A 42-year old loggerhead sea turtle suffered from a septicemic cutaneous ulcerative disease (SCUD) for the past 4 years, over time, the infection became resistant to most antibiotic treatments. Through collaboration between veterinarians and researchers from several universities, phage therapy in combination with antibiotics was administered using multiple routes of application over an 8 month period, leading to a reduction in clinical symptoms.

Here I provide a recap of the recent paper detailing this case: The Use of Bacteriophages and Immunological Monitoring for the Treatment of a Case of Chronic Septicemic Cutaneous Ulcerative Disease in a Loggerhead Sea Turtle Caretta caretta, finishing with a ‘behind the paper’ Q&A with Dr. Whitney Greene, the lead veterinarian on the case!

The clinical case

In 2014, a 42-year old female loggerhead sea turtle was diagnosed with Septicemic Cutaneous Ulcerative Disease (SCUD), a bacterial shell infection of aquatic turtles. Clinical symptoms of SCUD included loss of pigmentation of the shell, lesions and soft spots of the shell. The gram-negative bacterium, Citrobacter freundii, was isolated from the infection sites and displayed multidrug resistance to various treatment options. Antibiotics were administered previously, however, were not favourable as they require consistent dosing and were not effective at clearing the infection. It is also worth noting that the carapace of a sea turtle is made of bone, which is a primary reason why this disease was difficult to treat, as many drugs can’t penetrate bone to therapeutic levels. Therefore, the infection in the sea turtle presented a promising case for compassionate phage therapy.

Phage therapy treatment & monitoring

Phage therapy was chosen as the therapeutic choice for the SCUD infection in the sea turtle as phages continue to increase in titre when the bacterial host is present, reducing the number of repeated doses. Phages were also chosen as they are generally highly specific, only lysing the bacteria of concern, which has been proven in a previous study where phages did not disrupt the normal bacterial communities in the sea turtle gut. This is important as sea turtles rely on their good gut bacteria to extract nutrients from their food.

Isolation of phages

A total of 10 phages were isolated from raw sewage or wastewater and were mixed together in various combinations as a ‘phage cocktail’ for administration.

Administration of phages

A high titre of phage cocktails (10^12 PFU/mL) were administered using multiple formulations and application routes. In addition to phages, dual therapy with numerous antibiotics (oral and subcutaneous) were administered throughout the treatment period.

• Intravenous: Phages were administered directly into the cervical sinus (vein present in the neck)
• Topical: Phages were administered topically either via (i) syringed into trephined holes of the shell (Figure 1), (ii) syringed into deep pockets while the sea turtle was under anesthesia, or (iii) coated onto the shell using an aerosolized sprayer. In all topical administrations, a minimum of 20 minute contact time was allocated and petroleum jelly was applied prior to water submersion to increase the phage contact time.
• Water: Prior to the dissemination of phages into the water, sodium thiosulfate was added to remove chlorine, as high chlorine concentrations have been shown to reduce phage infectivity in water. Once high titre phages were immersed in the water, the water was not changed for 24 hours to allow for phage absorbance to the sea turtle infection.

Figure 1. White arrows showing holes drilled into the sea turtle shell for topical phage administration.

Treatment monitoring

Prior, during and after phage therapy treatment, blood samples were routinely collected to assess blood counts and chemistry, as well as examine immune markers. Photographs of the turtle cell were also recorded periodically. Culture swabs were also collected for bacterial culturing and bacterial susceptibility to antibiotics.

The outcomes of compassionate phage therapy on sea turtle infection

• Following treatment, majority of the lesions of the shell healed, with the presence of new epithelium and pigment, and the absence of soft spots (Figure 2).
• Even with the improved of clinical symptoms, C. freundii was recovered periodically, alongside a number of other bacterial species, including Enterococcus faecalis and Pseudomonas aeruginosa.
• Interestingly, compared to the high multidrug resistance profile of the C. freundii isolate prior to treatment, the C. freundii isolated at week 35 of treatment was susceptible to numerous antibiotics.

Figure 2. Photographs of shell healing throughout phage therapy treatment in affected sea turtle.

Conclusion

The use of compassionate phage therapy showed to improve the clinical signs of SCUD in a 42-year old sea turtle, with no adverse side effects. The dual therapy of phages and antibiotics in combination resulted in a change to the susceptibility patterns of the C. freundii isolates from high multidrug resistance to susceptible. While further work is required, phage therapy represents an effective treatment avenue for MDR infections in sea turtles and aquatic animals.

Behind the paper interview with Dr. Whitney Greene

We ask Dr. Whitney Greene, DVM, MS (veterinarian) some questions from behind the scenes of the paper!

Q1: What was the process from realising you wanted to try phage therapy to administering phages to the sea turtle?

A: I had no experience with bacteriophage therapy prior to this case.  I came into this case after the turtle had already been through treatment for SCUD for four years. Other clinicians had tried virtually every treatment option available but her infection progressed over time despite all treatments.  Her infection started as a region on her carapace the size of a dime and continued to spread and progress despite extensive treatment with antibiotics, surgery, other oral medications, and many topical treatments. Citrobacter was consistently the bacteria that was isolated, but its susceptibility pattern changed over time. Once it became multidrug resistant, we knew we had to think even more outside of the box than we had been and my mentor suggested bacteriophages. I knew nothing about it but found Dr. Steffanie Strathdee out of UC San Diego and she introduced me to a whole network of extremely intelligent phage researchers.  This collaboration was absolutely key to the success of this project.

Q2: What did you learn about the immune response to phages and the consequences for phage therapy from this study?

A: Difficult to answer this as we do not know much about sea turtle immunology and we suspect that Shelley may have an altered immune response. We did lots of different immune assay tests to try and learn more and compare her response to a control. One interesting finding is there was no seroconversion to the phages during the treatment period. This suggests that phage treatment could be continued without antibody interference in effectiveness.

Q3: How is the sea turtle, Shelley, doing now?

Shelley is doing well!  Though I no longer work with her, I periodically receive updates and have not heard of any recurrence of the clinical signs.

Want to learn more?

• Watch the recording of PHAVES #24, which we hosted Nov 10, where Dr. Greene and Dr. Grose dive further into this case:

YouTube video

Many thanks to Atif Khan and Stephanie Lynch for finding and summarizing this week’s phage news, jobs and community posts!