Characterisation of microbial communities and quantification of antibiotic resistance genes in Italian wastewater treatment plants using 16S rRNA sequencing and digital PCR

Abstract

The spread of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in humans, animals and environment is a growing threat to public health. Wastewater treatment plants (WWTPs) are crucial in mitigating the risk of environmental contamination by effectively removing contaminants before discharge. However, the persistence of ARB and ARGs even after treatment is a challenge for the management of water system. To comprehensively assess antimicrobial resistance dynamics, we conducted a one-year monitoring study in three WWTPs in central Italy, both influents and effluents. We used seasonal sampling to analyze microbial communities by 16S rRNA, as well as to determine the prevalence and behaviour of major ARGs (sul1, tetA, bla_TEM, bla_OXA-48, bla_{CTX-M-1 group}, bla_KPC) and the class 1 Integron (int1). Predominant genera included in order: Arcobacter, Acinetobacter, Flavobacterium, Pseudarcobacter, Bacteroides, Aeromonas, Trichococcus, Cloacibacterium, Pseudomonas and Streptococcus. A higher diversity of bacterial communities was observed in the effluents compared to the influents. Within these communities, we also identified bacteria that may be associated with antibiotic resistance and pose a significant threat to human health. The mean concentrations (in gene copies per liter, gc/L) of ARGs and int1 in untreated wastewater (absolute abundance) were as follows: sul1 (4.1 × 10⁹), tetA (5.2 × 10⁸), bla_TEM (1.1 × 10⁸), bla_OXA-48 (2.1 × 10⁷), bla_{CTX-M-1 group} (1.1 × 10⁷), bla_KPC (9.4 × 10⁵), and int1 (5.5 × 10⁹). The mean values in treated effluents showed reductions ranging from one to three log. However, after normalizing to the 16S rRNA gene (relative abundance), it was observed that in 37.5 % (42/112) of measurements, the relative abundance of ARGs increased in effluents compared to influents. Furthermore, correlations were identified between ARGs and bacterial genera including priority pathogens. This study improves our understanding of the dynamics of ARGs and provides insights to develop more effective strategies to reduce their spread, protecting public health and preserving the future efficacy of antibiotics.

Keywords: 16S rRNA sequencing; Antibiotic resistance bacteria; Antibiotic resistance gene; Antimicrobic resistance; Digital PCR; Microbial community; Wastewater treatment plant.