Molecular characterization of extended spectrum beta lactamase producing Escherichia coli in two different wastewater treatment plants in Hatay Province, Türkiye – Nature

Report on the Efficacy of Wastewater Treatment Plants in Mitigating Antimicrobial Resistance

Executive Summary

This report evaluates the performance of two municipal wastewater treatment plants (WWTPs) in Hatay, Türkiye, in eliminating multidrug-resistant bacteria, specifically extended-spectrum beta-lactamase producing Escherichia coli (ESBL-EC). The study highlights a significant public health concern directly impacting the achievement of Sustainable Development Goal 3 (Good Health and Well-being) and Sustainable Development Goal 6 (Clean Water and Sanitation). Findings indicate that while both conventional and advanced biological treatment processes reduce ESBL-EC counts, they fail to achieve complete elimination, resulting in the release of these hazardous bacteria into the environment. The prevalence of clinically significant resistance genes, such as bla_CTX-M-15, in the discharged effluent underscores the role of WWTPs as critical hotspots for the dissemination of antimicrobial resistance (AMR). This report concludes that current wastewater management practices are insufficient to safeguard public and environmental health, necessitating urgent upgrades and integrated surveillance strategies aligned with the 2030 Agenda for Sustainable Development.

1.0 Introduction: Antimicrobial Resistance as a Barrier to Sustainable Development

1.1 The Global Health Crisis of AMR and its Link to SDG 3

Antimicrobial resistance (AMR) represents a formidable threat to global public health, undermining the core objectives of SDG 3 (Good Health and Well-being). The proliferation of multidrug-resistant bacteria, such as ESBL-EC, renders common infections untreatable, increasing morbidity, mortality, and healthcare costs. ESBL-EC are of particular concern due to their resistance to critical beta-lactam antibiotics and their frequent association with community and hospital-acquired infections. The dissemination of these pathogens into the environment creates a continuous cycle of exposure and infection, challenging global health security.

1.2 Wastewater as a Vector for AMR and a Challenge to SDG 6

Wastewater treatment plants (WWTPs) are recognized as critical nexuses for the concentration and exchange of antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs). The discharge of inadequately treated effluent contaminates receiving water bodies, directly contravening the targets of SDG 6 (Clean Water and Sanitation), which aims to improve water quality and halve the proportion of untreated wastewater. This study was initiated to assess and compare the efficacy of a conventional WWTP and an advanced biological WWTP in removing ESBL-EC, thereby evaluating their role as control points in preventing environmental contamination and protecting public health, which also impacts SDG 14 (Life Below Water).

2.0 Methodology

2.1 Study Design and Sample Collection

A total of 24 wastewater samples were collected from two municipal WWTPs in Hatay, Türkiye, between March and May 2021. The facilities represented two different technological approaches:

• Plant 1: Conventional Treatment Process
• Plant 2: Advanced Biological Treatment Process

Samples were collected from both the influent (n=12) and effluent (n=12) stages of each plant to enable a comparative performance analysis.

2.2 Laboratory Analysis

A comprehensive analysis was conducted to characterize the presence and nature of ESBL-EC. The key procedures included:

1. Enumeration and Isolation: Quantification of ESBL-EC in water samples. A total of 66 ESBL-EC isolates were confirmed and selected for further characterization.
2. Antimicrobial Susceptibility Testing: The Kirby-Bauer disk diffusion method was used to determine the resistance profiles of the isolates against a panel of antibiotics.
3. Genetic Analysis: Polymerase Chain Reaction (PCR) was employed to identify key resistance genes, including those conferring resistance to beta-lactams (bla_CTX-M, bla_TEM), quinolones, and disinfectants.
4. Clonal Relationship Assessment: Pulsed-field gel electrophoresis (PFGE) was used to determine the genetic relatedness of the ESBL-EC isolates.

3.0 Results: Inadequate Containment of a Public Health Threat

3.1 Performance of Wastewater Treatment Plants in ESBL-EC Removal

The study revealed a significant, yet incomplete, reduction of ESBL-EC by both WWTPs, posing a direct challenge to achieving SDG 6.

• Influent Water: The average concentration of ESBL-EC was high, at 3.86 log cfu/ml.
• Effluent Water: The average concentration was reduced to 2.20 log cfu/ml, indicating that a substantial number of resistant bacteria are still being discharged.
• Comparative Efficacy: The advanced biological treatment plant demonstrated a statistically significant higher reduction in ESBL-EC counts compared to the conventional plant. However, neither facility achieved complete elimination.

The persistence of identical ESBL-EC strains in both influent and effluent samples, as confirmed by PFGE, provides definitive evidence of the plants’ failure to contain these pathogens.

3.2 Molecular Characterization of Resistant Isolates

The genetic profile of the isolates highlights a severe public health risk, undermining SDG 3 by contributing to the environmental reservoir of clinically important resistance mechanisms.

• Beta-Lactam Resistance: The bla_CTX-M gene was detected in 86.36% of isolates. Critically, the globally pandemic bla_CTX-M-15 subtype was the most prevalent, found in 86.36% of these isolates.
• Multidrug Resistance: High rates of co-resistance were observed for other important antibiotic classes, including tetracyclines (63.63%) and fluoroquinolones (43.93%).
• Disinfectant Resistance: Each isolate carried at least two disinfectant resistance genes, with ydgE (84.84%) and qacEΔ1 (81.81%) being the most common. This suggests that treatment processes may inadvertently select for co-resistant strains.

3.3 Genetic Diversity

PFGE analysis revealed high genetic diversity among the 66 isolates, with 49 identified as unique strains (singletons). This indicates that a wide variety of ESBL-EC strains are circulating in the community and entering the wastewater system, complicating control efforts.

4.0 Conclusion and Recommendations for Achieving Sustainable Development Goals

This investigation confirms that municipal WWTPs in the studied region act as conduits for the release of multidrug-resistant ESBL-EC into the environment. This failure to contain AMR directly threatens progress towards SDG 3 (Good Health and Well-being) and SDG 6 (Clean Water and Sanitation). The superior performance of the advanced biological treatment plant suggests a clear path for technological improvement, but even this is insufficient for complete removal.

To align wastewater management with the 2030 Agenda for Sustainable Development, the following actions are recommended:

1. Enhance Treatment Infrastructure: Prioritize investment in upgrading WWTPs to include advanced biological and tertiary treatment technologies capable of more effective pathogen and ARG removal. This is crucial for meeting the targets of SDG 6 and SDG 11 (Sustainable Cities and Communities).
2. Establish AMR Surveillance in Wastewater: Implement routine monitoring of ARB and ARGs in WWTP effluent as part of national AMR action plans. This “One Health” approach provides critical data for public health interventions under SDG 3.
3. Integrate AMR into Water Safety Policies: National and local policies for water and sanitation must be updated to include specific targets for the reduction of AMR determinants in wastewater discharges.
4. Promote Antibiotic Stewardship: Address the root cause of AMR by strengthening antibiotic stewardship programs in human and veterinary medicine to reduce the load of antibiotics and resistant bacteria entering wastewater systems, in line with SDG 12 (Responsible Consumption and Production).

Analysis of Sustainable Development Goals (SDGs) in the Article

1. Which SDGs are addressed or connected to the issues highlighted in the article?

1. SDG 3: Good Health and Well-being

   The article directly connects to SDG 3 by focusing on antibiotic resistance (AMR) as a “serious global concern” and a “significant public health concern.” It highlights how extended spectrum beta lactamase producing Escherichia coli (ESBL-EC) from wastewater poses a threat to human health. The introduction mentions that AMR adds a “financial burden on health-care systems” and cites “59,900 reported deaths worldwide in 2019 linked to 3rd generation cephalosporin-resistant E. coli infections,” underscoring the goal of ensuring healthy lives.

2. SDG 6: Clean Water and Sanitation

   This is the central SDG addressed in the article. The study’s main objective is to “evaluate and compare the performance of two municipal WWTPs in eliminating ESBL-EC.” This directly relates to the sustainable management of water and sanitation. The article investigates whether conventional and advanced wastewater treatment plants (WWTPs) are effective in removing dangerous pathogens, a key aspect of providing clean and safe water and treating wastewater before it is discharged into the environment.

3. SDG 14: Life Below Water

   The article connects to SDG 14 by discussing the consequences of inadequately treated wastewater. The conclusion states that despite treatment, a “significant number of bacteria still release into the receiving water bodies (river and sea).” This discharge of pollutants, specifically multidrug-resistant bacteria, from land-based sources like WWTPs contributes to the pollution of marine and aquatic ecosystems, which SDG 14 aims to prevent.

2. What specific targets under those SDGs can be identified based on the article’s content?

• SDG 3: Good Health and Well-being

  • Target 3.3: By 2030, end the epidemics of AIDS, tuberculosis, malaria and neglected tropical diseases and combat hepatitis, water-borne diseases and other communicable diseases. The spread of multidrug-resistant bacteria like ESBL-EC through wastewater is a major challenge to combating communicable diseases, as it makes infections harder to treat.
  • Target 3.9: By 2030, substantially reduce the number of deaths and illnesses from hazardous chemicals and air, water and soil pollution and contamination. The release of insufficiently treated wastewater containing pathogenic, drug-resistant bacteria is a form of water pollution that directly leads to illness and death.
  • Target 3.d: Strengthen the capacity of all countries, in particular developing countries, for early warning, risk reduction and management of national and global health risks. Monitoring wastewater for antibiotic-resistant bacteria, as done in this study, is a method for surveillance and risk management of the global health threat of AMR.

• SDG 6: Clean Water and Sanitation

  • Target 6.3: By 2030, improve water quality by reducing pollution, eliminating dumping and minimizing release of hazardous chemicals and materials, halving the proportion of untreated wastewater and substantially increasing recycling and safe reuse globally. The article’s core focus is on the effectiveness of WWTPs in reducing pollution (ESBL-EC), directly aligning with this target. It compares two treatment methods to see which is better at improving water quality before discharge.

• SDG 14: Life Below Water

  • Target 14.1: By 2025, prevent and significantly reduce marine pollution of all kinds, in particular from land-based activities, including marine debris and nutrient pollution. The discharge of WWTP effluent containing ESBL-EC into rivers and the sea is a clear example of pollution from a land-based activity that this target aims to reduce.

3. Are there any indicators mentioned or implied in the article that can be used to measure progress towards the identified targets?

Yes, the article provides specific quantitative data that can serve as indicators to measure progress:

• For Target 6.3: The primary indicator is the effectiveness of wastewater treatment. The article measures this by comparing the concentration of ESBL-EC in influent versus effluent water.
  • Indicator: Reduction in ESBL-EC counts post-treatment. The article quantifies this: influent water had a mean of 3.86 log cfu/ml, while effluent had a mean of 2.20 log cfu/ml. It also notes that the advanced biological treatment plant achieved a reduction of “more than 2 log cfu/ml,” providing a clear metric of treatment efficiency.
• For Target 3.9 and 14.1: An indicator for water pollution is the concentration of harmful substances in the water discharged into the environment.
  • Indicator: Abundance of ESBL-EC in effluent water. The study reports a mean concentration of 2.20 ± 0.27 log cfu/ml in effluent samples. This value serves as a direct measure of the pollution load being released into receiving water bodies.
• For Target 3.3 and 3.d: An indicator for monitoring public health risks is the prevalence of specific antibiotic resistance genes.
  • Indicator: Frequency of specific antibiotic resistance genes. The article states that the “bla_CTX-M gene was the most frequently detected gene in the isolates (86.36%), among which the bla_CTX-M-15 type (86.36%) was the most frequently detected.” Tracking the prevalence of such genes in wastewater can serve as an early warning system for public health threats.

4. Summary Table of SDGs, Targets, and Indicators

Source: nature.com