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Sequencing Uncovers Genetic Source of Pathogen's Resistance to More Than Two Dozen Antibiotics

NEW YORK (GenomeWeb) – By sequencing the genome of a pathogen resistant to more than two dozen antibiotics, researchers pinpointed the genes responsible for its lack of susceptibility to those drugs.

A US Centers for Disease Control and Prevention-led team sequenced and analyzed the genome of a Klebsiella pneumoniae isolate obtained from a patient in the US that was resistant to all 26 antibiotics tested — one of the first such strains reported to the CDC. Antibiotic resistance is an increasing public health concern, as it leads to about 23,000 deaths in the US each year.

As they reported today in mBio, the researchers' sequencing analysis enabled them to home in on the genes that conferred this high level of resistance and identify similar strains, giving clues to where the isolate arose.

"Evaluation of multidrug-resistant organisms using advanced molecular detection, including whole-genome sequencing, is needed to better understand the origins, acquisition, and spectrum of antimicrobial resistance mechanisms and their combination within one bacterial host," wrote the CDC's Alison Laufer Halpin and her colleagues in their paper.

The K. pneumoniae sample was isolated from a hip abscess from a woman in her 70s who was admitted to a Nevada hospital with systemic inflammatory response syndrome. She had, in the two years before being hospitalized in the US, been treated in India for a fracture of her right femur. At the Nevada hospital, the patient developed septic shock in September 2016 and died.

The researchers performed antimicrobial testing on the K. pneumonia strain isolated from the hip abscess to find it was resistant to all 26 antibiotics on the panel, including to beta-lactams like ampicillin and fluoroquinolones like ciprofloxacin.

Halpin and her colleagues also sequenced the strain using a combination of Pacific Biosciences long reads and Illumina short reads to generate a 5.8-megabase genome that included a 5.4-megabase chromosome and three smaller plasmids. Based on multilocus sequence typing, the researchers noted that this sample belongs to strain 15 (ST15), which is often resistant to beta-lactams and fluoroquinolones and has spread globally.

The researchers then searched through the strain's genome for antibiotic resistance genes. They identified four resistance genes for beta-lactams, which are broad-spectrum antibiotics. The IncA/C2 plasmid harbored both blaNDM-1 carbapenemase and blaCMY-6 class C beta-lactamase genes, while the blaCTX-M-15 and blaSHV-28 genes resided on the strain's chromosome. The researchers noted that the presence of these four genes could account for the strain's resistance to beta-lactams, including the new combination drug ceftazidime-avibactam.

At the same time, they uncovered two chromosomal mutations — in gyrA and parC — that would lead to the inactivation of fluoroquinolones and a frameshift mutation in ramR that leads to the overexpression of the AcrAB efflux pump and resistance to tigecycline, tetracycline, fluoroquinolones, and chloramphenicol.

The researchers likewise uncovered the mph(A) gene, which provides macrolide resistance, on a plasmid. However, they noted that as macrolides were not part of the resistance panel used, they were unsure whether the strain exhibited resistance to them.

Halpin and her colleagues also compared this strain's genome sequence to other K. pneumoniae samples in the National Center for Biotechnology Information database. This sample was most similar to 140 other ST15 isolates from the US, Europe, and South Asia, particularly an isolate found at Massachusetts General Hospital. The IncA/C2 plasmid, meanwhile, was similar to 13 others, including one found in a US patient who had also recently travelled in India and in a sample from Kenya.

These relationships, the researchers noted, could help them piece together where this strain came from and how it may spread.

"Bacteria are going to continue to evolve," Halpin said in a statement. "We cannot stop resistance because it's part of biology. But we do want to slow it."