The research team began with a substantial collection of clinical specimens gathered from patients at Al-Hussein Teaching Hospital in Al-Muthanna governorate. Out of 350 samples analyzed, 83 isolates, or 23.71 percent, were confirmed as E. faecalis. The statistical analysis of the clinical data revealed a significant association between these isolates and patients with urinary catheters and pyelonephritis, with a p-value of 0.045, underscoring the organism’s particular affinity for the urinary tract environment where catheterization and compromised kidney function create favorable conditions for colonization and infection. This epidemiological grounding matters because it ties the molecular findings directly to the kinds of infections clinicians actually encounter on the ward.

Antimicrobial susceptibility testing painted a sobering picture of the resistance landscape. The isolates showed their highest level of resistance to amoxicillin/clavulanate, with 95.2 percent of strains unaffected by this beta-lactam combination, a figure that reflects the widespread dissemination of beta-lactamase enzymes among local enterococcal populations. Resistance to vancomycin, the glycopeptide antibiotic long reserved as a last-line treatment for serious gram-positive infections, was present in 13.3 percent of the isolates, a level that remains lower than rates reported in many European surveillance studies but is nonetheless clinically significant. Notably, the isolates showed almost no intrinsic resistance to sitagliptin itself, with only 2.4 percent affected, which positioned the drug as a candidate for combination strategies rather than monotherapy.

Molecular analysis confirmed the genetic basis of these phenotypes. Polymerase chain reaction screening detected the vanA gene, the archetypal determinant of high-level vancomycin resistance, and the blaTEM gene, which encodes a beta-lactamase enzyme capable of degrading penicillin-class antibiotics. The researchers then went a step further and sequenced selected isolates to probe the fine structure of these genes. In vanA, they identified two single nucleotide polymorphisms, G521A and C560T, while blaTEM carried a G244A substitution. Each of these point mutations translates into an amino acid change in the corresponding protein, and such substitutions can alter enzyme kinetics, substrate affinity, or the structural conformation of the resistance machinery. Documenting these variants in a local clinical population provides a molecular fingerprint that can be compared with sequences deposited from around the world.

The quantitative gene expression experiments formed the heart of the study. Using quantitative reverse transcription PCR, the team measured how the two resistance genes responded to different chemical environments. When the bacteria were exposed to vancomycin, vanA expression was up-regulated 1.47-fold, consistent with the known biology of the vancomycin resistance system, in which the VanS sensor kinase detects the presence of the glycopeptide and activates the VanR response regulator to switch on the resistance operon. Exposure to sitagliptin produced a different and more surprising pattern, with vanA expression reaching 3.66-fold, indicating that the drug perturbs the regulatory circuitry of the resistance system in ways that are not yet fully understood. The blaTEM gene told a complementary story: its expression was strongly induced to 1.95-fold under antibiotic pressure, but fell to 0.75-fold, a clear down-regulation, when sitagliptin was present instead.

This differential modulation is precisely what makes the findings interesting from a therapeutic standpoint. A resistance-modifying agent does not need to kill bacteria on its own; it needs to disarm the defenses that make conventional antibiotics ineffective. The down-regulation of blaTEM under sitagliptin exposure suggests the drug could sensitize resistant strains to beta-lactam therapy, and the checkerboard assays performed by the team support this interpretation, revealing a synergistic effect when sitagliptin was combined with amoxicillin. In such assays, fractional inhibitory concentrations of each agent are plotted against one another, and synergy indicates that the combination achieves more than the simple sum of its parts. For a pathogen in which 95.2 percent of isolates resist amoxicillin/clavulanate, restoring susceptibility even partially would carry real clinical weight.

The study also incorporated computational structural work to explore how sitagliptin might interact with the resistance proteins at the molecular level. Using homology modeling approaches of the kind implemented in servers such as SWISS-MODEL, the researchers built three-dimensional models of the mutant proteins and examined the structural context of the observed amino acid substitutions. This in silico component reflects a broader trend in antimicrobial research, where drug repurposing pipelines use virtual screening and docking to identify approved compounds that bind bacterial targets or regulatory proteins. Sitagliptin itself has a growing record in this arena, with previous studies reporting that it attenuates virulence in Serratia marcescens and Porphyromonas gingivalis, and that gliptins as a class exhibit anti-quorum-sensing activity against Pseudomonas aeruginosa and Staphylococcus aureus. The new findings extend this repurposing narrative into the enterococci, a genus where the therapeutic pipeline is desperately thin.

Phylogenetic analysis of 16S rRNA sequences added an evolutionary dimension to the work. The Iraqi isolates clustered closely with E. faecalis strains previously reported from China, Hungary, and Tunisia, a pattern consistent with the international dissemination of successful lineages through travel, food chains, and healthcare networks. Resistance genes such as vanA typically reside on mobile genetic elements, including the transposon Tn1546 and various plasmids, which facilitate horizontal transfer between strains and even between bacterial species. The close genetic relatedness of isolates from geographically distant countries is a reminder that antimicrobial resistance is inherently a global problem, and that local surveillance data, such as the SNP profiles documented in this study, contribute to a worldwide picture of how resistance determinants evolve and spread.

The authors are careful to frame sitagliptin as a potential adjunct rather than a standalone cure. The drug showed no clear bactericidal activity against the isolates, meaning it does not kill the bacteria directly, and the up-regulation of vanA expression under sitagliptin exposure raises questions that will need to be resolved before any clinical translation. Whether the elevated vanA transcript levels translate into functional glycopeptide resistance, or whether they reflect a stress response without phenotypic consequence, remains an open issue. What the study does establish is a proof of concept: an FDA-approved oral anti-diabetic agent, already taken daily by millions of patients with a well-characterized safety profile, can measurably alter the expression of the two genes that define resistance in multidrug-resistant E. faecalis, and can act synergistically with a beta-lactam antibiotic in vitro.

The implications reach beyond a single drug and a single pathogen. As vancomycin-resistant enterococci continue to pose an ongoing challenge in hospitals across Europe, the Middle East, and beyond, strategies that preserve the utility of existing antibiotics are increasingly viewed as essential complements to new drug development. Resistance modifiers, anti-virulence agents, and combination therapies all belong to this emerging toolkit, and repurposed medications offer the advantage of bypassing the slowest and most expensive phases of drug development. The Iraqi team’s work, grounded in local clinical isolates yet connected to global sequence data, illustrates how a modestly resourced laboratory can contribute meaningful evidence to this effort. The next steps will involve confirming the modulatory effects across larger and more diverse isolate collections, elucidating the mechanism by which sitagliptin interferes with resistance gene regulation, and testing whether the synergy observed in checkerboard plates holds up in animal models of infection. If those results are positive, clinicians may one day reach for a diabetes pill not to lower blood sugar, but to strip a deadly bacterium of its chemical armor.

Subject of Research: Repurposing sitagliptin to modulate vanA and blaTEM antibiotic resistance gene expression in Enterococcus faecalis

Article Title: Molecular Characterization of vanA and blaTEM Mutant Genes in Enterococcus faecalis: In silico and in vitro investigation of Sitagliptin as a potential Resistance Modulator

Article References: Alabdali, Y. A. J., Munahi, M. G., Dawood, Y. A., Kudhair, B. K., Al-Hejjaj, M. Y., & Aziz, Z. N. (2026). Molecular Characterization of vanA and blaTEM Mutant Genes in Enterococcus faecalis: In silico and in vitro investigation of Sitagliptin as a potential Resistance Modulator. Molecular Biology Reports, 53(1), Article 1676. https://doi.org/10.1007/s11033-026-12827-2

Image Credits: AI Generated

DOI: 10.1007/s11033-026-12827-2

Keywords: Enterococcus faecalis, antimicrobial resistance, vanA, blaTEM, sitagliptin, drug repurposing, vancomycin resistance, qRT-PCR, single nucleotide polymorphisms, checkerboard synergy assay, 16S rRNA phylogenetics, Iraq

The research team, led by Jouman W. Hassan and Issmat I. Kassem of the University of Georgia’s Center for Food Safety, together with Walid G. Al Hakeem and USDA Agricultural Research Service scientist Michael J. Rothrock Jr., sampled eleven pastured poultry farms across the region, collecting material from 41 broiler flocks. The sampling strategy was deliberately comprehensive: feces and soil from the pasture itself, cecal contents from the birds’ intestines, and carcass rinses collected after processing. This farm-to-fork design allowed the investigators to track whether resistant bacteria appeared at multiple points along the production chain, from the living bird to the final product destined for consumers’ kitchens.

From those samples, the team isolated 560 E. coli strains and screened them for antibiotic susceptibility. Twenty-seven of the isolates proved to be multidrug-resistant, meaning they withstood multiple classes of clinically important antibiotics. That number may sound modest, but the implications are outsized. E. coli is a common inhabitant of the intestinal tract of poultry and a widely used sentinel organism for monitoring antimicrobial resistance in food animals and the environment. When MDR strains of this bacterium appear in pastured systems, they signal that resistance is not confined to intensive, antibiotic-heavy operations.

To understand what made these 27 isolates tick, the researchers turned to whole-genome sequencing, an approach that reads out the complete DNA blueprint of each strain. The genomic analysis covered four dimensions at once: the resistome, the full complement of antibiotic resistance genes; the virulome, the set of genes that equip bacteria to cause disease; the plasmidome, the collection of mobile DNA circles that shuttle genes between cells; and the sequence type, a genetic fingerprint that reveals how related the strains are to one another. Multi-locus sequence typing revealed 19 different sequence types among the 27 MDR isolates, a striking degree of genetic diversity that suggests the resistant bacteria were not the product of a single clone spreading farm to farm, but rather of independent resistance emergence or acquisition across a genetically varied E. coli population.

The resistome analysis showed that every one of the MDR isolates carried between two and seven antibiotic resistance genes. These genes encoded resistance to five major antibiotic classes: beta-lactams, the family that includes penicillins and cephalosporins; tetracyclines, workhorse antibiotics used in both human medicine and agriculture; folate pathway antagonists such as sulfonamides; amphenicols; and aminoglycosides. The breadth of this resistance profile matters because multidrug resistance narrows the treatment options available if such bacteria, or the genes they carry, make their way into human pathogens.

One gene stood out from the rest. blaTEM, which encodes a beta-lactamase enzyme capable of degrading penicillin-class antibiotics, was detected in 16 of the MDR E. coli, and crucially, it turned up in isolates recovered from both pre-harvest samples, such as feces, soil, and ceca, and post-harvest carcass rinses. That pattern indicates that beta-lactam resistance persisted through the production continuum, from the pasture floor to the processed bird. Beta-lactam antibiotics are among the most important drug classes in human medicine, and enzymes like the one encoded by blaTEM are a major mechanism by which bacteria defeat them. The presence of this gene across the farm-to-fork spectrum raises questions about how consumers and farmworkers might encounter resistant bacteria through contact with pastured poultry products or environments.

Perhaps the most consequential finding came from the plasmid analysis. In several isolates, the resistance genes and virulence genes were not embedded in the chromosome but instead rode on plasmids, self-replicating DNA elements that bacteria exchange readily, even across species boundaries. The identified plasmids included members of the IncI2α, IncFII, and IncFIB incompatibility groups, lineages well known in the world of antimicrobial resistance research for their role in disseminating resistance genes among Enterobacteriaceae. Plasmids are the molecular couriers of the resistance crisis, and their presence in these isolates meant the resistance genes had the potential to travel.

The team did not stop at identifying plasmids; they tested whether the genes could actually move. In laboratory conjugation assays, the classic experiment for demonstrating plasmid transfer, the researchers mixed the pastured-poultry E. coli with recipient bacteria and checked whether resistance genes changed hands. The results were unequivocal: blaTEM-carrying plasmids of the IncI2α and IncX2 types successfully transferred into both a laboratory strain of E. coli and Salmonella enterica serovar Enteritidis, a major foodborne pathogen. This is a critical demonstration, because it shows that the resistance genes found on these farms are not locked in place. They can hop into Salmonella, a bacterium that causes an estimated hundreds of thousands of foodborne illnesses in the United States each year, potentially creating newly resistant pathogens in the process.

The virulence side of the analysis added another layer of concern. Whole-genome screening for virulence genes revealed that some of the MDR isolates carried genetic determinants associated with the ability to cause disease, meaning these were not merely harmless commensal bacteria that happened to be resistant. The combination of virulence potential and transmissible resistance on the same mobile elements is precisely the scenario that public health authorities fear most, because it packages the capacity to sicken and the capacity to survive treatment into a single transferable package.

The authors emphasize that their findings highlight the need to assess the environmental and ecological drivers of antimicrobial resistance in pasture-raised poultry systems. Pastured flocks are exposed to soil microbiomes, wildlife, and environmental reservoirs of bacteria in ways that conventionally housed birds are not, and the open-air setting creates numerous opportunities for resistance genes to circulate between animals, soil, and potentially humans. The study’s genomic data, deposited in NCBI-GenBank under BioProject PRJNA1199014, provide a public resource for further surveillance work. As consumer demand for pasture-raised and antibiotic-free poultry continues to grow, this research serves as a reminder that antibiotic resistance is an ecosystem-level problem, one that does not respect the fence line between conventional and alternative agriculture, and that monitoring the pasture itself may be as important as monitoring the birds that roam it.

Subject of Research: Transmissible antibiotic resistance and virulence genes in multidrug-resistant E. coli from pastured poultry and their environment

Article Title: Transmissible antibiotic resistance and virulence genes in multidrug-resistant Escherichia coli isolated from pastured poultry and their environment in the Southeastern United States

Article References: Hassan, J. W., Al Hakeem, W. G., Rothrock, M. J., Jr, & Kassem, I. I. (2026). Transmissible antibiotic resistance and virulence genes in multidrug-resistant Escherichia coli isolated from pastured poultry and their environment in the Southeastern United States. Environmental Science and Pollution Research. https://doi.org/10.1007/s11356-026-38263-0

Image Credits: AI Generated

DOI: 10.1007/s11356-026-38263-0

Keywords: pastured poultry, Escherichia coli, antimicrobial resistance, multidrug resistance, blaTEM, plasmids, horizontal gene transfer, whole-genome sequencing, conjugation, Salmonella, food safety, One Health

The investigation focused on 14 drinking water treatment plants that draw raw water directly from the Upper Mahaweli River and its tributaries. Researchers collected 45 water samples across the system and cultured the bacteria they contained, ultimately isolating 167 strains of E. coli, the classic indicator organism for fecal contamination in water. Each isolate was tested against ten clinically important antibiotics using the Kirby-Bauer disk diffusion method, the standard laboratory technique in which antibiotic-impregnated discs are placed on bacterial lawns and zones of inhibition are measured to determine susceptibility.

The results were striking. Overall, 60.48 percent of the E. coli isolates exhibited resistance to at least one antibiotic, while multidrug resistance, defined as resistance to three or more antibiotic classes, was detected in 27.54 percent of isolates. The highest levels of resistance were observed for amoxicillin, a widely used penicillin-class drug, at 47.31 percent, followed by tetracycline at 26.95 percent and co-trimoxazole, a combination of sulfamethoxazole and trimethoprim, at 24.55 percent. Notably, resistance to four of the tested antibiotics showed seasonal variation, a pattern that researchers attribute to fluctuations in rainfall, river flow, and the runoff of contaminants from surrounding land during different times of the year.

Beyond the resistance phenotypes, the team went a step further and searched for the molecular machinery behind the phenomenon. Genomic DNA was extracted from the culturable bacteria in each of the 45 water samples and screened by polymerase chain reaction for 11 antibiotic resistance genes, or ARGs. Across 210 PCR assays, 42.42 percent returned positive detections. The most abundant genes were blaTEM, which encodes a beta-lactamase enzyme that degrades penicillin-type antibiotics, found in 80.0 percent of samples, followed by tetA at 66.67 percent, and tetM and qnrS, each detected in 62.22 percent of samples. The tet genes protect bacteria from tetracycline through efflux pumping and ribosomal protection respectively, while qnrS confers reduced susceptibility to fluoroquinolones, a class of critically important human medicines.

To quantify the overall burden of resistance, the researchers calculated two indices. The multiple antibiotic resistance index, or MAR index, which reflects the average number of antibiotics to which isolates are resistant, ranged from 0.00 to 0.80, with 44.91 percent of samples exceeding the 0.2 threshold that signals a high-risk source of contamination, typically one associated with human or animal waste. The antibiotic resistance index ranged from 0.00 to 0.32, with eight sampling points above the same threshold. These values suggest that the contamination entering the river is not random background noise but reflects sustained pressure from sources where antibiotics are used and excreted, such as households lacking adequate sanitation, livestock operations, and agricultural runoff.

Hierarchical cluster analysis, a statistical technique that groups sampling sites according to the similarity of their resistance profiles, revealed a clear spatial pattern. The majority of drinking water sources fell into an intermediate category of contamination, but a few stood out at the extremes. The Kotagala and Thalawakelle-Galkanda intakes showed the lowest resistance burdens, while Haragama, Paradeka, and Nawalapitiya emerged as hotspots with the highest levels. This variability mirrors the intensity of anthropogenic interference along the river, with densely populated towns, commercial activity, and agricultural land leaving distinct fingerprints on the microbial ecology of the water at each intake.

The Upper Mahaweli River is not a marginal water body. It is the longest river in Sri Lanka and the backbone of water supply for the central hill country, feeding treatment plants that serve towns and cities across the region, including greater Kandy. The finding that antibiotic-resistant bacteria and their genes are present upstream of drinking water treatment infrastructure matters because conventional treatment is not always designed to remove them. Chlorination, the most common disinfection step, can kill susceptible bacteria but has been shown in other studies to sometimes promote the horizontal transfer of resistance genes between surviving organisms, and free DNA carrying ARGs can persist in water even after the cells that hosted them are destroyed. Resistance genes are also mobile: they ride on plasmids and transposons that can shuttle between species, meaning that harmless environmental bacteria can serve as reservoirs from which resistance traits eventually reach pathogens.

The study’s authors emphasize that the presence of AR-E. coli and ARGs in these drinking water sources represents a genuine public health risk, particularly in a tropical South Asian setting where the environmental dimensions of antimicrobial resistance remain poorly documented. Antimicrobial resistance is globally recognized as one of the leading threats to modern medicine, and aquatic environments are increasingly understood as critical corridors through which resistance genes travel between humans, animals, and ecosystems. In Sri Lanka, national surveys have documented substantial antibacterial consumption in both human and veterinary medicine, and previous work in the country has already identified resistant bacteria in hospital wastewater, recreational river water, poultry operations, and the Kelani River, another major waterway. The new findings extend this picture to the drinking water supplies of the hill country itself.

What makes the results especially consequential is the seasonal dimension. The detection of seasonal variation in resistance to four antibiotics suggests that monsoon-driven hydrology plays a role in mobilizing contamination, washing resistance determinants from soils, latrines, and farms into the river during periods of heavy rain. As climate change alters rainfall patterns across South Asia, such episodic pulses of contamination may become more frequent or intense, compounding the challenge for water utilities that must deliver safe water under increasingly variable conditions.

The researchers conclude that proactive interventions are urgently needed to safeguard the long-term safety of drinking water sources along the Upper Mahaweli. Such measures would likely include improved sanitation and wastewater management in riparian communities, better regulation of antibiotic use in agriculture and livestock, routine surveillance of resistance in source waters, and evaluation of whether existing treatment trains adequately remove both resistant bacteria and free-floating resistance genes. In a world where the pipeline from environment to clinic grows shorter every year, the study stands as a warning that the battle against antimicrobial resistance will be fought not only in hospitals and pharmacies, but in the rivers from which people drink.

Subject of Research: Antibiotic-resistant E. coli and antibiotic resistance genes in drinking water sources of the Upper Mahaweli River, Sri Lanka

Article Title: Occurrence of antibiotic-resistant E. coli and antibiotic resistance genes from culturable bacteria in drinking water sources along the Upper Mahaweli River, Sri Lanka

Article References: Occurrence of antibiotic-resistant E. coli and antibiotic resistance genes from culturable bacteria in drinking water sources along the Upper Mahaweli River, Sri Lanka. (n.d.). https://doi.org/10.1007/s10661-026-15884-z

Image Credits: AI Generated

DOI: 10.1007/s10661-026-15884-z

Keywords: antibiotic resistance, E. coli, antibiotic resistance genes, drinking water, Mahaweli River, Sri Lanka, multidrug resistance, blaTEM, water quality, public health, environmental microbiology, antimicrobial resistance