Person holding a chicken

Fighting AMR starts with chicken microbiota

Antimicrobial resistance (AMR) is prevalent in all agriculture; including soil, water and various foods. AMR has been around forever, but only became a problem when antibiotics, the miracle drugs of the 20th century, began losing their effectiveness. Few researchers are developing new classes of antibiotics, but one genomics professor in Australia is attacking the issue from a different angle: In the gut microbiota of poultry. 

When genomics researcher and professor Dragana (Dana) Stanley, Ph.D., began her career, she was truly terrified of chickens. The fact that she is currently a leading expert in poultry microbiota and antimicrobial resistance (AMR) is a testament to her determination.

Her saga began when, as a young child, she was mercilessly pecked by a rooster. “I still have scars,” Stanley says. The fear lasted into adulthood.

But, she was also a realist. She had just received her Ph.D. and was the mother of two. Traveling a long distance to work wasn’t feasible. “I applied for a postdoc job under Robert Moore, who was a leader in microbiology and poultry research. The work involved studying gene expression and genetics – the exact areas in which I did my Ph.D.” The catch? It was at a chicken microbiology lab at Australian Animal Health Laboratory (now renamed to Australian Centre for Disease Preparedness) in Geelong, Victoria.

Undeterred, Stanley went to introduce herself. “We had a lovely interview. At the end, the interviewers asked if there was anything else I wanted to add. I said, ‘I faint when I see chickens.’ They laughed till they cried,” the academic recounts. But Stanley got the job.

Today, she heads a team of researchers at Central Queensland University (CQU) in Rockhampton, Australia, that delves into the interrelationships between human and animal health, including the global challenge of antimicrobial resistance. 

The use of antimicrobial growth promotors have been standard in the agricultural industry for decades. As has free-range farming and the separation of chicks from hens before hatching. Unfortunately, this has significantly contributed to antimicrobial resistance, or AMR, in poultry farms. Molecular microbiologist Dana Stanley, Ph.D., explains the issue and how she is fighting AMR at the root of the problem: By developing innovative ways to protect chickens from the inside out. 

There was never, ever a situation where we had millions of tons of antibiotics released in such a purified state into the nature.

Dana Stanley, Ph.D.
Professor at Central Queensland University Australia

The danger of antimicrobial resistance

Antimicrobial resistance occurs when microbes no longer respond to antibiotic treatments. Since the 1940s, when penicillin was first introduced, scientists have warned AMR would eventually occur. Soon after, the first resistance cases were documented.

Since then, AMR has escalated, spreading through agricultural animals and, through their manure, to soil, plants and humans throughout the world. 

Stanley explains that there are penguins and seal colonies in Antarctica that are physically isolated from the rest of the planet, yet nevertheless their gut bacteria carry antimicrobial resistance. 

Infections today are harder to treat than they were even a few years ago because antibiotics are losing their effectiveness against evolved microbes. Farm workers, who are closest to these antibiotic-resistant microbes, are at particular risk and experience more AMR-related deaths than any other group. 

They’re not the only people at risk, though. As AMR becomes increasingly prevalent, the chance of dying from a slight infection caused by a nick from a rose bush while gardening or a scraped knuckle, not to mention surgery, increases. 

We face the prospect of a world where antibiotics are no longer effective. In such an environment, surgeries, chemotherapy and even C-sections could become impossible from a safety standpoint. They are already becoming more dangerous. In 2022, the Lancet reported that 1.2 million people died because of antimicrobial resistance.

We can address the challenge of AMR, but “it requires global action,” Stanley says. Overuse of antibiotics in one region easily contaminates the rest of the world. “It’s like adding a drop of ink to a glass of water, ink will diffuse until it all becomes blue.”

Dana Stanley, Ph.D.

Dana Stanley, Ph.D. is a professor at Central Queensland University Australia, where she leads the Molecular Microbiology and Poultry Health research cluster in the Institute for Future Farming Systems. Stanley heads a team of researchers delving into interrelationships between human and animal health, including the global challenge of antimicrobial resistance.

Now more than ever, agricultural animals need proper disease control that does not overlap with clinical antibiotics that we use for human health.

Dana Stanley, Ph.D.
Professor at Central Queensland University Australia

AMR is as old as mummies

AMR is not a modern phenomenon, Stanley points out. “Resistant microbes existed in nature long before we discovered them, and mummies that are thousands of years old have antimicrobial resistance to all known antibiotics, including those just recently developed.”

Stanley’s fascination with AMR started at a conference. “My presentation was about microbiota,” she recalls, in line with the focus of the lab she’d recently set up at CQU to explore microbial genomics, gene expression of the host, and also human and animal cell cultures.

“Three of the speakers were top-level experts in antimicrobial resistance. We spent more than 10 days together and, after hearing their presentations multiple times, I was fascinated. AMR became a hot topic. I came back determined to look into it a bit deeper.”

Soon after that conference series, Stanley analyzed metagenomic sequencing data from the chicken gut. She was shocked when the analysis showed astonishingly high levels of antimicrobial resistance inside the chicken gut. 

“Friends in the field confirmed that it really is that bad. I started looking at manure and faecal samples to get a grasp of the issue. You can find hundreds of different resistant genes in the guts of livestock and poultry, but the human gut is among the worst. Ultimately, all faecal material is a possible source of AMR.”

Dana Stanley, Ph.D.
Stanley has built a strong working relationship with Australia’s leading egg producers. “I’ve done research on 100,000-bird projects, and a project where we hatched half a million birds when we worked in the hatchery,” she says. “This is part of an effort to control the core microbes in the gut to ensure reproducible outcomes for any treatments or vaccines.” The huge numbers of birds involved equate to more accurate results.

We use RNA-seq, which is the method that allows us to see the expression and change in expression of every single gene in the animal organ in response to use of phytogenic or other alternatives to antibiotic products.

Dana Stanley, Ph.D.
Professor at Central Queensland University Australia

Sequencing microbiota

Another way to tackle AMR is to replace the use of antibiotics in livestock production and other areas of agriculture with natural alternatives. At the moment one of the most promising and widely used alternatives to antibiotics are plant phytogenic products. These are active antimicrobial compounds from well known antimicrobial plants like garlic, oregano or eucalypts.”

“We use RNA-seq, which is the method that allows us to see the expression and change in expression of every single gene in the animal organ in response to use of phytogenic or other alternatives to antibiotic products.”

To analyze the RNA-seq data, she exports it to QIAGEN Ingenuity Pathway Analysis (IPA). IPA includes analysis methods that bring the investigation to another level, allowing the researcher to make reliable predictions based on the expression of marker genes. This way we were able to see that the use of these antibiotic alternatives protects from pathogens and AMR while also improving bird’s health and immunity in so many unexpected ways, Stanley says. “The output can be visualized immediately, and much more clearly than with other analysis methods.”

“IPA allows us to get a heatmap, for example, mapping existing diseases and seeing whether specific products will predispose the animal towards developing those diseases.”

Now, she’s aggregating all of the data they gather on antibiotic alternatives into IPA Analysis Match database to maximise the predications she can get from each dataset. 

The potential is exciting. “When we pinpoint the molecules in phytogenic antibiotic alternatives, that are responsible for the antimicrobial effect, we can then deliver a high amount of these molecules like we would deliver antibiotics. They often give us even stronger and better pathogen control, using IPA to predict overall health effects helps us to make sure that we will produce additional benefits for the bird health and welfare” explains Stanley.

tubes with plants
For thousands of years, plants have been used a for their antibiotic properties. . What’s different today is their scale and concentration. Rather than administering these plant chemicals, called phytogens, as a tea, modern scientists synthesize them to make more potent mixtures. Many phytogenic products are already commercialized as alternatives to antibiotics, and chicken producers are beginning to use them for prophylactic treatment. 

IPA is a powerful tool for predicting the benefits and side effects of treatments, as well as identifying potential interactions and pathways at the molecular level.

Dana Stanley, Ph.D.
Professor at Central Queensland University Australia

Natural antimicrobials and designer microbiota

The situation is serious, but not as dire as some suggest. “We’re not returning to the Middle Ages (of medicine), where a paper cut could kill you,” Stanley says. 

Stanley takes a multi-pronged approach: One is to develop natural, plant-based antimicrobial or antibiotic treatments. The other is to change the microbiota in the guts of farm animals to make them less susceptible to pathogens.

“Plants are experts at making antibiotics or antimicrobials,” Stanley points out. “Oregano, for example, is antibacterial, antiviral, antifungal, anti-insect and promotes the immune system.” That’s because one plant may have 50 different polyphenols, and therefore offers multiple benefits. 

In fact, one of her studies involving 40,000 pullets that, instead of antibiotics, were fed a phytogen supplement of menthol (found in peppermint and other mint plants), carvacrol (found in oregano) and carvone (found in caraway seeds, spearmint and dill) had a lower rate of mortality and produced higher-quality eggs, all without creating antimicrobial resistance. In a separate project involving 40,000 birds, Stanley reported the phytogenic blend also improved intestinal health and reduced obesity, cholesterol and cancer.

Stanley also is developing “designer microbiota” that are introduced to the birds as soon as they hatch to prevent access to pathogens in their first few days of life. The results appear positive. During the collaboration with a commercial hatchery, the research project included a half-million birds, while others involved 100,000 birds at a time. The overall goal, she says, “is to control colonization of the gut,” enabling more predictable, reproducible results when vaccinating or treating the birds later. “There’s great variability among birds,” Stanley points out. 

She’s performing similar work with pigs, but to remove AMR. One such project involves fragmenting the DNA in manure so there are no plasmids that can transfer AMR into the soil. 

These projects coalesce around reducing antimicrobial pressure on the environment by limiting the need for antibiotics. Evidence for this is promising; AMR levels were lower in regions – notably Scandinavia – where antibiotics are used much more sparingly, and only to treat ill livestock (and humans).

The most exciting aspect of her work, Stanley says, is problem-solving and engaging with people. “I like working with industry and knowing that the outcome of our experiments is applicable and interesting to producers and farmers because it makes their lives easier.”

And she has long overcome her fear of poultry, first by hatching and raising quail, and eventually raising exotic chickens at home in a sustainable chicken coop.

Dana Stanley, Ph.D.
By coupling IPA analysis with the identification of AMR genes in the intestinal tract using CLC Genomic Workbench and the Microbial Genomics plugin, we can achieve a comprehensive understanding of the effects of the treatment on AMR carriage”, explains Stanley. “This combination of tools enables my team to assess the impact of the treatment on the presence and expression of AMR genes in the microbial community and provides valuable insights into the treatment's effects on the host and the potential risks associated with AMR.

June 2023