Antibiotics | Transmission of ESKAPE Pathogens and Antibiotic Resistance Genes in Wastewater from Urban and Suburban Hospitals in Malaysia

This study systematically analyzed the antibiotic resistance profiles of ESKAPE pathogens in wastewater from urban and suburban hospitals in Malaysia, detecting various antibiotic resistance genes and residual antibiotics. It highlights the critical role of hospital wastewater as an environmental source of AMR, offering significant implications for public health and environmental management.

Literature Overview
The article titled 'City or Suburb, Resistance Flows: Wastewater-Borne ESKAPE and AMR Genes in Malaysian Hospitals', published in the journal Antibiotics, reviews and summarizes the distribution of ESKAPE pathogens and the prevalence of antibiotic resistance genes in wastewater from urban and suburban hospitals. The study also quantifies antibiotic residue levels in wastewater and assesses the multiple antibiotic resistance index (MARi), revealing the key role of hospital wastewater in AMR dissemination.

Background Knowledge
ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) are major multidrug-resistant causative agents of hospital-acquired infections (HAIs). Due to their intrinsic or acquired resistance to multiple antibiotics, they have become a global public health concern. Hospital wastewater, as a reservoir for antibiotic resistance genes (ARGs) and antibiotic-resistant bacteria (ARB), has garnered increasing attention in recent years. However, studies comparing AMR differences between hospitals in varying urbanization levels, particularly in Southeast Asia, remain limited. This study focuses on urban and suburban hospitals within the Klang Valley region of Malaysia, employing 16S rRNA PCR to identify ESKAPE pathogens and qPCR to detect resistance genes (e.g., ermB, VanA, BlaTEM), combined with LC-MS analysis for antibiotic residues. These findings highlight the potential environmental impact of hospital wastewater on AMR dissemination, providing scientific support for targeted wastewater management and antibiotic use policies.

Research Methods and Experiments
The research team selected four tertiary hospitals in the Klang Valley region of Malaysia along with one suburban hospital for comparative analysis. Hospital wastewater samples were collected from discharge points at treatment plants, followed by physical parameter testing (pH, conductivity, turbidity) to assess bacterial viability. ESKAPE pathogens were identified using 16S rRNA PCR, while antibiotic susceptibility testing was performed using the disk diffusion method. Furthermore, qPCR was employed to screen for antibiotic resistance genes (ARGs), and LC-MS was used to quantify residual antibiotics in the wastewater. Multiple antibiotic resistance indices (MARi) were calculated to evaluate resistance levels among bacterial isolates.

Key Conclusions and Perspectives

• Klebsiella pneumoniae and Enterobacter spp. were the most frequently isolated ESKAPE pathogens, with the ermB gene being the most prevalent (69% of ESKAPE isolates) in urban hospitals, compared to 45.5% in suburban hospitals.
• Although antibiotic residues were detected at relatively low levels, the presence of vancomycin (VA) and ciprofloxacin (CIP) residues suggests that high usage of these antibiotics in hospitals may exert selective pressure for environmental resistance.
• MARi analysis revealed that certain K. pneumoniae and E. faecium strains exhibited high or extreme resistance, particularly in urban hospitals.
• Antibiotic resistance levels of ESKAPE pathogens in urban hospitals were generally higher than in suburban hospitals, likely due to increased patient density and antibiotic consumption in urban settings.
• Although E. faecium was also detected in suburban hospitals, its resistance profile was comparatively lower, indicating weaker environmental selective pressure compared to urban hospitals.

Conclusion
This study represents the first systematic assessment of ESKAPE pathogens and their resistance profiles in hospital wastewater across urban and suburban settings in Malaysia, revealing widespread occurrence of resistance genes such as ermB, VanA, and BlaTEM, as well as residual antibiotics like vancomycin and ciprofloxacin. Findings indicate higher resistance levels and a greater prevalence of multidrug-resistant strains in urban hospital wastewater, suggesting that urban hospitals may serve as key environmental sources of AMR dissemination. These findings highlight the urgent need for improved hospital wastewater treatment systems and optimized antibiotic use strategies to minimize the spread of resistance genes into natural water systems. Future studies should examine gene flow between hospital wastewater and municipal treatment plants and explore effective disinfection and resistance gene removal technologies to reduce environmental release of resistant pathogens.

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