Municipal WWTPs are important reservoirs for harboring ARGs and ARB. The selective pressure exerted by antibiotic residues, co-selection by heavy metals, and conducive environment sustains ARGs and facilitates the emergence of ARB. The influent of a WWTP exclusively receiving hospital wastewater exhibits a markedly reduced richness and abundance of ARGs compared to WWTPs involved in municipal wastewater treatment [28]. With advancement in WWTP treatment technology, the sewage treatment has improved tremendously in terms of inorganic wastes, biological oxygen demand (BOD) and chemical oxygen demand (COD) but still, there is lacunae in assessing the ARG and ARB content in filtered water. In this study, we have investigated the ARG and ARB filtration efficiency of two different kinds of WWTPs, a traditional one (WWTPC) and an advanced one (WWTPA) on the same influent. Pseudomonadota or Proteobacteria typically dominates the microorganisms comprising the samples of WWTPs, and it was also the predominant phyla at each stage of treatment in the effluents of both WWTPC and WWTPA (Fig. 1 and Fig. S1 and S2) [29]. Proteobacteria are recognized for their significant contribution to the metabolic capacity for breaking down organic pollutants in bioreactors. Proteobacteria are the major carriers of ARGs in bacterial community harboured by the samples of WWTPs which was also true to our findings (Fig. 4). Acinetobacter johnsonii was identified as the predominant species which reduced with wastewater treatment. It has been earlier reported as the species thriving in WWTPs of warm areas and in extreme conditions such as Antarctica [11, 30]. The efflux pumps aid the adaptation of A. johnsonii to the challenging environment [31]. Acinetobacter spp. is associated with nosocomial infections but A. johnsonii is relatively rare with limited studies pertaining to antibiotic resistant strains [32]. It is prevalent in wastewater, as reported in WWTP at Košice in Slovakia and South Korea [29, 33]. Recently, A. johnsonii carrying blaNDM−1, blaOXA−58 and blaPER−1 was reported as an emerging high-risk clone with great resemblance to the global sewage strains [32]. The presence of the opportunistic pathogen Pseudomonas in effluents suggests the potential for carrying infections. Comparable microbiome richness and diversity between influent and effluent imply the inefficiency of WWTPs in bacterial filtration.
Remarkably, while the total number of ARGs decreased in both WWTPC and WWTPA effluents, the overall abundance of ARGs surged (Fig. 2). This increase is notably attributed to the heightened prevalence of qacL in the effluent WWTP-2, blaOXA−900 in the samples WWTP-3 and 4, and rsmA in the samples WWTP-6 and WWTP-7. In the influent, Lysobacter sp. H23M47 hosted qacL, while in the sample WWTP-2, Stenotrophomonas sp. 610A2 carried this gene. Shewanella putrefaciens harbored blaOXA−900. The transmission of rsmA involved multiple hosts, including Rheinheimera sp. MM224 in the effluent WWTP-5, Pseudomonas phenolilytica in the sample WWTP-6, and Stutzerimonas frequens and unclassified Pseudomonas in the sample WWTP-7. Although rsmA wasn't detected in the influent, its persistence highlights its ability to traverse the filtration process, including UV disinfection (Fig. 3). It's crucial to note that the ARG mobilization from diverse origin species seems intricately linked to the presence of Mobile Integron-Associated Site Elements (MISE) in the surrounding environment. Research underscores a pronounced prevalence of numerous MISEs in wastewater treatment plant influents and hospital effluents when compared to other environments. This heightened occurrence may be attributed to the preference for mobile genetic elements that harbor MISE, particularly those linked to a diverse array of mobile ARGs. Additionally, the greater diversity of Proteobacterial species in wastewaters, as opposed to the human gut, likely contributes to this trend, given that Proteobacterial species are recognized as being disproportionately involved in carrying MISE [34].
In this study, several ARGs of clinical concern such as ARGs against aminoglycoside (AAC(6')-Ib9, APH(3'')-Ib, APH(6)-Id), macrolide (EreD, mphE, mphF, mphG, mphN, msrE), lincosamide (lnuG), sulfonamide (sul1, sul2) and beta-lactamase (blaNDM−1) persisted through both conventional and advanced treatment processes (Figs. 2 and 3). This is in congruence with other studies reporting prevalence of these genes in the effluents of WWTPs in Europe, South Korea, Sri Lanka and Tokyo [11, 35–37]. The prevalence of sul1 and sul2 is an indicator of anthropogenic activity. The sul1 gene is consistently found in the 3'-conserved segment of class 1 integrons, emphasizing its significance in the capture and expression of gene cassettes [38]. The clinical class 1 integron-integrase gene stands out as a promising indicator for monitoring both the abundance and removal of antibiotic resistance genes in an urban wastewater treatment plant [39]. The acquisition and dissemination of ARGs in WWTP is a complex process influenced by multiple interconnected factors. While it is commonly believed that antibiotic residues play a crucial role in driving AMR in WWTP, a European surveillance study contradicts this notion, revealing no statistically significant correlation between antibiotic residues and AMR [11]. In WWTP, where the microenvironment is continuously altering with each successive step, predominance of microbial species signifies their adaptability and genome plasticity. Biofilm-forming antibiotic-resistant bacteria in WWTP effluent are the primary accumulators of ARGs [40]. The choice of filter media such as biologically activate carbon (BAC) can reduce the biofilm formation[41].
The prevalence of insertion sequences, such as IS_Pl3_3, ISPpu12, ISplu7D, and ISl2, are implicated in the dissemination of ARGs. These elements serve as mobile genetic components, facilitating the transfer of genetic material, including ARGs, among bacteria. Their inherent ability to move within and between bacterial genomes is a pivotal factor in the widespread distribution of antibiotic resistance determinants within microbial communities [42]. This mobility occurs through processes such as transposition, horizontal gene transfer, and recombination, fostering the transfer and dissemination of ARGs and contributing to the emergence of antibiotic-resistant bacterial strains. The abundance of virulence factors (VFs) originating from Pseudomonas aeruginosa observed at every stage, including the effluent, signifies the robust adaptability and fitness of these factors within WWTPs (Table S1) [43].
With advancement in wastewater treatment technologies, pathogens and ARGs are supposed to reduce efficiently. We observed more pronounced reduction in ARGs, MGEs by WWTPA involving UASB, solar-powered anodic oxidation and UV reactors but still, it didn’t remove all the ARGs. A concerning fact was the increased abundance of ARGs in the effluent and no significant change in microbiome abundance and diversity. A probable cause for the failure of UV irradiation in WWTPs is its dose which is generally lower in WWTPs [12]. It is crucial to account for seasonal variables such as spatiotemporal fluctuations, water temperature, and precipitation as they can significantly affect the destiny of antibiotic ARGs within aquatic ecosystems [44].
Effective containment of ARGs necessitates vigilant surveillance of WWTP effluents. This study emphasizes the critical need for establishing a standardized cut-off to govern acceptable ARG frequencies in WWTP effluents, particularly concerning their utilization in agriculture and other applications. Concurrently, advancements in technologies tailored for the comprehensive removal of both ARGs and ARB are indispensable. To address this, a multidisciplinary approach is proposed, encompassing studies that intricately investigate the interplay between ARGs, microbiome dynamics, MGEs, and VFs. Such investigations aim to identify robust indicators, quantifying the efficacy of ARB and ARG filtration processes. The proposed framework advocates for a holistic understanding of ARG dissemination, offering insights into developing strategies for mitigating the environmental impact of antibiotic resistance. Implementation of these measures will contribute to the optimization of WWTP operations, advancing our ability to curtail the proliferation of ARGs and combat the rising threat of antibiotic resistance in various sectors.