This experiment is part of the Wildlife Disease Association Challenge Grant Challenge Grant. Browse more projects

Do Pacific Northwest marine mammals carry antibiotic-resistant bacteria from land animals?

By Stephanie Norman, Susan Berta, Jessie Huggins, Cathy King DVM MS PhD, Dyanna Lambourn, Sandra M. Dubpernell, Jennifer Olson, Alysha Evans, and Victoria Souze
Backed by Wildlife Disease Association, Jay Norman, Wendy Clark, Steve Olson, Christi Norman Heal, Barbara Brock, Tim Cuchna, Tag Gornall, Debby Giard, Stefano Gioia, and 113 other backers
Orca Network
Bothell, Washington
BiologyMedicine
DOI: 10.18258/11694
Grant: Wildlife Disease Association Challenge GrantGrant: Wildlife Disease Association Challenge Grant
$9,010
Raised of $7,000 Goal
128%
Funded on 9/20/18
Successfully Funded
  • $9,010
    pledged
  • 128%
    funded
  • Funded
    on 9/20/18

Methods

Summary

A sampling study will be conducted to determine the prevalence (%) of antibiotic-resistant bacteria from samples of dead, stranded marine mammals in the inland waters of Washington State (Salish Sea). Specifically, two local species will be targeted for sampling: harbor seals (Phoca vitulina) and harbor porpoises (Phocoena phocoena). 

These species were chosen for the following reasons: 

  • They occur within the Salish Sea in the greatest number compared to other species 
  • Are the most commonly stranded species within the Sea  (Norman et al. 2004), 
  • Harbor seals and porpoises inhabiting the  Sea tend to stay more localized without traveling great distances, compared to their outer coast cohorts, 
  • These two species would be the most likely to carry antibiotic-resistant bacteria originating from terrestrial sources surrounding the Salish Sea, compared to more migratory or less common species within the Sea. 

Culture samples will be collected from approximately 69 seals and 69 porpoises as equally distributed between the northern half of the Salish Sea and the southern half as possible with as equal age class representation amongst the two species as feasible.

Challenges

Though this is a fairly straightforward study to perform, some potential challenges we might face include finding a sufficient number of fresh stranded carcasses to sample and provide the relative balance of equal numbers of samples by region and species. By including numerous volunteers from each of the team member organizations, a wide coverage area will be covered by the stranding network to ensure timely and successful recovery of carcasses. Based on historical average yearly stranding numbers and species within the Salish Sea, our project sampling goals should be reached. Another potential challenge is the overall study sample size. Small sample size statistical methods will be used to help address this issue. The number of total animals we have chosen to sample (138) should yield a relatively strong study. 

Pre Analysis Plan

We predict with the following hypotheses that antibiotic-resistant bacteria prevalence (%):

  • Will be less than 5%.
  • Will be different between harbor seals and harbor porpoises.
  • Will be different between the two (northern and southern) sampling regions.

The following analyses will be used to test these hypotheses:

  1.  Descriptive frequencies will be calculated for isolation of each aerobic bacterium from both collection regions (North and South Salish Sea) and for both species (harbor seals and harbor porpoises). 
  2. Chi-square tests will be used to compare resistance to each antibiotic by organism and to analyze resistance patterns by collection site (feces and any other lesions observed at necropsy). The proportion of resistance for each organism and for all organisms pooled will be compared between the two sampling regions. 
  3. Odds ratios with their 95% confidence limits will be calculated to estimate risk of antibiotic resistance between the two regions.

For each bacterial isolate, the following data will be collected: 

  • Animal and tissue of origin, 
  • Stranding location coordinates of sample collection, 
  • Taxonomic identification of isolate by the veterinary microbiological laboratory (Phoenix Labs, Mukilteo, Washington),
  • Sensitivity to each of the antibiotics tested.

Briefly, standard methods will be used to identify the bacteria, including growth on appropriate selective and differential media, gross colony appearance, morphology on gram stain, as well as biochemical reaction (CLSI 2018).

Next, individual profiles of antimicrobial susceptibility testing will be developed for each identified bacterial species. Antibiotic resistance will be determined using disk diffusion tests, such as the Kirby-Bauer method, against a suite of antibiotics that will include amikacin, amoxicillin/clavulanate, amoxicillin, cephalexin, cefpodoxime, cefovecin,ceftiofur, chloramphenicol, doxycycline, enrofloxacin, gentamicin, imipenem, marbofloxacin, ticarcillin, and trimethoprim-sulfamethoxazole, plus any others deemed necessary based on the identification of the bacterial species. The bacteria will be inoculated onto Mueller-Hinton agar plates. Discs impregnated with the antibiotic will be placed  onto the plate and incubated overnight at 37 degrees Celsius. The resulting zones of inhibition will be expressed in terms of sensitive, intermediate, or resistant, based on their size. 

The resulting dataset will then be used to obtain the following analytical information: 

  • Prevalence (%) of single and multiple antibiotic resistances across all isolates, 
  • Occurrence of antibiotic resistance within taxonomic groups of bacterial isolates, 
  • Effectiveness of each antibiotic against all bacterial isolates, 
  • Prevalence of multiple antibiotic resistances within different tissue groups across all animals. 

The proportion of drugs to which a particular isolate is resistant will generate a Multiple Antibiotic Resistance Index (MAR: range 0 to 1) (Krumperman 1983). An MAR value will be calculated for each tissue sampled in each animal by averaging MAR values for bacterial isolates from multiple swabs of the same tissue or MAR values for multiple bacteria isolated from a single swab.

To analyze differences in the level of antibiotic resistance between the two species, as well as by the two sampling regions, we will apply generalized linear models (GLM). Model development and evaluation will be performed. Analyses will be performed using standard statistical software programs (Stata©, College Station, Texas).

References:

Norman et al. 2004. Cetacean strandings in Oregon and Washington between 1930 and 2002. Journal of Cetacean Research and Management 6(1):87-99.

CLSI. 2018. Performance Standards for Antimicrobial Disk Susceptibility Tests, 13th Edition. CLSI standard M02. Clinical Laboratory Standards Institute, Wayne, PA, 92p.

Krumperman PH. 1983. Multiple antibiotic resistance indexing of Escherichia coli to identify high-risk sources of fecal contamination of foods. Applied and Environmental Microbiology 46(1):165-170.

Protocols

This project has not yet shared any protocols.