Relações clonais entre Pseudomonas aeruginosa multidroga resistentes de origem clínica e do efluente hospitalar

Submitted by Repositório Arca ([email protected]) on 2019-05-09T19:02:48Z No. of bitstreams: 1 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5)Approved for entry into archive by Simone Teixeira ([email protected]) on 2019-10-10T14:20:34Z (GMT) No. of bitstreams: 3 Tese_Catia_ Aparecida_Chaia_Miranda.pdf: 7790102 bytes, checksum: f40378f908acd0b91b42360c99680700 (MD5) Tese_Catia_ Aparecida_Chaia_Miranda.pdf: 7790102 bytes, checksum: f40378f908acd0b91b42360c99680700 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5)Made available in DSpace on 2019-10-10T14:20:34Z (GMT). No. of bitstreams: 3 Tese_Catia_ Aparecida_Chaia_Miranda.pdf: 7790102 bytes, checksum: f40378f908acd0b91b42360c99680700 (MD5) Tese_Catia_ Aparecida_Chaia_Miranda.pdf: 7790102 bytes, checksum: f40378f908acd0b91b42360c99680700 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2015Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde. Rio de Janeiro, RJ, Brasil.O lançamento de efluentes hospitalares, sem pré-tratamento adequado, em corpos receptores, é motivo de preocupação devido à disseminação de poluentes químicos e biológicos aos corpos receptores. A presença de Pseudomonas aeruginosa multidroga resistentes está associada à sua ampla colonização nos hospitais e consequentemente nos efluentes e ao meio ambiente. O objetivo deste estudo foi investigar a relação clonal entre os isolados de Pseudomonas aeruginosa de origem clínica e do efluente hospitalar, e a possível associação clonal à diminuição da susceptibilidade aos antibióticos, principalmente em relação aos beta-lactâmicos. Cento e setenta e sete isolados de P. aeruginosa recuperados do hospital (n=136) e seu efluente (n=41), foram identificados fenotipicamente e certificados pela amplificação do gene 16S rRNA. Quanto ao perfil de susceptibilidade, os isolados foram analisados frente a 17 antibióticos pelo Método de disco difusão. Dos 41 isolados da ETEH, 88% foram resistentes à fosfomicina, seguidos por ticarcilina/ácido clavulânico (71%) e ceftriaxona (63%). Os 136 isolados clínicos apresentaram maior valor de resistência a fosfomicina (100%), seguido por cefotaxima (79%) e ceftriaxona (76%). A produção de beta-lactamase foi verificada por meio de cultivo em Chromoagar – ESBL e pelo teste de Carba NP. Os genes codificadores de ESBL (blaPER, blaVEB, blaSHV, blaCTX-M-1, blaCTX-M-2, blaCTX-M-8, blaCTX-M-9, blaCTX-M-25, blaTEM e blaGES), de MBL (blaIMP, blaVIM, blaSPM e blaNDM) e de KPC (blaKPC) foram detectados pela PCR. Cento e onze isolados (63%) resistentes a pelo menos um antibiótico pertencente a três ou mais classes foram classificados como multirresistentes. Destes, 37 (33%) foram classificados como Multidroga Resistentes (MDR) e 74 (67%) Extensivamente Droga Resistentes (XDR). A relação genética dos isolados foi determinada através da ERIC-PCR e MLST, agrupando em 69 perfis distintos (HMLJ (n=49; ETEH (n=20)) e 51 ST (HMLJ (n=36); ETEH (n=15)), respectivamente. O ST 244 foi o único presente nos dois ambientes analisados. Os dados gerados a partir da árvore de MST, com isolados dos dois ambientes, mostraram que o ST595 e o ST1941 compartilham cinco alelos idênticos com o ST244 e devem ser incluídos no Complexo Clonal 244 (CC244). O CC244 demonstrou grande potencial patogênico deste clone por carrear cepas MDR e XDR de diferentes fontes, tais como clínica, água e efluente hospitalar. Nossos resultados nos permitem concluir que o tratamento terciário dispensado ao efluente hospitalar não foi totalmente eficiente, uma vez que foi demonstrado um aumento de linhagens XDR no efluente tratado. A grande diversidade genética de Pseudomonas aeruginosa recuperadas do hospital e seu efluente, constantemente liberadas no sistema aquático, contribui com a disseminação de genes de resistência entre microrganismos que compartilham o mesmo meio. Estas linhagens contribuem para a disseminação de microrganismos e genes de resistência podendo gerar impactos negativos ao meio ambiente e à saúde humana.The disposal of hospital waste without adequate pre-treatment in the environment is a concern due to the spread of chemical and biological pollutants to receiving water streams. Distribution of multidrug resistant Pseudomonas aeruginosa is associated with a broad colonization in hospitals and subsequent transmission to the effluents and the environment. The aim of this study was to investigate the clonal relationship among Pseudomonas aeruginosa isolates from clinical origin and hospital sewage, and the potential clonal association with decreased susceptibility to antibiotics, especially for β-lactams. One hundred seventy-seven isolates of P. aeruginosa, recovered from a hospital (n=136) and its HWTP (n=41), were identified phenotypically and confirmed by amplification of the 16S rRNA gene. Susceptibility profiles of isolates were analyzed against 17 antibiotics by disk diffusion Method. Of the 41 isolates of HWTP, 88% were resistant to fosfomycin, followed by ticarcillin/clavulanic acid (71%) and ceftriaxone (63%). One hundred thirty-six clinical isolates had greater value of resistance to fosfomycin (100%), followed by cefotaxime (79%) and ceftriaxone (76%). Production of beta-lactamase was investigated by screening in Chromoagar - ESBL and Carba NP test. Genes encoding ESBL (blaPER, blaVEB, blaSHV, blaCTX-M-1, blaCTX-M-2, blaCTX-M-8, blaCTX-M-9, blaCTX-M-25, blaTEM and blaGES), MBL (blaIMP, blaVIM, blaSPM and blaNDM) and KPC (blaKPC) were screened by PCR. One hundred and eleven isolated (63%) were resistant to at least one antibiotic belonging to three or more classes and were classified as multiresistant. Thirty seven (33%) were classified as MDR and 74 (67%) XDR. Genetic relationships of the isolates were determined by ERIC-PCR and MLST, clustering into 69 distinct profile (Hospital (n=49; HWTP (n=20)) and 51 ST (Hospital (n=36); HWTP (n=15)), respectively. Sequence Type 244 was the only ST present in both environments analyzed. Data generated from the MLST tree with isolates from the two environments showed that ST595 and ST1941 share five identical alleles with ST244 and should be included in Clonal Complex 244 (CC244). CC244 showed a great pathogenic potential carrying MDR and XDR strains from different sources such as clinical, water and hospital effluent. Our findings allow us to conclude that the tertiary treatment dispensed to the hospital effluents was not very efficient, because of the high proportion of XDR strains found in treated effluent. The great genetic diversity of Pseudomonas aeruginosa recovered from the hospital and its effluent, constantly released into the water system, contributes to the spread of resistance genes between organisms sharing the same environment. These strains contribute to the spread of microorganisms and resistance genes, which may generate negative impacts on the environment and human health

Isolation of aerobic cultivable cellulolytic bacteria from different regions of the gastrointestinal tract of giant land snail Achatina fulica

Submitted by Alexandre Sousa ([email protected]) on 2016-03-10T17:24:02Z No. of bitstreams: 1 fmicb-06-00860.pdf: 1456467 bytes, checksum: 8046b0dd63675ecfbdcf95d9a719403a (MD5)Approved for entry into archive by Alexandre Sousa ([email protected]) on 2016-03-10T17:45:42Z (GMT) No. of bitstreams: 1 fmicb-06-00860.pdf: 1456467 bytes, checksum: 8046b0dd63675ecfbdcf95d9a719403a (MD5)Made available in DSpace on 2016-03-10T17:45:42Z (GMT). No. of bitstreams: 1 fmicb-06-00860.pdf: 1456467 bytes, checksum: 8046b0dd63675ecfbdcf95d9a719403a (MD5) Previous issue date: 2015Instituto Nacional de Metrologia, Qualidade e Tecnologia. Rio de Janeiro, RJ, Brasil / Universidade Federal do Rio de Janeiro. Instituto de Biofísica Carlos Chagas Filho. Rio de Janeiro, RJ, Brasil.Instituto Nacional de Metrologia, Qualidade e Tecnologia. Rio de Janeiro, RJ, Brasil.Instituto Nacional de Metrologia, Qualidade e Tecnologia. Rio de Janeiro, RJ, Brasil.Instituto Nacional de Metrologia, Qualidade e Tecnologia. Rio de Janeiro, RJ, Brasil / Centro Universitário Estadual da Zona Oeste. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde. Departamento de Microbiologia. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde. Departamento de Microbiologia. Rio de Janeiro, RJ, Brasil.Instituto Nacional de Metrologia, Qualidade e Tecnologia. Rio de Janeiro, RJ, Brasil / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Departamento de Bioquímica e Biologia Molecular. Rio de Janeiro, RJ, Brasil.Instituto Nacional de Metrologia, Qualidade e Tecnologia. Rio de Janeiro, RJ, Brasil / Universidade Federal do Rio de Janeiro. Instituto de Biofísica Carlos Chagas Filho. Rio de Janeiro, RJ, Brasil.Instituto Nacional de Metrologia, Qualidade e Tecnologia. Rio de Janeiro, RJ, Brasil / Universidade Federal do Rio de Janeiro. Instituto de Biofísica Carlos Chagas Filho. Rio de Janeiro, RJ, Brasil.The enzymatic hydrolysis of cellulose by cellulases is one of the major limiting steps in the conversion of lignocellulosic biomass to yield bioethanol. To overcome this hindrance, significant efforts are underway to identify novel cellulases. The snail Achatina fulica is a gastropod with high cellulolytic activity, mainly due to the abundance of glycoside hydrolases produced by both the animal and its resident microbiota. In this study, we partially assessed the cellulolytic aerobic bacterial diversity inside the gastrointestinal tract of A. fulica by culture-dependent methods and evaluated the hydrolytic repertoire of the isolates. Forty bacterial isolates were recovered from distinct segments of the snail gut and identified to the genus level by 16S rRNA gene sequence analysis. Additional phenotypic characterization was performed using biochemical tests provided by the Vitek2 identification system. The overall enzymatic repertoire of the isolated strains was investigated by enzymatic plate assays, containing the following substrates: powdered sugarcane bagasse, carboxymethylcellulose (CMC), p-nitrophenyl-beta-D-glucopyranoside (pNPG), p-nitrophenyl-beta-D-cellobioside (pNPC), 4-methylumbelliferyl-beta-D-glucopyranoside (MUG), 4-methylumbelliferyl-beta-D-cellobioside (MUC), and 4-methylumbelliferyl-beta-D-xylopyranoside (MUX). Our results indicate that the snail A. fulica is an attractive source of cultivable bacteria that showed to be valuable resources for the production of different types of biomass-degrading enzymes

Detection of antimicrobial resistance genes in beta-lactamase- and carbapenemase-producing Klebsiella pneumoniae by patient surveillance cultures at an intensive care unit in Rio de Janeiro, Brazil

ABSTRACT Introduction: The increasing incidence of multi-resistant microorganisms has been considered a public health problem. One of the routines included in hospital practice is the screening of colonized and/or infected patients. Objective: The aim of this study was to evaluate the genetic variability and clonal relationships of extended-spectrum beta-lactamase (ESBL)-producing K. pneumoniae, from surveillance cultures, at an intensive care unit, in Rio de Janeiro, Brazil. Material and methods: Seventy K. pneumoniae isolates were obtained from rectal swabs (March 2013 to March 2014). Antimicrobial susceptibility was assessed by VITEK 2 System. Resistant genes blaSHV, blaTEM, blaOXA-1, blaKPC, blaOXA-48, blaCTX-M-15, blaVIM, blaIMP and blaNDM were investigated by polymerase chain reaction (PCR); genetic diversity, by Enterobacterial Repetitive Intergenic Consensus-PCR (ERIC-PCR). Results: Strains showed high resistance rates to cefepime (94%), ceftazidime (96%), ertapenem (61%), imipenem (54%) meropenem (43%) and ciprofloxacin (69%). The most prevalent genes were blaSHV (69%), blaTEM (63%), blaOXA-1 (60%), blaKPC (57%), blaCTX-M-15 (47%), blaOXA-48 (16%). Genes blaVIM, blaIMP and blaNDM were not detected. Twenty nine profiles of resistance genes were observed, with 23% carrying at least five genes. A great genetic diversity (68 ERIC profiles) was also observed among the strains. Conclusion: Although no clonal relationship was observed within the isolates, this study revealed alarming data on the antimicrobial resistance deficiently monitored for preventive purposes in Brazil. Our data allow us to conclude that the inclusion of surveillance cultures in health facilities is a recommended strategy aiming particularly at preventing the spread of resistance genes in the hospital environment and, consequently, reducing morbidity and mortality

Detection of antimicrobial resistance genes in betalactamase- and carbapenemase-producing Klebsiella pneumoniae

ABSTRACT Introduction: The increasing incidence of multi-resistant microorganisms has been considered a public health problem. One of the routines included in hospital practice is the screening of colonized and/or infected patients. Objective: The aim of this study was to evaluate the genetic variability and clonal relationships of extended-spectrum beta-lactamase (ESBL)-producing K. pneumoniae, from surveillance cultures, at an intensive care unit, in Rio de Janeiro, Brazil. Material and methods: Seventy K. pneumoniae isolates were obtained from rectal swabs (March 2013 to March 2014). Antimicrobial susceptibility was assessed by VITEK 2 System. Resistant genes blaSHV, blaTEM, blaOXA-1, blaKPC, blaOXA-48, blaCTX-M-15, blaVIM, blaIMP and blaNDM were investigated by polymerase chain reaction (PCR); genetic diversity, by Enterobacterial Repetitive Intergenic Consensus-PCR (ERIC-PCR). Results: Strains showed high resistance rates to cefepime (94%), ceftazidime (96%), ertapenem (61%), imipenem (54%) meropenem (43%) and ciprofloxacin (69%). The most prevalent genes were blaSHV (69%), blaTEM (63%), blaOXA-1 (60%), blaKPC (57%), blaCTX-M-15 (47%), blaOXA-48 (16%). Genes blaVIM, blaIMP and blaNDM were not detected. Twenty nine profiles of resistance genes were observed, with 23% carrying at least five genes. A great genetic diversity (68 ERIC profiles) was also observed among the strains. Conclusion: Although no clonal relationship was observed within the isolates, this study revealed alarming data on the antimicrobial resistance deficiently monitored for preventive purposes in Brazil. Our data allow us to conclude that the inclusion of surveillance cultures in health facilities is a recommended strategy aiming particularly at preventing the spread of resistance genes in the hospital environment and, consequently, reducing morbidity and mortality

Phylogenetic tree of bacterial clones from environmental bulk DNA.

<p>Reference sequences from GenBank are shown in bold. OTUs were defined by using a distance level of 3% by the furthest neighbor algorithm in MOTHUR. Access number from each OTU is displayed. Tree topology is based on neighbor joining and bootstrap analysis was performed with 1000 replications. Bootstrap value <50 are not shown.</p

Physico-chemical data from sampling stations.¹

*<p><i>MiliSiemens</i> – mS/cm; ** Nefelométric Turbidity Unity – NTU; *** mg/L.</p>1<p>Additional information on physico-chemical parameters of the sampling sites can be found at INEA (<a href="http://www.inea.rj.gov.br" target="_blank">http://www.inea.rj.gov.br</a>).</p

Phylogenetic tree of bacterial clones from BHI enriched culture.

<p>Reference sequences from GenBank showcased in bold. OTUs were defined by using a distance level of 3% by using the furthest neighbor algorithm in MOTHUR. Access number from each OTU is displayed. Tree topology is based on neighbor joining and bootstrap analysis was performed with 1000 replications. Bootstrap value <50 are not shown.</p

Bacterial taxonomic classes in environmental and cultured libraries.

<p>Sequences obtained from 16S rRNA gene libraries retrieved from environmental samples (black) and from enrichment cultures (grey) taxonomic assignment was performed through the RDP Classifier tool.</p

Antibiotic resistance profile of isolated bacteria.

1<p>Salt Manitol Agar.</p>2<p>Cetrimide Agar.</p>3<p>Blood Agar.</p>4<p>Mac Conkey Agar.</p><p>R, resistant and S, susceptive to antibiotic.</p

Environmental distribution of bacterial orders.

<p>Canonical correspondence analysis of sampling sites libraries from JM(e), BL(e) and JOA(e). Environmental parameters included in this analysis are: total phosphorus (Ptotal), ammonia (NH₃), turbidity (Turb), nitrite (NO₂⁻), temperature (Temp), salinity (Sal), pH, nitrate (NO₃⁻), dissolved oxygen (OD) and orthophosphate (PO₄⁻).</p