NEW YORK (GenomeWeb) – Researchers with Cincinnati Children's Hospital have developed a metagenomic sequencing protocol for fecal samples to detect bacteria resistant to multiple drugs, in order to prevent the spread of infection with such bugs.
The researchers examined the fecal microbiomes of both high-risk and low-risk inpatients and outpatients as well as from control samples. They also performed standard culturing methods to compare that to sequencing.
In a proof of principal published this week in the Journal of Clinical Microbiology, the team found that "key microbiome features such as diversity and relative anaerobe abundance, in addition to the detection of MDR bacteria" may be able to "better identify patients at increased risk of a MDR infection," the authors wrote.
The researchers sequenced the fecal microbiomes of 17 inpatient samples, who were deemed at a higher risk to becoming infected with drug resistant bacteria due to prolonged hospital stays, as well as 11 outpatient samples, who were deemed at a lower risk. In addition, they also used 30 adult and 25 pediatric public fecal microbiomes from the Human Microbiome Project as control samples.
Metagenomic sequencing was performed on the Illumina HiSeq 2500. An analysis of the results indicated that there was little overlap between the three groups, "indicating distinct species and antibiotic resistance gene composition between all groups," the authors wrote.
While they identified over 2,000 distinct bacterial species, further analysis revealed that 79 species tended to classify samples into their respective cohorts. Looking at antibiotic resistance genes, they identified 60 that best classified the samples, although there was no difference in the abundance of drug-resistance genes between groups.
The inpatient group tended to have less microbiome diversity, which the authors expected, while the outpatient group and controls had no difference in diversity. Interestingly, they found that anaerobic species composed 92 percent of the microbiome of outpatients, but only 25 percent of that of the inpatients.
The inpatient and outpatient cohorts had a somewhat surprisingly similar proportion of antibiotic-resistance genes. In particular, the authors were "surprised to find a relatively high prevalence of clinically important bacterial species and antibiotic-resistance genes" in outpatient samples, despite not being on antibiotics at the time. Nearly 73 percent of outpatient samples had MDR bacteria compared to none in the control cohort.
The authors noted that fecal colonization with MDR bacteria is not that uncommon in otherwise healthy children in a community setting, but added that this study showed that children who have not been on antibiotics for at least six months tended to have little to no colonization with MDR bacteria — and they speculated that the MDR bacteria identified in the outpatient samples was the result of prior antibiotic use.
The researchers did identify differences between the inpatient and outpatient groups when looking at specific types of antibiotic-resistance genes. For instance, two inpatient samples, or 24 percent, had the vancomycin regulatory resistance gene, while none had that gene in the outpatient group. Meanwhile, more than half of the outpatient cohort had an AmpC resistance gene, while 24 percent of inpatient samples had that gene.
The authors wrote that the small sample size may have played a role for not identifying differences in multi-drug resistance between the two patient groups.
When comparing the sequencing method to culture, the researchers found that the culture missed several drug-resistance genes and was "cumbersome and required additional testing given the growth of isolates not intended to be selected on all types of screening media."
By contrast, metagenomic sequencing was a "single diagnostic test with precise and clear quantitative detection of clinically important species and antibiotic-resistance genes of MDR bacteria relative to the abundance of all other species and antibiotic-resistance genes within the gut microbiota."
The researchers noted that their next step would be to develop prediction models in order to better stratify patients according to their risk of developing a multi-drug resistance bacterial based on their metagenomic sequencing profile.