Raw Milk Emerges as Reservoir of Virulent, Drug-Resistant Listeria in Iranian Dairy Study

A sweeping surveillance study of the dairy supply chain in Yazd province, Iran, has revealed that raw milk is the principal reservoir of Listeria monocytogenes, a foodborne pathogen that ranks among the deadliest threats circulating in the global food system. Researchers collected 370 samples spanning raw milk, pasteurised commercial milk, milking equipment, transport containers, and the hands and gowns of dairy workers between 2022 and 2023, and confirmed ten isolates of the bacterium using standardised biochemical and molecular techniques. Although the overall prevalence of 2.7 percent may sound modest, the distribution of those positives tells a far more consequential story about where the pathogen hides and how it travels through the products that reach consumers.

Raw milk accounted for nearly all of the contamination. Nine of the 170 raw milk samples drawn from dairy cooperatives and collection centres tested positive, a prevalence of 5.29 percent, while a single positive emerged from the 60 samples taken from milking apparatus and transit containers. Strikingly, none of the 90 pasteurised milk samples and none of the 50 worker-related swabs harboured the organism. Eight of the ten raw milk collection centres surveyed showed at least some contamination, with the highest rates reaching 10 percent of samples at individual cooperatives. The pattern is unambiguous: the early, unprocessed stages of the milk chain are where L. monocytogenes persists, and the industrial treatments applied downstream are doing their job.

The complete absence of the pathogen from pasteurised milk is a direct endorsement of the high-temperature, short-time protocol used by the commercial dairies in the region, which heat milk to between 72 and 75 degrees Celsius for 15 seconds before packaging. L. monocytogenes is a non-spore-forming bacterium and is highly vulnerable to these time-temperature combinations, provided that strict hygiene is maintained after treatment to prevent recontamination. The finding matters because listeriosis, the disease the organism causes, carries hospitalisation and mortality rates that are exceptionally high among infants, the elderly, pregnant women, and immunocompromised patients. Unlike many foodborne infections that produce unpleasant but self-limiting illness, listeriosis can invade the bloodstream and central nervous system, making every point of control in the dairy chain a matter of clinical consequence.

What elevates the study beyond a routine prevalence survey is the molecular characterisation of the recovered isolates. Using polymerase chain reaction, the team screened the bacteria for five major virulence genes that govern the organism’s ability to invade and survive inside human cells. Every one of the ten isolates carried prfA, the master transcriptional regulator that switches on Listeria Pathogenicity Island 1, along with inlA, an internalin essential for breaching the intestinal barrier, and iap, a marker associated with invasion and virulence. The gene inlB, which works with inlA to drive uptake into host cells, was present in 90 percent of isolates, and hlyA, which encodes listeriolysin O, the pore-forming toxin that lets the bacterium escape the phagosome and spread cell to cell, was found in 90 percent as well. In practical terms, the strains circulating in Yazd’s raw milk carry the full genetic toolkit for invasive human disease.

One isolate lacked hlyA entirely, a detail with real diagnostic implications. Strains missing the gene for listeriolysin O are considered atypical and often non-haemolytic, meaning they fail the blood-agar tests that many laboratories rely on for routine identification. Such organisms can slip through phenotype-based screening undetected, yet they may possess alternative invasion mechanisms or acquire virulence factors through horizontal gene transfer. Their presence in the food supply is a recognised challenge for public health monitoring, because the diagnostic net designed to catch L. monocytogenes has holes exactly where these variants swim.

The antimicrobial resistance picture is more nuanced, and in some respects reassuring. The isolates showed the expected intrinsic resistance to nalidixic acid, penicillin G, ceftazidime, ceftriaxone, and cefepime, a pattern long documented in this species and the reason cephalosporins are never used to treat listeriosis. More encouragingly, all ten strains were fully susceptible to gentamicin, amikacin, levofloxacin, ciprofloxacin, vancomycin, chloramphenicol, trimethoprim-sulfamethoxazole, ampicillin-sulbactam, and erythromycin, the workhorses of clinical therapy. Resistance to tetracycline appeared in 30 percent of isolates, and imipenem showed a split profile, with half the isolates resistant and half intermediate. Twenty percent of isolates exceeded the multiple antibiotic resistance index threshold of 0.2, a value interpreted as a signature of environments with heavy antibiotic exposure, and the highest indices belonged to strains resistant to imipenem, clindamycin, and tetracycline simultaneously.

The genotyping of resistance determinants uncovered a finding that may prove the most strategically important in the entire study: several resistance genes were present in isolates that remained phenotypically susceptible. The lincosamide resistance genes lnuA and lnuB were detected in 90 and 80 percent of isolates respectively, the macrolide-lincosamide-streptogramin genes ermA and ermB in 40 and 30 percent, the aminoglycoside gene aadB in 30 percent, and the tetracycline genes tetA and tetM, which mediate efflux and ribosomal protection, in 30 percent each. Yet clindamycin susceptibility remained at 90 percent and erythromycin at 100 percent. This silent carriage of resistance genes, likely due to non-functional promoters, regulatory mutations, or the absence of environmental triggers needed for expression under laboratory conditions, means these strains hold the genetic blueprint to flip to a resistant phenotype through gene activation or horizontal transfer. Phenotypic testing alone would miss the threat entirely, which is why the authors argue that molecular surveillance must run alongside conventional susceptibility testing.

The coexistence of a complete virulence gene complement with a broad array of resistance determinants in the same small set of isolates paints a picture of strains that are both capable of causing severe disease and equipped to survive therapeutic pressure. The researchers contextualise their findings against international data: prevalence figures of 18.46 percent in South African milk samples, 28.26 percent in milk from aborted cows in Iraq, and 2.48 percent in bovine milk in Southwest China all confirm that milk is a substantial and recurring reservoir for this pathogen worldwide. Comparable genomic surveys in dairy products across India and China have likewise documented the ubiquitous presence of prfA, inlA, inlB, iap, and hlyA alongside multidrug resistance, suggesting that the Yazd findings reflect a global pattern rather than a local anomaly.

The study is not without limits, and the authors acknowledge them candidly. The low isolation rate yielded only ten confirmed strains, a sample too small to generalise precise resistance percentages across the entire province. Nevertheless, the detailed molecular characterisation provides a baseline for future large-scale monitoring in a region where such data have been scarce. The contamination of 80 percent of raw milk collection points points to hygiene failures at the earliest stage of the chain, where faecal contamination during milking, mastitic glands, and environmental reservoirs such as silage, soil, and water all offer the bacterium entry routes. The single positive from milking equipment reinforces the well-established capacity of L. monocytogenes to form resilient biofilms on food-contact surfaces, which complicates sanitation in processing facilities.

The practical message for consumers and regulators alike is clear. Pasteurisation works, and the zero detection rate in commercial pasteurised milk demonstrates that the technology, properly applied, eliminates the pathogen from the final product. The risk concentrates in raw milk, which is sometimes consumed directly or used in traditional dairy products, exposing vulnerable populations to strains that are simultaneously highly virulent and increasingly drug-resistant. The authors call for continuous monitoring of L. monocytogenes, rigorous hygiene in raw milk production, judicious antibiotic use in veterinary medicine, and routine molecular surveillance to prevent the circulation of pathogenic and resistant strains. As antimicrobial resistance continues its global advance, studies like this one demonstrate that the front line of food safety runs directly through the dairy farm, and that the tools to track the threat, from PCR screens for virulence genes to resistance indices, are already in hand.

Subject of Research: Prevalence, virulence genes, and antimicrobial resistance of Listeria monocytogenes in the dairy supply chain

Article Title: Molecular Characterisation and Antimicrobial Resistance Patterns of Listeria monocytogenes From Dairy Sources

Article References: Heidari, M. R., Shakerian, A., Chaleshtori, R. S., & Rahimi, E. (2026). Molecular Characterisation and Antimicrobial Resistance Patterns of Listeria monocytogenes From Dairy Sources. Veterinary Medicine and Science, 12(6), Article e71265. https://doi.org/10.1002/vms3.71265

Image Credits: AI Generated

DOI: 10.1002/vms3.71265

Keywords: Listeria monocytogenes, raw milk, foodborne disease, antimicrobial resistance, virulence genes, pasteurisation, dairy safety, PCR, MAR index, listeriosis, Iran, public health