Biofilms and Mare Infertility

A mare’s reproductive tract and immune system are designed to protect her against infection. However, biofilm can form in a mare’s uterus when bacterial breach her other lines of defense against reproductive tract infection.

Photo: Photos.com

Biofilm. No, it’s not a documentary playing at your local art-house theater or the newest sci-fi flick, but it might be what’s standing between your mare and pregnancy.

Bacterial infection of a mare’s reproductive tract, or endometritis, is a significant cause of equine infertility. The condition is often chronic and resistant to treatment, which is frustrating for mare owners, veterinarians, and researchers and can cause significant economic loss for the horse industry, said Ryan Ferris, DVM, MS, Dipl. ACT, of Colorado State University’s (CSU) Department of Clinical Sciences.

With an eye on chronic endometritis in the mare, Ferris has spent the past two years studying biofilms—a group of any microorganisms that stick to each other and a surface—and their possible relationship to mare infertility. He presented his team’s preliminary research findings at the 2014 American College of Theriogenology (Animal Reproduction) Symposium held Aug. 6-9 in Portland, Oregon.

Biofilm: Stronger Together

For the past 100 years, Ferris said, veterinarians and doctors have thought of bacteria, which are single-celled organisms, individually. These lone bacteria live in a planktonic state, floating around by themselves, and in some cases propelled by flagella (think of the bacterium as a raft and the flagella as the oars).

More recently, however, researchers have discovered bacteria and other microorganisms (such as fungi) can colonize, and live together as a biofilm. Researchers now consider the biofilm state a “more prevent lifestyle for bacteria (compared to the planktonic state),” Ferris said, with an estimated 99% of the world’s overall biomass living in biofilm.

Within a biofilm, microorganisms literally stick together via a self-formed exopolysaccharide matrix (or structure) that can also contain DNA, RNA, lipids, and proteins. Once they reach the biofilm, self-propelled bacteria give up or shed their flagella and become immobile when joining the colony and its protection. This matrix, which Ferris described as “goolike,” protects the innermost organisms from outside assaults.

Stained biofilm on pegs in the laboratory.

Photo: Ryan Ferris, DVM, MS, Dipl. ACT

“Research shows that bacteria residing in a biofilm can be up to 1,000 times more resistant to treatment with antibiotics as compared to free-living bacteria,” Ferris said.

That’s likely because forming a biofilm allows bacteria to go unrecognized by the host’s immune system (the host body doesn’t “see” the microorganisms within the biofilm), prevents exposure of the bacteria to antibiotics, and the horizontal exchange of beneficial genetic material between the bacteria.

“Within a biofilm community there are millions of bacteria,” Ferris explained. “If one of these bacteria are resistant to an antibiotic, this resistance can be transferred to all the bacteria in the colony.”

This leads to antibiotic resistance. “In 2012, 80% of infections in humans are associated with biofilm, a $20 million health care cost in in human medicine,” he added. “In equine medicine, we have just started investigating the role of biofilms in chronic infections.

In the Mare

A mare’s reproductive tract and immune system are designed to protect her against infection. However, Ferris described how biofilm can form in a mare’s uterus when bacterial breach her other lines of defense against reproductive tract infection. Those defenses include:

  1. Physical barriers, comprising her vulva, vagino-vestibular sphincter muscle, and cervix, which block feces, air, and environmental pathogens from reaching the uterus;
  2. Her innate immune system, which results in inflammation an fluid production; and
  3. Mechanical uterine clearance.

“A single alteration to any of these defense mechanisms of a mare may allow for colonization of the uterus with a bacterial pathogen, leading to chronic infection,” he said.

That chronic infection, researchers believe, might be related to the establishment of a biofilm. “However, to date, in vivo (in the body) biofilm production and identification has not occurred in the endometrium of the mare,” Ferris pointed out. “And, unfortunately, no clinical diagnostic tests are available for the detection of biofilm-related infections.”

Biofilm growing at the liquid/air interface in a test-tube.

Photo: Ryan Ferris, DVM, MS, Dipl. ACT

The Preliminary Study

In unpublished work from 2014, Ferris and his team at CSU isolated bacteria from the equine uterus, and their preliminary findings suggest that Streptococcus equi subspecies zooepidemicus, Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumonia are capable of producing biofilm in vitro (in a laboratory setting).

In both human and veterinary medicine, antibiotic administration alone has been unable to eliminate chronic infections suspected of involving biofilm, Ferris said. So, he and his team further investigated the use of non-antibiotic treatment for biofilms, such as acetylcysteine, Ceragyn, tris-EDTA, DMSO, hydrogen peroxide solutions, tricide, and hypochlorous acid in vitro on strains of P. aeruginosa, E. coli, K. pneumonia, and S. equi subsp. zooepidemicus.

Work in other species has shown that tris-EDTA (a buffering chelating agent) might help break biofilms, Ferris said, by perforating the biofilm matrix and making it more susceptible to antimicrobial treatment. However, his team found that tris-EDTA alone, was unable to consistently disrupt preformed biofilm in vitro.

Hydrogen peroxide is a commonly used antiseptic in human and veterinary medicine, Ferris said, and a 1% solution has been suggested to be helpful in treating mare endometritis. His study found that a 1% hydrogen peroxide disrupted the biofilm 50% of the time in vitro.

Take-Home Message

Unfortunately, in cases of endometritis that might be caused by biofilm, treatment failure is common, Ferris said. “Identification of the organism present in a mare’s uterus is required to select the appropriate agent to help in degradation of a biofilm,” he concluded.

About the Author

Michelle N. Anderson, TheHorse.com Digital Managing Editor

Michelle Anderson serves as The Horse's digital managing editor. In her role, she produces content for our web site and hosts our live events, including Ask the Vet Live. A lifelong horse owner, Anderson competes in dressage and enjoys trail riding. She's a Washington State University graduate (Go Cougs!) and holds a bachelor's degree in communications with a minor in business administration and extensive coursework in animal sciences. She has worked in equine publishing since 1998. She currently lives with her husband on a small horse property in Central Oregon.

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