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:
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.
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.
We predict with the following hypotheses that antibiotic-resistant bacteria prevalence (%):
The following analyses will be used to test these hypotheses:
For each bacterial isolate, the following data will be collected:
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:
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).
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.
This project has not yet shared any protocols.