Insights into Antibiotic Resistance Through Metagenomic Approaches

Robert Schmieder; Robert Edwards

Disclosures

Future Microbiol. 2012;7(1):73-89. 

In This Article

Detection of Antibiotic Resistance

Antibiotic resistance is a highly selectable phenotype and can be detected using growth inhibition assays performed in broth or by agar disc diffusion. In a dilution-based growth inhibition assay, the MIC of an antibiotic can be calculated for each bacterial isolate and the organism is typically interpreted as being susceptible or resistant to the antibiotic. Of course, there is a gradation of resistance and some classification schemes include one or more intermediate levels. For clinically important bacteria, diagnostic laboratories perform phenotypic-based analyses using standardized susceptibility testing methods, usually in accordance with those published by the Clinical and Laboratory Standards Institute.[102]

To produce results, culture-based approaches can take 1–2 days for fast-growing bacteria, such as Escherichia coli or Salmonella, but several weeks for slow-growing bacteria, such as Mycobacterium tuberculosis. Moreover, culturing only works for a fraction of microbes; although most pathogens can be cultured because of our years of experimental experience, the vast majority of microbes cannot grow outside their host environment, including pathogens such as Chlamydia or Trypanosomes. Newer molecular detection techniques for resistance such as quantitative PCR (qPCR)[12] or microarrays[13,14] are able to determine the presence of specific resistance genes and have improved diagnosis by providing results within hours. However, these culture-independent approaches only target well-studied pathogens or resistance-causing genes and cannot easily be used for broad-spectrum screening.

Research targeted towards antibiotics for treating infectious diseases and the risks to human health posed by antibiotic resistance have focused mainly on the clinical setting.[15,16] To fully understand the development and dissemination of resistance, we need to address the study of antibiotics and their resistance genes not just in clinics but in natural (nonclinical) environments as well.[17–19]

Before next-generation sequencing, antibiotic resistance genes were typically isolated from environmental samples by cloning from cultured bacteria or by PCR amplification.[20] Those methods ignored potential antibiotic resistance reservoirs because most bacteria are not culturable, and PCR detection depends on primers that are based on known resistance genes and does not readily allow for the discovery of novel genes. The development of culture-independent techniques was required to identify novel resistance genes and access the genetic diversity of most bacteria.

Metagenomics is one of the more modern approaches that overcome the limitations of methods based on culturing or amplification (Box 1).[21] This approach is a powerful tool to describe the genetic potential of a community and to identify the types of microbes present in a community, as well as the presence or absence of genes or genetic variations responsible for antibiotic resistance. Using metagenomics, several novel antibiotic resistance genes have been identified, including resistance to β-lactams,[22,23] tetracycline,[24] aminoglycosides[20,23] and bleomycin.[25] In the next sections, we describe the different metagenomic approaches that are used to identify antibiotic-resistance genes.

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