New Diagnostic Methods Differentiate Escherichia marmotae From Escherichia coli in Clinical Isolates

At the 2025 American Society for Microbiology 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 2 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%. This divergence has clinical implications, yet current diagnostic systems frequently misidentify E marmotae as E coli, including matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (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. “It's the one that diverged the furthest, and it is about 10% 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 polymerase chain reaction (PCR) assay targeting the uidA and uidB genes, with ADK used as a positive control. The assay showed 100% 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 results found that E marmotae was misidentified as E coli with a median in vitro diagnostic (IVD) confidence score of 99.9%. Although research use only scores for E marmotae were significantly lower (median, 0%) than those for E coli (median, 87.4%, P < .0001), the systems failed to differentiate between the species in clinical practice. A spectral peak between a mass to charge ratio of 7260 to 7268 consistently identified E marmotae, whereas E coli presented peaks from 7268 to 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% 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.

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

“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 the bioMérieux MALDI-TOF mass spectrometry—that still lack 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 the prevalence and the pathogenic potential of E marmotae, which is still currently understudied,” she said.

Oladipo concluded, “So that’s why our laboratory has been able to develop 2 high-identification methods. No. 1 method 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.”

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 posted online July 10, 2025. doi:10.1101/2025.07.10.25331261