Antibiotics and multidrug-resistant bacteria in sewage and receiving surface waters: risks of spreading genes encoding ESBL, carbapenemases and metal tolerance in the context of one health

Author(s)

Guilherme Sgobbi Zagui

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Escola de Enfermagem de Ribeirão Preto (PCARP/BC)
• Defense date: 2022-12-21

Examining board members

Susana Segura Muñoz; Brisa Maria Fregonesi; Cristina Filomêna Pereira Rosa Paschoalato; Danilo Vitorino dos Santos

• Advisor: Susana Segura Muñoz; Leonardo Neves de Andrade

Abstract

Antibiotics are among the most successful classes of drugs in medicine, by treating bacterial infections and consequently increasing the life expectancy of the population. However, their exacerbated use since their discovery has promoted the insertion of antibiotics into the environment, as well as global dispersion of resistant bacteria. As a measure to the scarcity of new antibiotics, metals have been considered as potential antimicrobial agents, however, tolerance mechanisms have been reported, but with unknown epidemiological distribution. In this sense, the objectives of the present work were to evaluate the occurrence of antibiotics, resistant bacteria, and determinants of antibiotic resistance and metal tolerance in samples of hospital wastewater, urban wastewater, and surface water, in the context of One Health. In total, 10 antibiotics of different classes were evaluated in ultra-performance liquid chromatography coupled to mass spectrometry (UPLC-MS) and resistant bacteria were quantified and isolated from chromogenic media selective for bacteria producing ESBL and carbapenemases. The isolates obtained were subjected to antibiotic susceptibility testing, as well as detection of antibiotic resistance and metal tolerance genes by Polymerase Chain Reaction (PCR). The antibiotics amoxicillin and ampicillin were found in high concentrations, ranging between 131.5 and 1896.29 ng L-1 and between 366.35 and 1113.07 ng L-1 , respectively. The antibiotics ciprofloxacin, sulfamethoxazole, and tetracycline were also detected. The removal percentage of the antibiotics in the treated effluent consists of 53.43% for amoxicillin, 16.88% for ampicillin, 95.6% for sulfamethoxazole, and 65.5% for tetracycline. The concentration profiles of phenotypically ESBL- and carbapenemase-producing bacteria were similar, with higher occurrence in hospital wastewater. It should be noted that the municipal sewage treatment system did not show efficiency in removing these bacteria and high concentrations were detected in the treated effluent (ESBL: 3.80E+08, KPC: 6.17E+08 CFU/100mL). It was possible to verify that the concentrations of resistant bacteria downstream of the WWTP were higher in relation to the concentrations of bacteria upstream, showing a possible impact of the discharge of the treated effluent into the water body. Regarding the resistance phenotype, the isolates showed resistance mostly to β-lactam antibiotics, followed by quinolone class antibiotics. The multiple antibiotic resistance index (MAR Index) was calculated, where it was verified that all sampling points configured as high-risk sources for bacterial resistance. Regarding the molecular detection of antibiotic resistance genes, it was found that 51% of isolates (n = 55/108) carried the carbapenemase encoding gene blaKPC and 33% of isolates (n =36/108) harbored the ESBL encoding gene blaCTX-M from groups 1 or 8. In smaller percentages, blaGES (2.8%), blaNDM (1.9%) and blaVIM (0.9%) genes were detected, these last two genes encoding carbapenemases of the metallo-β-lactamase type that are considered infrequent. Regarding metal tolerance genes, genes conferring tolerance to silver, copper and mercury were detected in 52%, 50% and 28% of isolates, respectively. A strong correlation was verified in the coexistence between silver tolerance (silA) and copper tolerance (pcoD) genes (r = 0.85). The results point to the spread of antibiotic-resistant bacteria in the evaluated matrices, with a predominance of genes encoding carbapenemases. In terms of public health, the data raised point to a health emergency, in which mitigation measures need to be urgently adhered to. In this sense, and in line with the One Health approach, the need for treatment of the hospital effluent is proposed, as well as the addition of tertiary treatment in the WWTP. The integrated collaboration between the public health, veterinary, and environmental sectors are fundamental to curb this \"silent pandemic\". (AU)

• FAPESP's process: 19/05938-3 - Antibiotics, toxic metals and multiresistant bacteria in hospital and urban sewage and their potential impact on receiving water ecosystems
• Grantee: Guilherme Sgobbi Zagui
• Support Opportunities: Scholarships in Brazil - Doctorate