Insights into Antibiotic Resistance Through Metagenomic Approaches

Robert Schmieder; Robert Edwards


Future Microbiol. 2012;7(1):73-89. 

In This Article

Antibiotic Resistance in Human-associated Microbes

It is estimated that there are ten-times as many microbes in and on any given human as there are human cells in that person's body, and 100-times as many unique genes than in our own genome.[36] The majority of these microbes reside in the gut, with an estimated 800–1000 different bacterial species in the gut community.[37] Those microbes are crucial for human life. However, approximately 80% of the gut microbiota have not yet been cultured.[38]

In recent years, large-scale projects have been started as part of the International Human Microbiome Consortium to investigate the human microbiota. The Human Microbiome Project (HMP), an initiative of the NIH, was launched in 2008 with the mission to sequence, characterize, and analyze as much of the human microbiota as possible in 5 years.[39,40] Part of the HMP is to learn about microbes through metagenomics by sampling from five body sites: the mouth, nasal cavity, skin, gut and (in women) vagina of healthy donors. The HMP also funded 15 demonstration projects for 1 year to look at the microbiome in correlation to human health and awarded continued funding to eight of the projects. In addition to the HMP, the Metagenomics of the Human Intestinal Tract (MetaHIT) project[41] applies metagenomics to the study of the human intestinal tract of European and Asian populations.

A recent MetaHIT metagenomic study of 124 individuals suggests the existence of a common core human gut microbiome,[41] but this core may exist more at the level of shared functional genes rather than shared taxa.[42] An open question in microbial ecology is whether selection operates at the level of taxa or the level of the functions that those taxa perform. Despite the core microbiome, there is a subject-to-subject variability in the composition of the gut microbiota among humans. Numerous internal and external factors, including diet, geography, host physiology, disease state, and features of the gut itself contribute to the community composition of the gut microbiota, and the HMP and related studies will significantly extend our understanding of these relationships.[36,43,44]

Humans have directly exposed their bodies to antibiotics in medicines and indirectly through agriculture and cleaning or beauty products. On average, an adult in the USA makes two outpatient visits per year and 15.3% of these visits result in the prescription of an antibiotic.[45] Antibiotics prescribed during clinical visits may have long-term consequences on the human microbiota; community level studies demonstrated that the gut microbiota recovery is incomplete after repeated antibiotic perturbation and that treatment might cause a shift to a different, but stable community.[46]

Antibiotic-resistant strains persist in the human host environment in the absence of selective pressure.[4,47] For example, Sommer et al. characterized the functional resistance reservoir in two unrelated healthy individuals who had not been treated with antibiotics for at least 1 year.[48] The subject's microbiota was analyzed using functional screening of metagenomic DNA and genomic DNA from 572 bacterial isolates. The sequencing of inserted clones conferring resistance to 13 different antibiotics revealed 95 unique inserts that were evolutionarily distant from known resistance genes. This diverse gene pool of resistance genes in the commensal microbiota of healthy individuals could potentially lead to the emergence of new resistant pathogenic strains. It may also suggest that barriers exist to lateral gene transfer between the bacteria with the novel resistance genes and readily cultured human pathogens.[49] We do not know where the resistance mechanisms come from, but they are easily acquired. For example, a study with a newborn showed that methicilin and flouroquinolone resistance genes and multidrug efflux pumps could be found in the meconium 3 days after birth.[50] At day 6, teicoplanin resistance genes were found in the infant fecal sample. In this particular individual, a fever at day 92 was followed by the presence of β-lactamase genes and a reduction in bacterial load in the stool, even though antibiotics were not reported to be used at that time.

Similar to the human gut microbiata, an important attribute of the oral microbiota is its ability to act as a reservoir of antibiotic resistant organisms. Oral bacteria can easily reach other body sites by swallowing or via the bloodstream and spread to other individuals by, for example, coughing and kissing. Therefore, resistant oral bacteria have the opportunity for rapid dissemination.

A novel tetracycline resistance determinant, tet37, was identified by Diaz-Torres et al. who constructed metagenomic libraries from the microbiota of the human oral cavity,[24] and then 3 years later, they applied functional metagenomics to identify genes encoding antibiotic resistance in the oral microbiota of 60 adult humans.[51] Clones resistant to tetracycline and amoxycillin were detected in each library, and the former was the most common resistance in each library. They did not report on their patient's antibiotic usage, and so it is not known how that affected their experiments.

Our oral and fecal microbiota are distinctly different, both in the organisms present and the resistance mechanisms found in those organisms. Seville et al. investigated the presence and prevalence of various tetracycline and erythromycin resistance genes in the metagenomic DNA isolated from human oral and fecal samples from six different European countries.[52] Although the subjects did not receive antibiotics for 3 months, the profile of resistance genes detected in the fecal metagenome differed considerably from those detected in the oral samples, again suggesting that exposure to antibiotics has long term affects on the microbiota.

The fate of antibiotics used in human medicine is largely unknown. After secretion from the human, some antibiotics are broken down by resistance, some are light, or temperature labile, while others may exist for a long time in the environment. As we shall see later, antibiotics in the environment may promote the development of novel mechanisms of antibiotic resistance.