Application of microarray and functional-based screening methods for the detection of antimicrobial resistance genes in the microbiomes of healthy humans.
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), blaTEM, 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.
Department of Bacteriology, Animal Health and Veterinary Laboratories Agency, Addlestone, Surrey, United Kingdom.
Roderick M
RM
Card
Department of Microbial Diseases, Eastman Dental Institute, University College London, London, United Kingdom.
Philip J
PJ
Warburton
Department of Bacteriology, Animal Health and Veterinary Laboratories Agency, Addlestone, Surrey, United Kingdom.
Nikki
N
MacLaren
Department of Microbial Diseases, Eastman Dental Institute, University College London, London, United Kingdom.
Peter
P
Mullany
Department of Microbial Diseases, Eastman Dental Institute, University College London, London, United Kingdom.
Elaine
E
Allan
Department of Bacteriology, Animal Health and Veterinary Laboratories Agency, Addlestone, Surrey, United Kingdom.
Muna F
MF
Anjum
D000595:Amino Acid Sequence; D000890:Anti-Infective Agents / Q000494:pharmacology; D004094:Dihydropteroate Synthase / Q000737:chemistry / Q000235:genetics; D004352:Drug Resistance, Microbial / Q000235:genetics*; D005243:Feces / Q000382:microbiology; D064368:Healthy Volunteers; D006801:Humans; D054892:Metagenome; D046228:Microarray Analysis / Q000379:methods; D008826:Microbial Sensitivity Tests; D064307:Microbiota; D008969:Molecular Sequence Data; D012463:Saliva / Q000382:microbiology; D016415:Sequence Alignment
D000890:Anti-Infective Agents; D004094:Dihydropteroate Synthase