New Diagnostic Methods Differentiate Escherichia marmotae from E coli in Clinical Isolates

At the 2025 ASM Microbe conference, Pelumi (Magret) Oladipo, a fourth-year PhD candidate in immunology and microbiology at Wayne State University School of Medicine, presented a study describing two new diagnostic methods that distinguish Escherichia marmotae from Escherichia coli. The study also documents the first confirmed case of human infection with E marmotae in North America.

Although E marmotae has been classified as a “cryptic clade” of E coli, whole genome sequencing reveals it diverges by approximately 10 percent. This divergence has clinical implications, yet current diagnostic systems frequently misidentify E marmotae as E coli, including MALDI-TOF-MS platforms used in clinical laboratories.

“E marmotae is also known as HRA, Clade V, and it is one of the E coli cryptic clades that has already diverged from E coli,” Oladipo said in an interview. “And it's—it's—it's the one that diverged the farthest, and it is about 10 percent different from E coli based on its own genome sequence.”

For years, E marmotae was primarily detected in environmental reservoirs, including dogs, bats, and raccoons. Recently, it has been isolated from human infections, including septicemia, urinary tract infections, pyelonephritis, and sepsis.

“But the challenge now is that E marmotae cannot be distinguished from E coli based on standard diagnostic tests—biochemical assays as well as colony morphology, coliform profiles—only by nucleotide differences,” Oladipo explained. “So due to this phenotypic similarity to E coli, it is possible that this species must have been overlooked in the past, misidentified as E coli, thereby underestimating its clinical significance as a potential pathogen.”

To address this diagnostic gap, Oladipo’s team developed a TaqMan PCR assay targeting the uidA and uidB genes, with adk used as a positive control. The assay showed 100 percent specificity for E marmotae, with no cross-reactivity to E coli or other Escherichia species.

The team also developed a mass spectrometry–based biomarker to improve detection using MALDI-TOF-MS. Using the bioMérieux VITEK system, the study found that E marmotae was misidentified as E coli with a median IVD confidence score of 99.9 percent. Although RUO scores for E marmotae were significantly lower (median 0 percent) than those for E coli (median 87.4 percent, p<.0001), the systems failed to differentiate between the species in clinical practice. A spectral peak between m/z 7260–7268 consistently identified E marmotae, while E coli presented peaks from 7268–7280, with no overlap (p<.001).

Application of both methods to 176 clinical isolates identified the first confirmed case of E marmotae in a human infection in North America. The isolate had initially been identified as E coli based on a 99.1 percent IVD confidence score but was later confirmed as E marmotae through PCR and whole genome sequencing. This isolate contained multiple antibiotic resistance gene markers and lacked motility at 37 °C, which is not typically observed in clinical E coli strains.

“And this misidentification means that, you know, we don't know what—how it causes disease, how virulent it is compared to E coli, or whether different treatments might work better for infections—infections—infections caused by E marmotae,” Oladipo said.

“And so we have the MALDI-TOF mass spectrometry, which is mostly used in clinical laboratories. They use that in identifying bacteria, and that also isn't reliable to distinguish this species,” she added. “So the first E marmotae spectrum in the Bruker database was included in 2021, but that also couldn't even distinguish E marmotae from E coli. And there are other systems—like we have the bioMérieux MALDI-TOF mass spectrometry—that also still lacks E marmotae in their database.”

“So, as a result, many clinical laboratories lack the necessary tools or protocols to identify this species accurately, and this misdiagnosis has critical implications, including inaccurate infection tracking, suboptimal antibiotic treatment decisions, and an incomplete understanding of, you know, the prevalence and, I would say, the pathogenic potential of E marmotae, which is still currently understudied,” she said.

Oladipo concluded, “So that’s why our lab has been able to develop two high-identification methods. Number one method, which is the TaqMan PCR assay using species-specific primers. And the other method is the MALDI-TOF mass spectrometry–based biomarker, where we were able to identify a unique spectral peak that reliably distinguishes E marmotae from other species.”

Stay tuned for part 2 of our interview.

Reference

Oladipo PM, Tibbetts RJ, Sivertsen A, et al. New diagnostic methods for Escherichia marmotae and the first report of its identification in clinical isolates in North America. medRxiv. Preprint. Published July 10, 2025. doi:10.1101/2025.07.10.25331261