The culturable soil antibiotic resistome: a community of multi-drug resistant bacteria

Abstract

Understanding the soil bacterial resistome is essential to understanding the evolution and development of antibiotic resistance, and its spread between species and biomes. We have identified and characterized multi-drug resistance (MDR) mechanisms in the culturable soil antibiotic resistome and linked the resistance profiles to bacterial species. We isolated 412 antibiotic resistant bacteria from agricultural, urban and pristine soils. All isolates were multi-drug resistant, of which greater than 80% were resistant to 16-23 antibiotics, comprising almost all classes of antibiotic. The mobile resistance genes investigated, (ESBL, bla NDM-1, and plasmid mediated quinolone resistance (PMQR) resistance genes) were not responsible for the respective resistance phenotypes nor were they present in the extracted soil DNA. Efflux was demonstrated to play an important role in MDR and many resistance phenotypes. Clinically relevant Burkholderia species are intrinsically resistant to ciprofloxacin but the soil Burkholderia species were not intrinsically resistant to ciprofloxacin. Using a phenotypic enzyme assay we identified the antibiotic specific inactivation of trimethoprim in 21 bacteria from different soils. The results of this study identified the importance of the efflux mechanism in the soil resistome and variations between the intrinsic resistance profiles of clinical and soil bacteria of the same family.

Figure 1. The antibiotic resistance profiles of all the study bacteria from ten soils.

Isolates were individually screened for resistance to each of 23 antibiotics.

Figure 2. Resistance spectrum of the bacterial soil isolates.

Antibiotic resistance was measured as growth on 20 µg/ml of antibiotic.

Figure 3. The antibiotic resistance profiles and percentage mediated by efflux of all the study bacteria.

The antibiotics are color coded according to class. The total bar is the percentage resistance of all isolates to each antibiotic. The lightly shaded bar is the percentage of resistance mediated by efflux for each antibiotic.

Figure 4. The phylogenetic distribution and relatedness of the soil bacteria.

The 16S rRNA genes were sequenced from all bacteria and the phylogenetic tree was created using these sequences and the ARB program package. Branches of the tree are color coded by bacterial orders, with each line at the branch end representing an individual bacterial isolate. The orders with the largest number of lines indicate the largest number of bacterial members within this study. The relatedness of the orders is defined by the distance from one branch to the next. The unclassified bacteria are therefore, closest to the Pseudomonadales.

Figure 5. Enzyme inhibition assay using trimethoprim (20 µg/ml) and trimethoprim susceptible S. aureus ATCC 25923.

An agar plate section containing trimethoprim (20 µg/ml), was inoculated with trimethoprim susceptible S. aureus ATCC 25923. An agar plug containing the trimethoprim inhibiting soil bacteria were placed into the inoculated trimethoprim agar. The soil bacteria producing the enzyme inhibiting trimethoprim is located in the center circle (A). The ring of growth of trimethoprim susceptible S. aureus, which has been protected from the inhibitory effects of trimethoprim by the soil bacteria enzyme (B). There is no S. aureus growth outside the zone of trimethoprim inhibition by the soil bacteria (C).