Application of Microarray and Functional-Based Screening Methods for the Detection of Antimicrobial Resistance Genes in the Microbiomes of Healthy Humans

Abstract

The aim of this study was to screen for the presence of antimicrobial resistance genes within the saliva and faecal microbiomes of healthy adult human volunteers from five European countries. Two non-culture based approaches were employed to obviate potential bias associated with difficult to culture members of the microbiota. In a gene target-based approach, a microarray was employed to screen for the presence of over 70 clinically important resistance genes in the saliva and faecal microbiomes. A total of 14 different resistance genes were detected encoding resistances to six antibiotic classes (aminoglycosides, β-lactams, macrolides, sulphonamides, tetracyclines and trimethoprim). The most commonly detected genes were erm(B), bla TEM, and sul2. In a functional-based approach, DNA prepared from pooled saliva samples was cloned into Escherichia coli and screened for expression of resistance to ampicillin or sulphonamide, two of the most common resistances found by array. The functional ampicillin resistance screen recovered genes encoding components of a predicted AcrRAB efflux pump. In the functional sulphonamide resistance screen, folP genes were recovered encoding mutant dihydropteroate synthase, the target of sulphonamide action. The genes recovered from the functional screens were from the chromosomes of commensal species that are opportunistically pathogenic and capable of exchanging DNA with related pathogenic species. Genes identified by microarray were not recovered in the activity-based screen, indicating that these two methods can be complementary in facilitating the identification of a range of resistance mechanisms present within the human microbiome. It also provides further evidence of the diverse reservoir of resistance mechanisms present in bacterial populations in the human gut and saliva. In future the methods described in this study can be used to monitor changes in the resistome in response to antibiotic therapy.

Impact of Ciprofloxacin and Clindamycin Administration on Gram-Negative Bacteria Isolated from Healthy Volunteers and Characterization of the Resistance Genes They Harbor

“…This indicates that multidrug resistance is possibly common in the microbiota of the healthy human population from Sweden, a finding which we previously noted from the United Kingdom (6). Commonly detected genes included bla TEM and sul2, both of which usually reside on plasmids or transposons and are widespread in the human microbiota (5,6,8,10,12,25). Many of the other microarray-positive genes have been described previously in the microbiota of healthy humans (5,6,8,10,12,25).…”

“…The purified isolates represented many of the natural inhabitants of the human oral and fecal microbiota. All of the fecal anaerobes were identified as Bacteroides, one of the most abundant genera in the human gut (12,23). The majority of the isolated fecal aerobes were E. coli strains, with other Gram-negative species recovered in low numbers, reflecting the normal microbial diversity in the human gut (5,23).…”

“…The majority of the isolated fecal aerobes were E. coli strains, with other Gram-negative species recovered in low numbers, reflecting the normal microbial diversity in the human gut (5,23). The saliva samples mainly yielded Gram-negative isolates that grew anaerobically and were from genera naturally resident in the human oral microbiota (12,24). Only one aerobic isolate per participant per time point was selected based on colony morphology, but we believe this was representative of the most prevalent isolate at that time point, and the recovery of clonal isolates at different time points supports this view.…”

“…Commonly detected genes included bla TEM and sul2, both of which usually reside on plasmids or transposons and are widespread in the human microbiota (5,6,8,10,12,25). Many of the other microarray-positive genes have been described previously in the microbiota of healthy humans (5,6,8,10,12,25). The majority of the ␤-lactamase genes detected in non-E. coli aerobes are likely genes chromosomally located in these species, e.g., bla SHV in K. pneumoniae, bla CMY in C. freundii, and bla ACC in H. alvei (26).…”

“…Furthermore, antibiotic treatment can alter the composition of the natural microbiota and the prevalence of antimicrobial resistance (AMR) genes (2)(3)(4)(5)(6). The human microbiota also serves as a reservoir of AMR genes (7)(8)(9)(10)(11)(12) that may be acquired by susceptible pathogenic or commensal bacteria, contributing to the spread of multidrug-resistant bacteria.…”