick and non-invasive test which measures the maximal respiratory pressures (MRS). Guidelines recommend the use of a digital manovacuometer due to its high accuracy. The purpose of this study was to assess the test-retest reliability and concurrent validity of a digital manovacuometer in measuring the maximal inspiratory and expiratory pressures (MIP/MEP) and nasal inspiratory pressure while sniffing (SNIP). A total of 30 healthy subjects were assessed (20-30 years old) using the UFMG and MicroRPM(r) (Micro Medical, UK) digital manovacuometers. To assess reliability, Intraclass Correlation Coefficient (ICC) and Student's t test it was used for dependent samples. For the validity assessment, the following were used: Pearson correlation, Student's t test for dependent samples, linear regression and the Bland-Altman method. The level of significance was set at 5% (p<0.05). The ICC values were significant and showed a good magnitude (0.76 to 0.89) and no significant differences were found between the means of the variables of the UFMG digital manovacuometer analyzed within two days (p>0.05); the correlation between observed values from the two instruments was of high magnitude for all variables (0.82 to 0.85); no significant difference was found between the values obtained for both instruments (p>0.05); a strong association was observed between measures of MIP and MEP obtained by the two methods and Bland-Altman analysis showed no systematic overestimation or underestimation of maximal respiratory pressures and SNIP. In conclusion, the results suggest that the UFMG manovacuometer is a reliable and valid instrument for assessing MIP, MEP and SNIP in healthy subjects

A avaliação do desenvolvimento infantil: um desafio interdisciplinar

OBJETIVO: Avaliar o desenvolvimento de crianças de 2 meses a 2 anos de idade por meio da Atenção Integrada às Doenças Prevalentes na Infância (AIDPI), no contexto do Programa de Educação pelo Trabalho em Saúde (PET-Saúde). MÉTODO: Estudo transversal realizado com 122 crianças, com idades entre 2 meses e 2 anos, da área de abrangência do Centro de Saúde São Bernardo (CSSB) - Belo Horizonte (MG), em 2009. Os dados relativos ao desenvolvimento foram obtidos através da aplicação de dois questionários: AIDPI e Caderneta de Saúde da Criança (CSC). Foram comparadas as classificações do desenvolvimento pela AIDPI e pela CSC, a associação entre atraso do desenvolvimento e as variáveis estudadas. RESULTADOS: As características com maior frequência na população estudada foram a baixa escolaridade das mães (62,1%), seguida de parentes com deficiência mental (71,3%) e problemas na gestação (71,3%). A AIDPI evidenciou que 61,5% da população estudada encontra-se normal com fator de risco, 16,4% normal sem fator de risco, 11,5% com possível atraso e 10,7% com provável atraso do desenvolvimento infantil. A concordância observada entre a classificação da AIDPI e da CSC foi de 0,34, coeficiente Kappa igual a - 0,12 (p = 0,98). Não houve associação estatisticamente significativa entre as variáveis analisadas (frequenta creches; convívio com problemas emocionais; escolaridade da mãe; idade gestacional; e peso ao nascer) e atraso possível/provável do desenvolvimento identificado pela AIDPI. CONCLUSÃO: O PET-Saúde, como proposta de integração da educação pelo trabalho, permitiu uma oportunidade de convivência e troca de experiências entre alunos e profissionais de diferentes áreas de atuação, trabalhando em um projeto comum