Análise microbiológica de isolados de Pseudomonas spp. provenientes das cinco regiões geográficas brasileiras sob uma perspectiva de saúde única
Data
2024-11-29
Autores
Neves, Raíssa Fidelis Baêta 
Orientadores
Da Silva, Rodrigo Cayô
Tipo
Dissertação de mestrado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
A relevância dos genes de resistência aos antimicrobianos (ARGs) mediados por elementos genéticos móveis (MGE) não se restringe à sua presença em patógenos clínicos, mas envolve todas as bactérias, sejam elas patogênicas, comensais ou ambientais, capazes de atuar como reservatório desses genes. Devido à sua versatilidade metabólica e capacidade de colonizar diversos ambientes, Pseudomonas se destaca como um gênero bacteriano de grande interesse, relevante no contexto clínico, ambiental e biotecnológico. O presente estudo teve por objetivo identificar, analisar e caracterizar cepas de Pseudomonas spp. isoladas em diferentes matrizes ambientais nas cinco regiões geográficas brasileiras, sob os preceitos de saúde única, investigando o resistoma bacteriano. Um total de 462 isolados de Pseudomonas spp. foram recuperados de amostras de solo, água e fezes (humanas e animais) coletadas pelos centros participantes do Grupo GUARANI. Os isolados foram cultivados em meio cromogênico após incubação em meio de cultura líquido suplementado com ceftriaxona, ceftazidima e meropeném (2 µg/mL cada), acrescidos de vancomicina (4 µg/mL). A identificação bacteriana foi realizada por espectrometria de massa, seguida por testes de sensibilidade pelo método de diluição em ágar (BrCAST/EUCAST) utilizando os mesmos antimicrobianos da seleção inicial. Foi realizada a triagem molecular de ARGs para β-lactâmicos, aminoglicosídeos e fluoroquinolonas pela técnica de reação em cadeia da polimerase (PCR). Os ARGs detectados foram confirmados por sequenciamento de Sanger e a análise clonal foi realizada por ERIC-PCR. Os isolados carreadores de ARGs com resultados confirmados pelo sequenciamento de Sanger tiveram as CIMs determinadas pela técnica de diluição em ágar para os principais antimicrobianos de importância clínica. A sensibilidade à piperacilina/tazobactam foi determinada pelo método de disco-difusão.
Os isolados recuperados estavam distribuídos entre as matrizes da seguinte forma: fezes de suínos (n=138), fezes humanas (n=98), solo (n=72), fezes de frango (n=60), água (n=45) e fezes de bovinos (n=45). As cidades de Ji-Paraná (n=137) e Fortaleza (n=115) forneceram o maior número de isolados, seguidos por Blumenau (n=94), Bragança Paulista (n=60), Castanhal (n=28) e Dourados (n=28). As espécies predominantes foram P. aeruginosa (43%) e P. putida (16%). Um total de 14 diferentes ARGs foram detectados em 92 isolados (19,9%). Estes incluíram blaCTX-M-1/2-like (n=3), blaKPC-like (n=11), blaBIC-like (n=2), blaBKC-like (n=7), blaOXA-1-like (n=1), blaOXA-23-like (n=3), blaOXA-24/40-like (n=1), blaOXA-46-like (n=8) e blaGIM-like (n=9) entre os ARGs para β-lactâmicos; aac(6')-Ib-cr (n=4), crpP (n=41) e qnrD (n=5) para resistência às fluoroquinolonas; e aac(6')-Ib (n=7) e rmtE (n=4) para aminoglicosídeos. Alguns dos ARGs detectados estavam restritos a algumas localidades, tais como blaCTX-M-1/2-like em Bragança Paulista e blaBIC-like em Blumenau, enquanto outros foram encontrados em mais de uma localidade, como foi o caso de blaGIM-like e qnrD (Ji-Paraná e Fortaleza), rmtE (Blumenau e Ji-Paraná), blaBKC-like (Blumenau e Fortaleza) e blaKPC-like (Castanhal, Blumenau, Fortaleza e Ji-Paraná). A análise clonal revelou poucos isolados relacionados, geralmente originários da mesma amostra submetida a diferentes pressões seletivas. A fase final dos testes de sensibilidade ficou prejudicada em decorrência da perda dos ARGs detectados por PCR e confirmados por sequenciamento de Sanger em muitos isolados. Ainda assim, o estudo revelou uma diversidade de ARGs em diferentes matrizes e regiões do Brasil, incluindo genes ainda não descritos em isolados de Pseudomonas spp. no país, reforçando seu papel como reservatórios de resistência. O estado do Ceará se destacou pela grande diversidade de ARGs encontrados e confirmados por sequenciamento. Além disso, apesar da alta frequência do gene crpP verificada na maioria das regiões brasileiras, o fenótipo de resistência obtido corrobora com os estudos que sugerem que a proteína CrpP não é capaz de reduzir a sensibilidade às fluoroquinolonas nas espécies de Pseudomonas. O estudo destaca o papel crucial dos plasmídeos na disseminação da RAM, reforçando a necessidade de investigações sobre sua propagação sob uma ótica capaz de integrar a saúde humana, animal e ambiental.
The relevance of antimicrobial resistance genes (ARGs) mediated by mobile genetic elements (MGEs) is not limited to their presence in clinical pathogens but extends to all bacteria—whether pathogenic, commensal, or environmental—that can act as reservoirs of these genes. Due to their metabolic versatility and ability to colonize diverse environments, Pseudomonas emerges as a bacterial genus of significant interest, relevant in clinical, environmental, and biotechnological contexts. This study aimed to identify, analyze, and characterize Pseudomonas spp. strains isolated from different environmental matrices across Brazil's five geographical regions under the One Health framework, investigating the bacterial resistome. A total of 462 Pseudomonas spp. isolates were recovered from soil, water, and fecal samples (human and animal) collected by the centers participating in the GUARANI Group. The isolates were cultured on chromogenic media following incubation in liquid culture media supplemented with ceftriaxone, ceftazidime, and meropenem (2 µg/mL each), along with vancomycin (4 µg/mL). Bacterial identification was conducted using mass spectrometry, followed by susceptibility testing using the agar dilution method (BrCAST/EUCAST) with the same antimicrobials as in the initial selection. Molecular screening for β-lactam, aminoglycoside, and fluoroquinolone ARGs was performed via polymerase chain reaction (PCR). Detected ARGs were confirmed by Sanger sequencing, and clonal analysis was performed using ERIC-PCR. Isolates carrying ARGs confirmed by Sanger sequencing underwent MIC determination using the agar dilution method for key clinically relevant antimicrobials. Susceptibility to piperacillin/tazobactam was evaluated using the disk diffusion method. The recovered isolates were distributed among the matrices as follows: swine feces (n=138), human feces (n=98), soil (n=72), poultry feces (n=60), water (n=45), and cattle feces (n=45). The cities of Ji-Paraná (n=137) and Fortaleza (n=115) yielded the highest number of isolates, followed by Blumenau (n=94), Bragança Paulista (n=60), Castanhal (n=28), and Dourados (n=28). The predominant species were P. aeruginosa (43%) and P. putida (16%). A total of 14 different ARGs were detected in 92 isolates (19.9%). These included blaCTX-M-1/2-like (n=3), blaKPC-like (n=11), blaBIC-like (n=2), blaBKC-like (n=7), blaOXA-1-like (n=1), blaOXA-23-like (n=3), blaOXA-24/40-like (n=1), blaOXA-46-like (n=8), and blaGIM-like (n=9) among β-lactam ARGs; aac(6')-Ib-cr (n=4), crpP (n=41), and qnrD (n=5) for fluoroquinolone resistance; and aac(6')-Ib (n=7) and rmtE (n=4) for aminoglycosides. Some ARGs were restricted to specific locations, such as blaCTX-M-1/2-like in Bragança Paulista and blaBIC-like in Blumenau, while others were found in multiple locations, including blaGIM-like and qnrD (Ji-Paraná and Fortaleza), rmtE (Blumenau and Ji-Paraná), blaBKC-like (Blumenau and Fortaleza), and blaKPC-like (Castanhal, Blumenau, Fortaleza, and Ji-Paraná). Clonal analysis revealed few related isolates, usually originating from the same sample subjected to different selective pressures. The final phase of susceptibility testing was impaired due to the loss of ARGs detected by PCR and confirmed by Sanger sequencing in many isolates. Nonetheless, the study unveiled a diversity of ARGs across different matrices and regions in Brazil, including genes not previously described in Pseudomonas spp. isolates in the country, reinforcing their role as resistance reservoirs. Ceará stood out for the high diversity of ARGs detected and confirmed by sequencing. Moreover, despite the high frequency of the crpP gene observed in most Brazilian regions, the resistance phenotype obtained aligns with studies suggesting that the CrpP protein does not reduce fluoroquinolone susceptibility in Pseudomonas species. This study highlights the crucial role of plasmids in the dissemination of antimicrobial resistance, emphasizing the need for further investigations into their propagation through an integrated human, animal, and environmental health perspective.
The relevance of antimicrobial resistance genes (ARGs) mediated by mobile genetic elements (MGEs) is not limited to their presence in clinical pathogens but extends to all bacteria—whether pathogenic, commensal, or environmental—that can act as reservoirs of these genes. Due to their metabolic versatility and ability to colonize diverse environments, Pseudomonas emerges as a bacterial genus of significant interest, relevant in clinical, environmental, and biotechnological contexts. This study aimed to identify, analyze, and characterize Pseudomonas spp. strains isolated from different environmental matrices across Brazil's five geographical regions under the One Health framework, investigating the bacterial resistome. A total of 462 Pseudomonas spp. isolates were recovered from soil, water, and fecal samples (human and animal) collected by the centers participating in the GUARANI Group. The isolates were cultured on chromogenic media following incubation in liquid culture media supplemented with ceftriaxone, ceftazidime, and meropenem (2 µg/mL each), along with vancomycin (4 µg/mL). Bacterial identification was conducted using mass spectrometry, followed by susceptibility testing using the agar dilution method (BrCAST/EUCAST) with the same antimicrobials as in the initial selection. Molecular screening for β-lactam, aminoglycoside, and fluoroquinolone ARGs was performed via polymerase chain reaction (PCR). Detected ARGs were confirmed by Sanger sequencing, and clonal analysis was performed using ERIC-PCR. Isolates carrying ARGs confirmed by Sanger sequencing underwent MIC determination using the agar dilution method for key clinically relevant antimicrobials. Susceptibility to piperacillin/tazobactam was evaluated using the disk diffusion method. The recovered isolates were distributed among the matrices as follows: swine feces (n=138), human feces (n=98), soil (n=72), poultry feces (n=60), water (n=45), and cattle feces (n=45). The cities of Ji-Paraná (n=137) and Fortaleza (n=115) yielded the highest number of isolates, followed by Blumenau (n=94), Bragança Paulista (n=60), Castanhal (n=28), and Dourados (n=28). The predominant species were P. aeruginosa (43%) and P. putida (16%). A total of 14 different ARGs were detected in 92 isolates (19.9%). These included blaCTX-M-1/2-like (n=3), blaKPC-like (n=11), blaBIC-like (n=2), blaBKC-like (n=7), blaOXA-1-like (n=1), blaOXA-23-like (n=3), blaOXA-24/40-like (n=1), blaOXA-46-like (n=8), and blaGIM-like (n=9) among β-lactam ARGs; aac(6')-Ib-cr (n=4), crpP (n=41), and qnrD (n=5) for fluoroquinolone resistance; and aac(6')-Ib (n=7) and rmtE (n=4) for aminoglycosides. Some ARGs were restricted to specific locations, such as blaCTX-M-1/2-like in Bragança Paulista and blaBIC-like in Blumenau, while others were found in multiple locations, including blaGIM-like and qnrD (Ji-Paraná and Fortaleza), rmtE (Blumenau and Ji-Paraná), blaBKC-like (Blumenau and Fortaleza), and blaKPC-like (Castanhal, Blumenau, Fortaleza, and Ji-Paraná). Clonal analysis revealed few related isolates, usually originating from the same sample subjected to different selective pressures. The final phase of susceptibility testing was impaired due to the loss of ARGs detected by PCR and confirmed by Sanger sequencing in many isolates. Nonetheless, the study unveiled a diversity of ARGs across different matrices and regions in Brazil, including genes not previously described in Pseudomonas spp. isolates in the country, reinforcing their role as resistance reservoirs. Ceará stood out for the high diversity of ARGs detected and confirmed by sequencing. Moreover, despite the high frequency of the crpP gene observed in most Brazilian regions, the resistance phenotype obtained aligns with studies suggesting that the CrpP protein does not reduce fluoroquinolone susceptibility in Pseudomonas species. This study highlights the crucial role of plasmids in the dissemination of antimicrobial resistance, emphasizing the need for further investigations into their propagation through an integrated human, animal, and environmental health perspective.
Descrição
Citação
NEVES, Raíssa Fidelis Baêta. Análise microbiológica de isolados de Pseudomonas spp. provenientes das cinco regiões geográficas brasileiras sob uma perspectiva de saúde única. 2024. 177 f. Dissertação (Mestrado em Infectologia) – Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP). São Paulo, 2024.