Uncovering linezolid resistance in Enterococcus isolates from Tunisian traditional healthy chickens: A One health surveillance study

In the quiet corners of rural Tunisia, a different kind of farming thrives. It is not the vast, industrial operations seen in many parts of the world, but small-scale, traditional poultry keeping where families raise chickens for their own tables or local sale. These birds live in close contact with their owners and the surrounding environment, a setup that defines a significant part of the local economy. Within the guts of these healthy chickens live bacteria called enterococci. For the most part, these microbes are harmless residents, part of the natural community inside the animal. However, some strains of enterococci can cause serious infections in humans, and a growing worry is that these bacteria are learning to ignore the medicines doctors use to treat them. Among the most powerful tools in a doctor's arsenal is an antibiotic called linezolid, a drug reserved for the toughest infections when other treatments fail. If bacteria learn to resist this last line of defense, the consequences for public health could be severe. The question researchers face is whether these resistant bacteria are hiding in the places we least expect, such as the traditional farms that supply food to local communities.

A team of scientists set out to investigate this possibility by looking directly at the intestines of chickens from eight different traditional farms in northern Tunisia. Between October 2020 and July 2021, they collected samples from the ceca, which are the pouches at the end of the chicken's intestine, from 107 healthy birds. The researchers did not use any anesthesia or special procedures; they simply gathered the samples immediately after the chickens were slaughtered for food, a routine part of local practice. They then grew the bacteria from these samples in a laboratory, identifying the specific types of enterococci present and testing how they reacted to a wide range of antibiotics. The goal was to see if any of these bacteria had developed resistance to linezolid or other drugs, and to understand the genetic makeup of any resistant strains they found.

The study revealed that while the chickens were healthy, their gut bacteria were not entirely innocent. Out of the 105 distinct bacterial strains identified, a small but significant number showed resistance to linezolid. Specifically, seven samples, representing about 6.5 percent of the total, contained bacteria that could survive the presence of this powerful drug. These resistant bacteria belonged to three different species: Enterococcus faecalis, Enterococcus faecium, and Enterococcus gallinarum. What made this discovery particularly important was the genetic mechanism behind the resistance. Every single one of these seven resistant strains carried a specific gene called optrA. This gene acts like a shield, allowing the bacteria to pump the antibiotic out of their cells before it can do any harm. The researchers did not find other known resistance genes, such as cfr or poxtA, suggesting that optrA was the sole driver of this resistance in these particular chickens.

The spread of these resistant bacteria was not limited to a single location. The seven resistant strains were found across three different farms, some of which were separated by a distance of 57 kilometers. In one striking instance, two chickens from farms this far apart both carried Enterococcus faecium bacteria that were not only resistant to linezolid but were also genetically identical, belonging to a newly identified family type known as ST2516. This suggests that the same strain of bacteria is moving between different farms, likely through the environment or the movement of people and animals. The researchers also noted that these resistant bacteria were not just resistant to linezolid; they were often resistant to many other common antibiotics as well, including tetracycline and ciprofloxacin, making them what is known as multidrug-resistant.

Beyond the antibiotic resistance, the researchers looked at whether these bacteria carried genes that make them more dangerous to humans, known as virulence factors. They found that most of the linezolid-resistant strains carried a gene called hyl, which produces an enzyme capable of breaking down tissue barriers in the body. This ability helps bacteria invade and spread within a host. One of the resistant strains also carried a gene called agg, which helps bacteria stick together and attach to cells. While the chickens themselves showed no signs of illness, the presence of these aggressive traits in bacteria that can survive powerful drugs raises concerns about how easily they could move from the farm to a human. The farmers reported that they never used antibiotics on these chickens, yet the resistance was still there. This points to the possibility that the bacteria are picking up resistance genes from the environment, perhaps through contaminated water sources or food waste that the chickens consume, rather than from direct medical treatment.

The findings paint a picture of a "low noise" spread of resistance, where dangerous bacteria are circulating quietly in traditional farming systems that often go unmonitored. Unlike large industrial farms where antibiotic use is tracked, these small, family-run operations operate with less oversight, creating a hidden reservoir for resistant bacteria. The study highlights that the resistance to linezolid, a drug of last resort, is not confined to hospitals or intensive farming but is present in the traditional rural economy. The researchers emphasize that without better surveillance and control measures in these environments, these resistant bacteria could continue to spread, eventually reaching humans through the food chain or direct contact. The discovery of the optrA gene in Enterococcus gallinarum in this region marks the first time this specific resistance has been found in this type of bacteria in African traditional poultry, adding a new layer to the global understanding of how antibiotic resistance travels.

Ultimately, the work underscores the need for a broader approach to health that connects the well-being of animals, the environment, and people. The presence of these resistant strains in healthy chickens suggests that the barrier between farm and human health is thinner than previously thought. By identifying these hidden threats in traditional farming, the study provides a crucial warning: to protect the effectiveness of life-saving medicines, monitoring must extend beyond the clinic and into the quiet, unregulated spaces where our food is raised. The data gathered here serves as a call to action for more rigorous surveillance, ensuring that the spread of these resistant bacteria can be detected and managed before it becomes a crisis for public health.