Caracterização molecular de bacilos gram-negativos isolados em unidade de terapia intensiva neonatal e detecção fenotípica e molecular de esbl e carbapenemases.
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Data
2020-02-20
Autores
Barbosa, Thaís Alves [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
A elevação das taxas de bactérias multirresistentes em ambientes destinados a prestação de cuidados à saúde é reconhecido como um problema de saúde pública mundial. Atualmente, as opções terapêuticas são limitadas em decorrência da produção de β-lactamases de espectro estendido (ESBL), β-lactamases do tipo AmpC e carbapenemases. Tais enzimas são capazes de se disseminar com facilidade entre patógenos, dificultando o controle e o tratamento das infecções nosocomiais. Sendo assim, o presente estudo teve como objetivo principal estudar micro-organismos Gram-negativos isoladas de aspirado traqueal (AT) e da mucosa nasal (MN) e anal (MA) de neonatos da UTIN pertencentes ao Hospital das Clínicas (HC) da Faculdade de Medicina de Botucatu (FMB). Os micro-organismos foram coletados previamente em um estudo de coorte prospectivo no período de novembro de 2013 a novembro de 2014, visando determinar a resistência aos antimicrobianos, fatores de virulência e pesquisa de clones prevalentes na Unidade. As enterobactérias e os bacilos gram-negativos não fermentadores (BGNF) previamente coletados foram submetidos à identificação genotípica, detecção de β-lactamases de espectro estendido (ESBL), β-lactamases do tipo AmpC e carbapenemases, produção de biofilme e detecção de marcadores de virulência específicos em Pseudomonas aeruginosa. Para a pesquisa de clones prevalentes na unidade, foi realizada a tipagem pela técnica de eletroforese em gel de campo pulsado (PFGE). Os resultados revelaram que as enterobactérias foram os micro-organismos predominantes em sítios de colonização e infecção. Este trabalho demonstrou maior colonização da MA seguida pela MN. Enterobacter cloacae foi a espécie isolada com maior frequência em amostras de colonização. Os dados aqui encontrados revelaram maiores índices de infecção por Klebsiella spp. e Serratia marcescens, causando infecção da corrente sanguínea e pneumonia, respectivamente. Nesta pesquisa, pôde ser observada elevada produção de biofilme, sendo que 95,0% das enterobactérias e 100% dos BGNF foram produtores, com maior proporção de enterobactérias mostrando média produção de biofilme, enquanto os BGNF demonstraram em sua maioria serem fortes produtores. Através da pesquisa dos genes codificadores de fatores de virulência específicos para Pseudomonas aeruginosa foi possível observar que a maioria dos isolados apresentaram genes codificadores de alginato, fosfolipase C hemolítica, exotoxina A e ramnolipídeos. A pesquisa fenotípica de ESBL revelou 16 (5,3%) dos isolados produtores dessas enzimas. Dentre esses, seis apresentaram genes de resistência, quatro (66,7%) apresentaram o gene codificador de CTX-M-9, três (50%) apresentaram o gene de TEM e um (16,7%) foi positivo para o gene de SHV e de CMY-2. A pesquisa de β-lactamases do Tipo AmpC revelou a produção fenotípica por 20 isolados e, destes, dois apresentaram o genótipo EBC. Através da análise do perfil genético, foi possível observar linhagens idênticas de espécies de enterobactérias em diferentes sítios de coleta de RNs distintos, evidenciando possível transmissão cruzada; além disso, foi possível identificar característica de persistência de algumas cepas dentro da unidade. Empregou-se uma análise uni e multivariada de regressão de Cox para verificar os fatores de risco de colonização e infecção por micro-organismos Gram-negativos. Os resultados da análise multivariada revelaram que, a cada semana a mais na idade gestacional, aumenta o risco diário em 7,0% para a colonização do RN por bacilos Gram-negativos, enquanto o uso de antimicrobianos nas primeiras 72 horas diminui o risco diário em 35% de colonização por esses patógenos. Além disso, a produção de biofilme por esses micro-organismos apresentou associação com a persistência da colonização do RN pelo mesmo patógeno e aumenta em 75% a chance diária do RN evoluir para infecção, assim como a produção de ESBL, que aumenta a chance diária em 46,8 vezes para o RN ter infecção. Os dados desta pesquisa são de fundamental importância para a saúde pública, pois permitem o conhecimento da epidemiologia molecular de espécies importantes de enterobactérias na UTIN de um hospital universitário terciário de alta complexidade, permitindo, assim, a intensificação do monitoramento e implantação de medidas de controle e precaução.
The increase rates of multidrug-resistant bacteria in healthcare environments are recognized as a worldwide public health problem. Currently, therapeutic options are limited due to the production of extended spectrum β-lactamases (ESBL), AmpC-type β-lactamases and carbapenemases. Such enzymes can spread easily among pathogens, making it difficult to control and treat nosocomial infections. Therefore, the present study aimed to study Gramnegative microorganisms isolated from tracheal aspirate (TA) and nasal mucosa (MN) and anal (MA) from NICU newborns belonging to the Hospital das Clínicas (HC) of the Faculty of Medicine of Botucatu (FMB). Microorganisms were previously collected in a prospective cohort study from November 2013 to November 2014, in order to determine resistance to antimicrobials, virulence factors and clone research prevalent in the Unit. Previously collected enterobacteria and non-fermenting gram-negative bacilli (NFGNB) were subjected to genotypic identification, detection of extended spectrum β-lactamases (ESBL), AmpC-type β-lactamases and carbapenemases, biofilm production and detection of specific virulence in Pseudomonas aeruginosa. For the research of clones prevalent in the unit, typing was performed using the pulsed field gel electrophoresis (PFGE) technique. The results revealed that enterobacteria were the predominant microorganisms in colonization and infection sites. This work demonstrated greater colonization of MA followed by MN. Enterobacter cloacae was the most frequently isolated species in colonization samples. The data found here revealed higher rates of infection by Klebsiella spp. and Serratia marcescens, causing bloodstream infection and pneumonia, respectively. In this research, high biofilm production could be observed, with 95.0% of enterobacteria and 100% of NFGNBs being producers, with a higher proportion of enterobacteria showing average biofilm production, while NFGNBs were mostly strong producers. Through the search for genes encoding specific virulence factors for Pseudomonas aeruginosa it was possible to observe that most of the isolates had genes encoding alginate, hemolytic phospholipase C, exotoxin A and ramnolipids. The ESBL phenotypic research revealed 16 (5.3%) of the isolates producing these enzymes. Among these, six had resistance genes, four (66.7%) had the CTX-M-9 coding gene, three (50%) had the TEM gene and one (16.7%) was positive for the gene of SHV and CMY-2. The search for AmpC-type β-lactamases revealed phenotypic production by 20 isolates and, of these, two presented the EBC genotype. Through the analysis of the genetic profile, it was possible to observe identical lineages of species of enterobacteria in different collection sites of different newborns, showing possible cross-transmission; in addition, it was possible to identify the persistence characteristic of some strains within the unit. A univariate and multivariate analysis of Cox regression was used to verify the risk factors for colonization and infection by Gram-negative microorganisms. The results of the multivariate analysis revealed that, each week more at gestational age, the daily risk increases by 7.0% for the colonization of newborns by Gram-negative bacilli, while the use of antimicrobials in the first 72 hours decreases the daily risk in 35% of colonization by these pathogens. In addition, the production of biofilm by these microorganisms was associated with the persistence of colonization of the newborn by the same pathogen and increases by 75% the daily chance of the newborn evolving to infection, as well as the production of ESBL, which increases the daily chance 46.8 times for the newborn to have infection. The data of this research are of fundamental importance for public health, as they allow the knowledge of the molecular epidemiology of important species of enterobacteria in the NICU of a tertiary university hospital of high complexity, thus allowing the intensification of the monitoring and implementation of control measures and precaution
The increase rates of multidrug-resistant bacteria in healthcare environments are recognized as a worldwide public health problem. Currently, therapeutic options are limited due to the production of extended spectrum β-lactamases (ESBL), AmpC-type β-lactamases and carbapenemases. Such enzymes can spread easily among pathogens, making it difficult to control and treat nosocomial infections. Therefore, the present study aimed to study Gramnegative microorganisms isolated from tracheal aspirate (TA) and nasal mucosa (MN) and anal (MA) from NICU newborns belonging to the Hospital das Clínicas (HC) of the Faculty of Medicine of Botucatu (FMB). Microorganisms were previously collected in a prospective cohort study from November 2013 to November 2014, in order to determine resistance to antimicrobials, virulence factors and clone research prevalent in the Unit. Previously collected enterobacteria and non-fermenting gram-negative bacilli (NFGNB) were subjected to genotypic identification, detection of extended spectrum β-lactamases (ESBL), AmpC-type β-lactamases and carbapenemases, biofilm production and detection of specific virulence in Pseudomonas aeruginosa. For the research of clones prevalent in the unit, typing was performed using the pulsed field gel electrophoresis (PFGE) technique. The results revealed that enterobacteria were the predominant microorganisms in colonization and infection sites. This work demonstrated greater colonization of MA followed by MN. Enterobacter cloacae was the most frequently isolated species in colonization samples. The data found here revealed higher rates of infection by Klebsiella spp. and Serratia marcescens, causing bloodstream infection and pneumonia, respectively. In this research, high biofilm production could be observed, with 95.0% of enterobacteria and 100% of NFGNBs being producers, with a higher proportion of enterobacteria showing average biofilm production, while NFGNBs were mostly strong producers. Through the search for genes encoding specific virulence factors for Pseudomonas aeruginosa it was possible to observe that most of the isolates had genes encoding alginate, hemolytic phospholipase C, exotoxin A and ramnolipids. The ESBL phenotypic research revealed 16 (5.3%) of the isolates producing these enzymes. Among these, six had resistance genes, four (66.7%) had the CTX-M-9 coding gene, three (50%) had the TEM gene and one (16.7%) was positive for the gene of SHV and CMY-2. The search for AmpC-type β-lactamases revealed phenotypic production by 20 isolates and, of these, two presented the EBC genotype. Through the analysis of the genetic profile, it was possible to observe identical lineages of species of enterobacteria in different collection sites of different newborns, showing possible cross-transmission; in addition, it was possible to identify the persistence characteristic of some strains within the unit. A univariate and multivariate analysis of Cox regression was used to verify the risk factors for colonization and infection by Gram-negative microorganisms. The results of the multivariate analysis revealed that, each week more at gestational age, the daily risk increases by 7.0% for the colonization of newborns by Gram-negative bacilli, while the use of antimicrobials in the first 72 hours decreases the daily risk in 35% of colonization by these pathogens. In addition, the production of biofilm by these microorganisms was associated with the persistence of colonization of the newborn by the same pathogen and increases by 75% the daily chance of the newborn evolving to infection, as well as the production of ESBL, which increases the daily chance 46.8 times for the newborn to have infection. The data of this research are of fundamental importance for public health, as they allow the knowledge of the molecular epidemiology of important species of enterobacteria in the NICU of a tertiary university hospital of high complexity, thus allowing the intensification of the monitoring and implementation of control measures and precaution
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Palavras-chave
Colonização, Infecção, β-lactamases de espectro estendido, AmpC, Carbapenemases, Colonization, Infection, Extended-spectrum β-lactamases,