In this study faecal samples were collected from ten selected whales for analysis of antimicrobial resistance. The study is a part of a larger project and therefore builds on previous experiments where a culture-based approach was used to investigate the MIC values of bacteria isolates from 55 whale samples and 5 seals (Venter, 2019). Here we have extended the culture-based approach by a non-culture approach. Hence, this study combines the strengths of both a culture-based approach where resistance phenotypes can be investigated, and non-culture-based approach, which is not limited to microorganisms that can only grow under laboratory conditions.

Findings from previous experiments were published by Venter in 2019. Faecal samples from 60 marine mammals (55 minke whales and 5 seals) were tested for phenotypic resistance to a selection of antibiotics (ampicillin, cefotaxime, vancomycin, kanamycin, tetracycline, ciprofloxacin, erythromycin and trimethoprim). MICs were determined for 239 isolates from whale samples and 46 isolates from seal samples. For each antibiotic, isolates which were capable of growing at the maximum concentration were tested (32 mg/L). The MIC values for ampicillin and tetracycline were low compared to studies of other environmental samples. The highest number of isolates with an MIC of ≥32 mg/L was seen for trimethoprim, which also had an overall MIC range which was high compared to similar studies. This was followed by molecular testing for a selection of antibiotic resistance genes (ARGs) using standard PCR methods. The selected ARGs were mecA, tetA, ermB, nptII, qnrs, dfrA1, vanA, as well as two genes which code for multidrug resistance (MDR) efflux pumps, acrB and mexD. Only nptII failed to produce any amplicons. There was not a strong association between high MIC values and the detection of resistance genes (except for vancomycin and VanA), though this is likely due to the small selection of genes screened for.

Based on the findings from the first part of the project (Venter, 2019), we decided to continue the research with the most interesting samples based on bacteria isolates with the highest or most interesting MIC values. These findings were correlated back to the original whale samples, and it was decided to select samples from ten whales. From these selected whales total microbial DNA was extracted for targeted PCR for selected ARGs; mecA, tetA, tetB, tetM, qnrS, erm(B), dfrA1, nptII, and nptIII. MDR efflux pumps; mexB, mexD, acrB and acrD, were also included in this essay, and several of the analyzed samples were positive for one or more genes coding for antibiotic MDR efflux.

In addition to targeted PCR, the extracted microbial DNA from the whale faecal samples was subjected to shotgun metagenomics analysis, using the HiSeq platform of Illumina. The findings from the metagenomic analysis shows that variants of van and tet genes, coffering resistance to vancomycin and tetracycline, respectively, were among the most prevalent. In addition, it was found one or more genes coding for antibiotic MDR efflux in the samples. The detected tet genes are different variants to those screened for in the targeted PCR analysis, and show the importance of combining different methodology in the case of environmental microbiology.

The results of this study reveal a snapshot of the antimicrobial susceptibility and resistance genes present in samples collected from marine mammals, and contribute to a growing body of evidence connecting the environment to dissemination of antibiotic resistance. The present study is limited in terms of scale, and continued research is required to fully understand the role of marine mammals in the context of maintenance and spread of antimicrobial resistance.

Download the full report as pdf.

Ullmann, I. F et al. (2020) Antimicrobial resistance in marine mammals: Targeted PCR and metagenomic analysis. Project report, M-1641|2020, GenØk, Tromsø, Norway.