Determinação de resíduos de antimicrobianos clinicamente importantes e verificação de microrganismos resistentes em águas das Represas Guarapiranga e Billings, SP
Data
2024-03-24
Autores
Santos, Felipe Rafael Guedes dos 
Orientadores
Moraes, Maria de Lourdes Leite de
Tipo
Dissertação de mestrado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
O uso indiscriminado de antibióticos na saúde humana e no crescimento de animais de criação levou à contaminação ambiental, especialmente das águas superficiais. Esse fato contribuiu para o aumento da resistência antimicrobiana, pois a exposição constante a subdoses de antibióticos pode criar uma pressão seletiva favorável à ocorrência e sobrevivência de bactérias resistentes, uma preocupação global da OMS. Novas abordagens que possibilitem o desenvolvimento de métodos de análise acessíveis e estudos associados à resistência antimicrobiana são prioridades para as agências ambientais estabelecerem programas de monitoramento desses compostos na água. Neste trabalho, foi desenvolvido um método utilizando cromatografia líquida de alta performance por absorção no ultravioleta e fluorescência (HPLC-DAD-FLD) para avaliação de antibióticos (amoxicilina, ceftazidima, cefalexina e ciprofloxacino) priorizados na região metropolitana de São Paulo e foi determinada a diversidade de espécies de bactérias gram-negativas resistentes a antimicrobianos clinicamente importantes nas águas das represas Billings e Guarapiranga. O método foi otimizado utilizando uma coluna RP18 Kinetex (150 mm x 4,6 mm, 5 µm). A fase móvel consistiu de água acidificada (pH 3,0):MeOH 25:75 (v/v), vazão 0,8 mLmin-1; 40ºC; detecção DAD a 237 nm e FLD (λex 350nm e λem 450nm). O LOD e LOQ variaram de 0,014 e
0,27 µg/mL e 0,044 e 0,83 µg/mL, respectivamente, para a faixa de 0,1-100 µg/mL. A avaliação da pré-concentração foi realizada por meio do SPE testando cartuchos HLB e Strata C-18 e a microextração em filme fino (TFME). As fases de extração PAN/C18; PAN/PS-DVB; PAN/C18/SCX e PAN/HLB foram testadas usando três solventes diferentes. PAN/PS-DVB apresentou melhor recuperação utilizando acetonitrila:água (25:75 v/v) como solvente de dessorção. Os testes de sensibilidade aos antimicrobianos foram realizados usando difusão em disco, de acordo com o BRCast. Espécies bacterianas foram selecionadas a partir de colônias recuperadas das represas e identificadas por MALDI-TOF. Cerca de 196 micro-organismos gram negativos foram isolados e identificados, com a presença de 6 diferentes famílias observadas. O perfil de sensibilidade das enterobactérias, considerado de prioridade crítica pela OMS, mostrou resistência a pelo menos um antimicrobiano de aminoglicosídeos, fluoroquinolonas e carbapenemas, respectivamente: Enterobacter spp. (24%, 54% e 67%), Escherichia coli (39%, 87% e 9,8%) e Klebsiella pneumoniae (100%, 50%, 77%). Foi observada triagem positiva para ESBL em isolados de E. coli (96%) e K. pneumoniae (46%). Essa abordagem permitiu a otimização dos esforços para o monitoramento de antibióticos na água, pois sua presença pode aumentar a taxa de transferência horizontal de genes de resistência entre diferentes cepas bacterianas, permitindo que bactérias sensíveis adquiram resistência aos antimicrobianos.
The indiscriminate use of antibiotics in human health and animal husbandry has led to environmental contamination, especially of surface waters. This fact has contributed to the increase in antimicrobial resistance, as constant exposure to subtherapeutic doses of antibiotics can create a selective pressure favorable to the occurrence and survival of resistant bacteria, a global concern of the WHO. New approaches that enable the development of accessible analysis methods and studies related to antimicrobial resistance are priorities for environmental agencies to establish monitoring programs for these compounds in water. In this work, a method was developed using high-performance liquid chromatography with ultraviolet absorption and fluorescence detection (HPLC-DAD-FLD) for the evaluation of prioritized antibiotics (amoxicillin, ceftazidime, cephalexin and ciprofloxacin) in the metropolitan region of São Paulo, and the diversity of species of gram-negative bacteria resistant to clinically important antimicrobials in the waters of the Billings and Guarapiranga reservoirs was determined. The method was optimized using a RP18 Kinetex column (150 mm x 4.6 mm, 5 μm). The mobile phase consisted of acidified water (pH 3.0):MeOH 25:75 (v/v), flow 0.8 mLmin-1; 40ºC; DAD detection at 237 nm and FLD (λex 350nm and λem 450nm). The LOD and LOQ varied from 0.014 and 0.27 μg/mL and 0.044 and 0.83 μg/mL, respectively, for the range of 0.1-100 μg/mL. The evaluation of the pre-concentration was performed by means of SPE, testing HLB and Strata C-18 cartridges and thin-film microextraction (TFME). The extraction phases PAN/C18; PAN/PS-DVB; PAN/C18/SCX, and PAN/HLB were tested using three different solvents. PAN/PS-DVB showed the best recovery using acetonitrile:water (25:75 v/v) as the desorption solvent. Antimicrobial sensitivity tests were performed using disk diffusion, according to the BRCast. Bacterial species were selected from colonies recovered from the reservoirs and identified by MALDI-TOF. About 196 gram-negative microorganisms were isolated and identified, with the presence of 6 different families observed. The sensitivity profile of the enterobacteria, considered of critical priority by the WHO, showed resistance to at least one antimicrobial of aminoglycosides, fluoroquinolones, and carbapenems, respectively: Enterobacter spp. (24%, 54%, and 67%), Escherichia coli (39%, 87%, and 9.8%) and Klebsiella pneumoniae (100%, 50%, 77%). Positive screening for ESBL in E. coli (96%) and K. pneumoniae (46%) isolates was observed. This approach allowed the optimization of efforts for monitoring antibiotics in water, as their presence can increase the rate of horizontal gene transfer of resistance among different bacterial strains, allowing sensitive bacteria to acquire resistance to antimicrobials.
The indiscriminate use of antibiotics in human health and animal husbandry has led to environmental contamination, especially of surface waters. This fact has contributed to the increase in antimicrobial resistance, as constant exposure to subtherapeutic doses of antibiotics can create a selective pressure favorable to the occurrence and survival of resistant bacteria, a global concern of the WHO. New approaches that enable the development of accessible analysis methods and studies related to antimicrobial resistance are priorities for environmental agencies to establish monitoring programs for these compounds in water. In this work, a method was developed using high-performance liquid chromatography with ultraviolet absorption and fluorescence detection (HPLC-DAD-FLD) for the evaluation of prioritized antibiotics (amoxicillin, ceftazidime, cephalexin and ciprofloxacin) in the metropolitan region of São Paulo, and the diversity of species of gram-negative bacteria resistant to clinically important antimicrobials in the waters of the Billings and Guarapiranga reservoirs was determined. The method was optimized using a RP18 Kinetex column (150 mm x 4.6 mm, 5 μm). The mobile phase consisted of acidified water (pH 3.0):MeOH 25:75 (v/v), flow 0.8 mLmin-1; 40ºC; DAD detection at 237 nm and FLD (λex 350nm and λem 450nm). The LOD and LOQ varied from 0.014 and 0.27 μg/mL and 0.044 and 0.83 μg/mL, respectively, for the range of 0.1-100 μg/mL. The evaluation of the pre-concentration was performed by means of SPE, testing HLB and Strata C-18 cartridges and thin-film microextraction (TFME). The extraction phases PAN/C18; PAN/PS-DVB; PAN/C18/SCX, and PAN/HLB were tested using three different solvents. PAN/PS-DVB showed the best recovery using acetonitrile:water (25:75 v/v) as the desorption solvent. Antimicrobial sensitivity tests were performed using disk diffusion, according to the BRCast. Bacterial species were selected from colonies recovered from the reservoirs and identified by MALDI-TOF. About 196 gram-negative microorganisms were isolated and identified, with the presence of 6 different families observed. The sensitivity profile of the enterobacteria, considered of critical priority by the WHO, showed resistance to at least one antimicrobial of aminoglycosides, fluoroquinolones, and carbapenems, respectively: Enterobacter spp. (24%, 54%, and 67%), Escherichia coli (39%, 87%, and 9.8%) and Klebsiella pneumoniae (100%, 50%, 77%). Positive screening for ESBL in E. coli (96%) and K. pneumoniae (46%) isolates was observed. This approach allowed the optimization of efforts for monitoring antibiotics in water, as their presence can increase the rate of horizontal gene transfer of resistance among different bacterial strains, allowing sensitive bacteria to acquire resistance to antimicrobials.
Descrição
Citação
SANTOS, Felipe Rafael Guedes dos. Determinação de resíduos de antimicrobianos clinicamente importantes e verificação de microrganismos resistentes em águas das represas Guarapiranga e Billings, SP. 2024. 102 f. Dissertação (Mestrado em Análise Ambiental Integrada) - Instituto de Ciências Ambientais, Químicas e Farmacêuticas, Universidade Federal de São Paulo, Diadema, 2024.