Carbapenem-resistant Enterobacterales, commonly abbreviated CRE, represent one of the most urgent threats in modern medicine. These Gram-negative bacteria, which include Escherichia coli and Klebsiella pneumoniae among others, have acquired the ability to withstand carbapenems, a class of antibiotics often reserved as a last line of defense against serious infections. The consequences are stark: mortality rates from CRE infections can climb as high as fifty percent, leaving clinicians with vanishingly few therapeutic options. The problem is also accelerating globally, with resistance prevalence rising from just one percent in 2013 to forty-three percent in 2020 in parts of North America, a trajectory that underscores how quickly these organisms can adapt and spread.

The engine driving this resistance is the production of carbapenemase enzymes, a diverse family of beta-lactamases grouped into distinct classes by the Ambler classification system. Class A enzymes such as KPC, Class B metallo-beta-lactamases including NDM, IMP and VIM, and Class D oxacillinases such as OXA-48 each hydrolyze carbapenems through different chemical mechanisms. Because phenotypic tests alone cannot reliably distinguish between these classes, molecular techniques such as multiplex polymerase chain reaction are essential for pinpointing which resistance genes are actually present. In the Indian context, where blaNDM-1 has become widespread, identifying these determinants is critical for predicting transmissibility, constructing empirical antibiograms and strengthening hospital infection control.

Recognizing that systematic molecular data from eastern India were virtually absent, the AIIMS Patna team designed a cross-sectional study conducted between July 2021 and July 2023 in the hospital’s Microbiology laboratory. The research, approved by the Institutional Ethics Committee under approval number AIIMS/Pat/IEC/2021/578 and performed in accordance with the Declaration of Helsinki, analyzed residual clinical isolates collected during routine diagnostic work, with a formal waiver of individual patient consent and no patient-identifiable data collected. To avoid duplication bias, the investigators included only the first isolate per patient per episode of infection, ensuring that repeated cultures from the same admission did not inflate the results.

The scope of the underlying resistance problem was formidable. During the study period, a total of 3,421 Enterobacterales were isolated, drawn overwhelmingly from urine specimens, followed by pus, blood and respiratory samples. Of these, 1,128 isolates, or 32.97 percent, were phenotypically confirmed as carbapenem resistant, a figure drawn from the team’s previously published phenotypic work at the same center. Resistance was markedly higher among inpatients, at 47.74 percent, compared with only 14.48 percent among outpatients. All 213 CRE isolates characterized in detail showed complete resistance to third-generation cephalosporins, near-universal resistance of 99.4 percent to beta-lactam and beta-lactamase inhibitor combinations such as piperacillin-tazobactam, and one hundred percent resistance to aztreonam, a sobering profile that leaves almost no conventional beta-lactam therapy intact.

To confirm which isolates were producing carbapenemase enzymes, the researchers deployed a battery of phenotypic assays, including the Modified Carbapenem Inactivation Method, or mCIM, its EDTA-supplemented variant eCIM designed to identify Class B metallo-beta-lactamases, and combination inhibition tests using phenylboronic acid, cloxacillin and EDTA. Of the 213 CRE isolates, 203 were confirmed carbapenemase producers by mCIM. From this positive pool, a consecutive subset of 98 isolates with viable stored stock and sufficient DNA yield was selected for multiplex PCR-based gene profiling using validated primers originally described by Poirel and colleagues, with amplification performed on a ProFlex thermocycler and amplicons resolved on agarose gels. The authors caution that all gene-detection rates apply to this genotyped subset, which represents 48.3 percent of the mCIM-positive isolates, and should not be extrapolated as prevalence estimates for the entire CRE cohort.

The molecular results were striking. Among the 98 profiled isolates, 60 were Escherichia coli, 33 were Klebsiella pneumoniae, and the remainder comprised Citrobacter freundii and Enterobacter species. blaNDM emerged as the most prevalent gene, detected in 63.27 percent of isolates, with E. coli and K. pneumoniae as the predominant carriers. blaOXA-48 followed closely at 61.22 percent, while blaIMP appeared in 10.20 percent, blaKPC in 5.10 percent and blaVIM in 3.06 percent. Perhaps most concerning was the degree of co-carriage: half of the genotyped K. pneumoniae isolates carried both NDM and OXA-48 simultaneously, and 27.27 percent of E. coli harbored the same dual combination. Individual isolates carrying three resistance genes, such as NDM, OXA-48 and KPC together, were also documented, illustrating how bacterial genomes can accumulate layered defensive armories.

The comparison between phenotypic and genotypic results revealed both reassuring agreement and instructive discrepancies. All isolates carrying blaKPC were mCIM positive and displayed Class A carbapenemase phenotypes, and every isolate harboring a metallo-beta-lactamase gene, whether blaNDM, blaIMP or blaVIM, was positive on both mCIM and eCIM, confirming Class B enzyme production. However, among the 60 isolates carrying blaOXA-48, only two exhibited the phenotypic signature of Class D carbapenemase, a gap the authors attribute partly to limitations in the EUCAST-recommended temocillin zone-diameter threshold used as an indirect confirmatory test. Unexpressed genes, undetected beta-lactamase families such as blaSPM or blaGIM, and PCR inhibitors may all contribute to such mismatches, and the researchers note that amplicons were not confirmed by sequencing, meaning allelic variants cannot be entirely excluded.

These findings carry direct implications for therapy. Given the overwhelming predominance of Class B metallo-beta-lactamases, particularly NDM, the authors argue that empirical treatment of suspected CRE infections in this region should prioritize agents with proven activity against Class B enzymes, such as cefiderocol or the combination of ceftazidime-avibactam with aztreonam. They also emphasize that neither phenotypic nor genotypic testing alone is sufficient, and that balancing both approaches offers the most complete picture of resistance. International comparisons in the study highlight how sharply gene distributions vary by geography, with Thailand reporting NDM rates of ninety percent, China dominated by KPC at 53.4 percent, and Saudi Arabia led by OXA-48 at 76.11 percent, reinforcing that local surveillance data are indispensable for guiding rational antibiotic use.

Beyond its immediate clinical relevance, the study fills a critical gap in India’s national antimicrobial resistance surveillance architecture. Bihar and neighboring eastern states carry enormous infectious disease burdens yet have historically lacked the molecular diagnostic capacity to characterize circulating resistance mechanisms, undermining targeted infection control interventions. By documenting the genotypic landscape of CRE in an underrepresented setting, the AIIMS Patna team supports the objectives of India’s National Action Plan on Antimicrobial Resistance, particularly those concerning laboratory strengthening and evidence-based surveillance. The researchers acknowledge limitations, including the subset-based genotyping design and the absence of sequencing-based strain typing, and they plan future work involving blaNDM allele subtyping and whole-genome sequencing to trace clonal spread. For now, their findings stand as a clear warning and a practical guide: the superbugs of eastern India are armed with a dangerous genetic repertoire, but knowing exactly which weapons they carry is the first step toward disarming them.

Subject of Research: Genotypic profiling of carbapenemase genes in carbapenem-resistant Enterobacterales at a tertiary care hospital in Bihar, India

Article Title: Deciphering the genotypic profiles of Carbapenem-resistant Enterobacterales: A study from a tertiary care hospital in Bihar, India

Article References: Pramurtajyoti, D., Prathyusha, K., Zeeshan, F. M., Asim, S., Pati Binod, K., & Bhaskar, T. (2026). Deciphering the genotypic profiles of Carbapenem-resistant Enterobacterales: A study from a tertiary care hospital in Bihar, India. New Microbes and New Infections, 73, Article 101850. https://doi.org/10.1016/j.nmni.2026.101850

Image Credits: AI Generated

DOI: 10.1016/j.nmni.2026.101850

Keywords: carbapenem-resistant Enterobacterales, antimicrobial resistance, blaNDM, blaOXA-48, carbapenemase genes, multiplex PCR, Klebsiella pneumoniae, Escherichia coli, metallobeta-lactamases, India, hospital surveillance, infection control

Urban sewage systems are more than just conduits for waste disposal; they are dynamic microbial hubs where bacteria from diverse origins intermingle, evolve, and interact. India’s extensive urban centers, characterized by dense populations and intensive antibiotic usage, offer a unique yet alarming environment to study these microbial interactions. The researchers led by Paul, Talukdar, Kapuganti, and colleagues meticulously analyzed sewage samples from multiple metropolitan cities, employing cutting-edge metagenomic sequencing and bioinformatics techniques to trace the trajectory of antibiotic compounds and resistant microbes.

One of the pivotal revelations of this study is the pervasive presence of multiple classes of antibiotic residues in sewage waters. These molecules, released from household waste, hospital effluents, and pharmaceutical industries, persist despite conventional treatment processes. Their continued presence serves as a selective pressure, fostering the survival and propagation of bacteria harboring resistance genes. The deep sequencing efforts mapped a diverse resistome landscape, revealing resistance determinants not only against commonly used antibiotics but also against last-resort drugs, raising profound concerns about treatment efficacy in clinical settings.

The environment within urban sewage acts as a hotspot for horizontal gene transfer (HGT), a fundamental evolutionary mechanism whereby bacteria exchange genetic material, including antibiotic resistance genes. This HGT catalyzes the spread of resistant traits across different bacterial species, sometimes crossing species barriers. The study identified an enrichment of mobile genetic elements such as plasmids, integrons, and transposons, all instrumental in facilitating these genetic exchanges. This genomic plasticity accelerates AMR proliferation, which could eventually manifest as superbug outbreaks beyond the confines of sewage systems.

Importantly, the research delves into the temporal dynamics of the sewage microbiome, demonstrating how seasonal variations and antibiotic consumption trends influence resistance patterns. Periods marked by increased antibiotic prescription, like post-infection outbreaks, corresponded with surges in resistance gene abundance and diversity. This cyclical pattern underscores the intimate connection between human behavior, pharmaceutical practices, and microbial ecology, reinforcing the concept that mitigation strategies must be systemic and multifaceted.

The authors also explored the efficacy of existing sewage treatment methodologies in mitigating antibiotic contamination and microbial resistance. Conventional treatments such as activated sludge processes and chlorination were found insufficient in fully removing antibiotic residues or eliminating resistant bacteria. In fact, sub-lethal concentrations of antibiotics post-treatment may further exacerbate resistance development. This finding calls for innovations in wastewater treatment technologies, emphasizing advanced oxidation processes, membrane filtration, and bioremediation strategies tailored to dismantle these molecular pollutants effectively.

Beyond the localized environmental perspective, the study highlights the regional and global implications of antibiotic contamination in urban sewage. With metropolises serving as nodes of human activity and travel, the resistant microbes harbored in their sewage can be disseminated through water bodies, soil, and even airborne particles, contributing to the broader ecological spread of AMR. This interconnectedness suggests that antibiotic resistance is not a contained problem but a planetary health crisis that necessitates international cooperation and policy alignment.

The interplay between antibiotic residues and microbial communities extends to complex microbial population shifts. The study observed that resistant bacterial taxa often dominated post-antibiotic exposure conditions, leading to reduced microbial diversity. This loss in biodiversity can alter nutrient cycling, biogeochemical processes, and overall ecosystem stability within urban wastewater environments. Such ecological perturbations have downstream effects on environmental quality and public health, as sewage effluents ultimately interact with natural water systems.

Notably, the research team employed novel computational models to predict future AMR trends based on current contamination levels and treatment practices. These predictive insights are crucial for public health officials and urban planners in designing proactive strategies. They stress the need for integrated surveillance systems that monitor antibiotic residues and resistance genes continuously, enabling timely risk assessments and targeted interventions to prevent AMR escalation.

The authors also revisit the role of antibiotic stewardship in curbing this menace. Reducing unnecessary antibiotic prescriptions, promoting rational drug use, and enhancing public awareness are pivotal pillars in this fight. Coupled with improved pharmaceutical waste management and robust sewage infrastructure upgrades, such stewardship can curb the influx of antibiotics into urban environments, thereby diminishing selective pressures driving resistance.

From a microbiological standpoint, the study also explores potential biotic interventions. The researchers investigated bacteriophage therapy and the use of competitive microbial consortia as innovative approaches to modulate sewage microbiomes and suppress resistant populations. While these strategies remain nascent, they offer promising tools to re-engineer microbial ecology in a way that controls resistance gene dissemination without resorting to heavier chemical interventions.

The comprehensive nature of this research also blends socio-economic considerations. Urban sewage management in India faces infrastructural challenges, resource constraints, and regulatory gaps, all of which compound the AMR problem. Addressing antibiotic contamination requires not only technological advancements but also systemic socio-political reforms, including investment in sanitation infrastructure, enforcement of environmental regulations, and community engagement initiatives.

Conclusively, this study serves as a poignant reminder that urban sewage systems are critical battlegrounds in the global fight against antibiotic resistance. The contamination of wastewater by antibiotics is not merely an environmental issue but an urgent public health threat that transcends national borders. Holistic strategies integrating environmental science, microbiology, public health policy, and socio-economic development are imperative to stem the rising tide of antimicrobial resistance emanating from our cities.

With the accelerating pace of urbanization and antibiotic consumption worldwide, the insights derived from this Indian urban sewage microbiome study resonate far beyond geographical confines. They underscore a universal imperative: to safeguard the efficacy of antibiotics—a cornerstone of modern medicine—efforts must extend into understanding and managing the environmental reservoirs of resistance. This paradigm shift is vital to avert a post-antibiotic era and secure sustainable health outcomes for future generations.

Subject of Research: Antibiotic contamination and antimicrobial resistance dynamics in urban sewage microbiomes.

Article Title: Antibiotic contamination and antimicrobial resistance dynamics in the urban sewage microbiome in India.

Article References:
Paul, D., Talukdar, D., Kapuganti, R.S. et al. Antibiotic contamination and antimicrobial resistance dynamics in the urban sewage microbiome in India. Nat Commun (2025). https://doi.org/10.1038/s41467-025-68034-3

Image Credits: AI Generated