Efecto del tratamiento de las aguas servidas del Gran Concepción, en la carga de Enterobacterales resistentes a cefalosporinas de tercera generación y/o carbapenémicos, y de genes codificantes de β-lactamasas de espectro extendido y/o carbapenemasas.
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Date
2025
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Universidad de Concepción
Abstract
La resistencia a cefalosporinas de tercera generación (C3G), y a carbapenémicos mediada por β-lactamasas de espectro extendido (BLEE) y, por carbapenemasas respectivamente, constituye un creciente problema a nivel mundial, por ser mecanismos de resistencia de prioridad critica, presentes principalmente, aunque no exclusivamente en Enterobacterales. La resistencia a tales antibacterianos se ha estudiado tradicionalmente durante décadas, desde una perspectiva antropocéntrica. Sin embargo, las evidencias más recientes indican que la fauna doméstica y silvestre, la flora, así como otros sustratos naturales, también se han perjudicado por dicho problema, por lo cual, se ha incentivado su estudio desde el enfoque “One health”.
Las plantas de tratamiento de aguas servidas (PTAS) constituyen importantes reservorios de múltiples contaminantes, en las cuales se han pesquisado por varios autores a nivel internacional, bacterias resistentes productoras de BLEE y carbapenemasas, así como genes codificantes para tales enzimas, directamente en muestras de aguas servidas (AS) y de lodos. No obstante, en Chile existen escasos reportes sobre resistencia a antibióticos en AS, por lo que el objetivo de esta tesis fue; evaluar diferencias en las cargas de Enterobacterales resistentes a C3G y/o carbapenémicos y en la carga de genes que codifican BLEE y/o carbapenemasas, por acción del tratamiento de las AS del Gran Concepción.
Se obtuvieron 8 muestras de AS, (4 de afluente (AF) y 4 de efluente (EF)), entre Enero y Julio de 2022, las que se sembraron en agar R2A, MacConkey (MCC), MacConkey suplementado con ceftriaxona (MCC+C) y MacConkey suplementado con meropenem (MCC+M), empleando diluciones seriadas para estimar recuentos bacterianos. Se aislaron un total de 205 Bacilos Gram negativos (BGN), de los cuales 50 correspondieron a Enterobacterales (48 de AF y 2 EF). A estos últimos, se les determinó la susceptibilidad a diferentes antibióticos mediante antibiograma, la detección fenotípica y genotípica de BLEE y carbapenemasas. Además, directamente desde las mismas muestras de AF y EF, se llevó a cabo detección genotípica de dichas enzimas.
Los recuentos bacterianos se redujeron significativamente en todas las muestras de AF en comparación a las de EF, y en todos los medios de cultivo empleados, sobre todo, los recuentos totales, que disminuyeron entre 5 a 6 logaritmos. De los Enterobacterales aislados, las especies más frecuentes fueron E. coli (16/50), K. pneumoniae complex (12/50), y K. oxytoca complex (6/50). En las cepas de Enterobacterales obtenidos de AF (48/50), 7 cepas demostraron índices MAR > 0,5, de las cuales; dos destacaron por poseer MAR de 0,81 y 0,75, presentando resistencia a 13 y 12 antibióticos de 16 probados. Otras tres presentaron un MAR de 0,63, (resistentes a 10/16 antibacterianos). Mientras que las dos restantes poseyeron un MAR de 0,56 (resistentes a 10/16 antibacterianos). Además, 8 cepas presentaron perfiles MDR.
En la detección fenotípica de BLEE se obtuvo positividad en 23/48 cepas de AF y 1/2 cepas de EF, mientras que 3/48 de AF resultaron positivas para detección fenotípica de carbapenemasas. En relación con los genes codificantes de BLEE, estos se detectaron en 23/48 cepas de AF y en 1/2 de EF, siendo blaCTX-M-1-like el prevalente entre los 50 Enterobacterales indagados. Luego, en 3/48 cepas de AF se detectó el gen codificante de carbapenemasas blaKPC-2. Mientras que, en las cepas de EF, no se detectaron genes codificantes de carbapenemasas. Por otra parte, directo de las AS, se detectaron los genes blaCTX-M-1-like, blaKPC, y blaOXA-48-like en AF y EF, mientras que blaVIM se encontró solo en las muestras de AF.
La reducción de los recuentos bacterianos demostró la eficacia del tratamiento de AS del Gran Concepción sobre la vida bacteriana, pero no así sobre los genes codificantes de BLEE y carbapenemasas disueltos en las AS. Los Enterobacterales estudiados representaron un bajo porcentaje (27%) de los 205 BGN totales, por lo que, los restantes 155 (73%) organismos podrían poseer una participación más importante en la mantención y diseminación de genes de resistencia antibiótica (GRAs) en AS. Las BLEE fueron las enzimas más prevalentes en las cepas estudiadas, siendo blaCTX-M-1-like el gen codificante más frecuente encontrado en estas, lo que conecta con el hallazgo de este en todas las muestras de AS.
Por lo tanto, la pesquisa de los variados genes codificantes de BLEE y de carbapenemasas, tanto en los Enterobacterales estudiados, como directamente desde las AS, demuestra la capacidad de estas últimas de almacenar y transmitir GRAs de alto impacto en salud pública.
The emergence of resistance to third-generation cephalosporins (TGC) and carbapenems, driven by extended-spectrum β-lactamases (ESBL) and carbapenemases respectively, represents a significant global challenge. These critical priority resistance mechanisms are primarily, though not exclusively, present in Enterobacterales. For decades, the study of resistance to antibacterials has been approached from an anthropocentric perspective. Recent evidence suggests that domestic and wild animals, plants, and other natural elements have also been impacted by this issue. Therefore, research in this area is promoted through the "One Health" approach. Wastewater treatment plants (WWTPs) are significant reservoirs of various contaminants. Several international studies have detected ESBL and carbapenemase-producing resistant bacteria, as well as genes encoding these enzymes, directly in wastewater (WW) and sludge samples. However, in Chile, there limited reports on antibiotic resistance in WW. Therefore, the objective of this thesis was to evaluate differences in the loads of Enterobacterales resistant to TGC and/or carbapenems and in the load of ESBL and/or carbapenemases-encoding genes, due to the treatment of WW in the Gran Concepción Metropolitan area. Between January and July 2022, eight WW samples were collected: four from influent (IF) and four from effluent (EF). The samples were cultured on R2A agar, MacConkey (MCC), MacConkey supplemented with ceftriaxone (MCC+C), and MacConkey supplemented with meropenem (MCC+M), using serial dilutions to estimate bacterial counts. A total of 205 Gram-negative bacilli (GNB) were isolated, including 50 Enterobacterales (48 from IF and 2 from EF). The fifty strains were tested for antibiotic susceptibility using antibiograms, and ESBL and carbapenemases detection was conducted phenotypically and genotypically. Genotypic detection of these enzymes was also performed directly from the same IF and EF samples. Bacterial counts in all IF samples were significantly lower than in EF samples, across all culture media used, with total counts dropping by 5 to 6 logarithms. Among the isolated Enterobacterales, the most frequent species were E. coli (16/50), K. pneumoniae complex (12/50), and K. oxytoca complex (6/50). In the Enterobacterales strains obtained from IF (48/50), 7 strains showed MAR indices > 0.5, of which two stood out with MAR of 0.81 and 0.75, showing resistance to 13 and 12 antibiotics out of 16 tested. Three others had an MAR of 0.63 (resistant to 10/16 antibacterials), while the remaining two had a MAR of 0.56 (resistant to 10/16 antibacterials). Furthermore, eight strains demonstrated multidrug-resistant (MDR) profiles. In phenotypic ESBL detection, 23/48 IF and 1/2 EF strains were positive. Additionally, 3/48 IF strains tested positive for carbapenemases. ESBL-encoding genes were found in 23 of 48 IF strains and 1 of 2 EF strains, with blaCTX-M-1-like being the most common among the 50 Enterobacterales studied. Furthermore, the blaKPC-2 gene, which encodes for carbapenemase, was identified in 3 out of 48 IF strains. No carbapenemase genes were found in the EF strains. Moreover, from the WW samples, blaCTX-M-1-like, blaKPC, and blaOXA 48-like genes were present in both IF and EF, while blaVIM was detected only in the IF samples. The reduction in bacterial counts demonstrated the effectiveness of the WW treatment in the Gran Concepción Metropolitan area on bacterial life, but not on the ESBL and carbapenemase-encoding genes dissolved in the WW. The studied Enterobacterales represented a low percentage (27%) of the total 205 GNB, suggesting that the remaining 155 (73%) organisms might play a more significant role in the maintenance and dissemination of antibiotic resistance genes (ARGs) in WW. ESBLs were the most prevalent enzymes in the studied strains, with blaCTX-M-1-like being the most frequent encoding gene, which correlates with its presence in all WW samples. Therefore, the detection of various ESBL and carbapenemase-encoding genes, both in the studied Enterobacterales and directly from the WW samples, demonstrates the latter's ability to store and transmit ARGs with a high impact on public health.
The emergence of resistance to third-generation cephalosporins (TGC) and carbapenems, driven by extended-spectrum β-lactamases (ESBL) and carbapenemases respectively, represents a significant global challenge. These critical priority resistance mechanisms are primarily, though not exclusively, present in Enterobacterales. For decades, the study of resistance to antibacterials has been approached from an anthropocentric perspective. Recent evidence suggests that domestic and wild animals, plants, and other natural elements have also been impacted by this issue. Therefore, research in this area is promoted through the "One Health" approach. Wastewater treatment plants (WWTPs) are significant reservoirs of various contaminants. Several international studies have detected ESBL and carbapenemase-producing resistant bacteria, as well as genes encoding these enzymes, directly in wastewater (WW) and sludge samples. However, in Chile, there limited reports on antibiotic resistance in WW. Therefore, the objective of this thesis was to evaluate differences in the loads of Enterobacterales resistant to TGC and/or carbapenems and in the load of ESBL and/or carbapenemases-encoding genes, due to the treatment of WW in the Gran Concepción Metropolitan area. Between January and July 2022, eight WW samples were collected: four from influent (IF) and four from effluent (EF). The samples were cultured on R2A agar, MacConkey (MCC), MacConkey supplemented with ceftriaxone (MCC+C), and MacConkey supplemented with meropenem (MCC+M), using serial dilutions to estimate bacterial counts. A total of 205 Gram-negative bacilli (GNB) were isolated, including 50 Enterobacterales (48 from IF and 2 from EF). The fifty strains were tested for antibiotic susceptibility using antibiograms, and ESBL and carbapenemases detection was conducted phenotypically and genotypically. Genotypic detection of these enzymes was also performed directly from the same IF and EF samples. Bacterial counts in all IF samples were significantly lower than in EF samples, across all culture media used, with total counts dropping by 5 to 6 logarithms. Among the isolated Enterobacterales, the most frequent species were E. coli (16/50), K. pneumoniae complex (12/50), and K. oxytoca complex (6/50). In the Enterobacterales strains obtained from IF (48/50), 7 strains showed MAR indices > 0.5, of which two stood out with MAR of 0.81 and 0.75, showing resistance to 13 and 12 antibiotics out of 16 tested. Three others had an MAR of 0.63 (resistant to 10/16 antibacterials), while the remaining two had a MAR of 0.56 (resistant to 10/16 antibacterials). Furthermore, eight strains demonstrated multidrug-resistant (MDR) profiles. In phenotypic ESBL detection, 23/48 IF and 1/2 EF strains were positive. Additionally, 3/48 IF strains tested positive for carbapenemases. ESBL-encoding genes were found in 23 of 48 IF strains and 1 of 2 EF strains, with blaCTX-M-1-like being the most common among the 50 Enterobacterales studied. Furthermore, the blaKPC-2 gene, which encodes for carbapenemase, was identified in 3 out of 48 IF strains. No carbapenemase genes were found in the EF strains. Moreover, from the WW samples, blaCTX-M-1-like, blaKPC, and blaOXA 48-like genes were present in both IF and EF, while blaVIM was detected only in the IF samples. The reduction in bacterial counts demonstrated the effectiveness of the WW treatment in the Gran Concepción Metropolitan area on bacterial life, but not on the ESBL and carbapenemase-encoding genes dissolved in the WW. The studied Enterobacterales represented a low percentage (27%) of the total 205 GNB, suggesting that the remaining 155 (73%) organisms might play a more significant role in the maintenance and dissemination of antibiotic resistance genes (ARGs) in WW. ESBLs were the most prevalent enzymes in the studied strains, with blaCTX-M-1-like being the most frequent encoding gene, which correlates with its presence in all WW samples. Therefore, the detection of various ESBL and carbapenemase-encoding genes, both in the studied Enterobacterales and directly from the WW samples, demonstrates the latter's ability to store and transmit ARGs with a high impact on public health.
Description
Tesis presentada para optar al grado de Magíster en Ciencias con mención en Microbiología
Keywords
Aguas servidas Purificación, Antibióticos lácticos Genética, Enterobacteriaceae