New Diagnostic Methods for Escherichia marmotae and the First Report of its Identification in Clinical Isolates in North America

New Diagnostic Methods for Escherichia marmotae and the First Report of its Identification in Clinical Isolates in North America | medRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-P4HH5NV'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Diagnostic Methods for Escherichia marmotae and the First Report of its Identification in Clinical Isolates in North America View ORCID Profile Pelumi M. Oladipo , Robert J. Tibbetts , Audun Sivertsen , Justin M. Barger , Torbjørn S. Bruvold , Alemu Fite , Matthew Sims , Marcus Zervos , Ali Jomaa , Jeffrey L. Ram doi: https://doi.org/10.1101/2025.07.10.25331261 Pelumi M. Oladipo 1 Department of Biochemistry, Microbiology, and Immunology, Wayne State University , Detroit, MI 48201 USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Pelumi M. Oladipo For correspondence: jeffram{at}wayne.edu hl8030{at}wayne.edu Robert J. Tibbetts 2 Department of Microbiology, Henry Ford Health System , Detroit, MI 48202 USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Audun Sivertsen 3 Department of Microbiology, Haukeland Hospital , Bergen, Norway Find this author on Google Scholar Find this author on PubMed Search for this author on this site Justin M. Barger 2 Department of Microbiology, Henry Ford Health System , Detroit, MI 48202 USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Torbjørn S. Bruvold 3 Department of Microbiology, Haukeland Hospital , Bergen, Norway Find this author on Google Scholar Find this author on PubMed Search for this author on this site Alemu Fite 4 Microbiology Laboratory, Corewell Health , Royal Oak, MI USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Matthew Sims 5 William Beaumont University Hospital , Corewell Health, Royal Oak, MI USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Marcus Zervos 6 Department of Infectious Diseases, Henry Ford Health System , Detroit, MI 48202 USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ali Jomaa 7 Department of Physiology, Wayne State University , Detroit, MI 48201 USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jeffrey L. Ram 1 Department of Biochemistry, Microbiology, and Immunology, Wayne State University , Detroit, MI 48201 USA 7 Department of Physiology, Wayne State University , Detroit, MI 48201 USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: jeffram{at}wayne.edu hl8030{at}wayne.edu Abstract Full Text Info/History Metrics Supplementary material Data/Code Preview PDF ABSTRACT Genomic sequences of E. marmotae and E. coli differ by 10%. Discovered as an environmental “cryptic clade” of Escherichia , E. marmotae also occurs in human infections. Microbiological and MALDI-TOF-MS methods frequently misidentify E. marmotae as E. coli. Our goal was to develop methods that reliably distinguish E. marmotae from E. coli to improve therapeutic decisions and treatments. A Taqman PCR method was developed to distinguish E. marmotae from E. coli based on sequences of uidA and uidB and a positive control targeting adk . uidA- and uidB species-specific PCR amplified DNA from E. marmotae with 100% specificity, and not from E. coli or other Escherichia species. The Biomérieux VITEK MALDI-TOF-MS consistently misidentified E. marmotae as E. coli , with median IVD confidence scores for both E. marmotae and E. coli of 99.9%; however, RUO scores for E. marmotae (median 0%) were significantly lower (P<0.0001) than for E. coli (median=87.4%). The spectral peak between m/z 7250 to 7280 consistently occurred between 7260 and 7268 in E. marmotae and only between 7268 and 7280 in E. coli , with no overlap (p<0.001). Application of these spectral criteria to 176 clinical isolates revealed the first isolate of E. marmotae from a human infection in North America. The isolate had originally been diagnosed as E. coli based on a 99.1% IVD confidence score. This first North American clinical isolate was confirmed as E. marmotae by Taqman-PCR and whole genome sequencing. This isolate had numerous antibiotic resistance gene markers and unlike most clinical E. coli , this E. marmotae isolate lacked motility at 37 °C. Clinical tests based on these methods of differentiating E. marmotae and E. coli may assist in therapeutic decisions. Moreover, discovering the identity of the structure underlying the shift in the MALDI-TOF-MS peak may help determine if it has a special role in infection. INTRODUCTION Escherichia marmotae was originally discovered as a “cryptic clade” of Escherichia , having identical metabolic and colony morphology profiles on standard microbiological tests but having an average pairwise difference from E. coli over its whole genome of about 10% ( 1 , 2 ). Although originally discovered in animal feces (e.g., marmots ( 3 ), racoons ( 4 ), and birds ( 2 ) and environmental samples ( 2 ), E. marmotae has now been identified in clinical cases as serious as those caused by E. coli. Clinical isolates of E. marmotae have been identified from human cases of septicemia ( 5 ), urinary tract infection ( 6 ), and thoracic spondylodiscitis, pyelonephritis, acute sepsis of unknown origin, and postoperative sepsis ( 7 ). However, within these reports, E. marmotae were always initially misclassified as E. coli by routine clinical tests. Techniques that distinguish these two species are often not applied to clinical isolates once the tentative diagnosis as E. coli has been made. Therefore, we have only limited knowledge about the prevalence of E. marmotae in human infections, its relative risk of causing serious disease, and whether different treatments than are currently used for E. coli infections may be more effectively used on E. marmotae infections. Rapid, convenient methods of distinguishing E. marmotae from E. coli clinically are needed to determine its prevalence and to guide its treatment. The identification of E. marmotae by routine clinical methods has proven challenging. One previous approach to identifying E. marmotae has been to use a set of PCR amplifications followed by agarose gel electrophoresis to identify PCR product sizes ( 5 ). Application of this method to 1081 strains from septicemic patients that were originally classified as E. coli during the COLIBAFI study in France ( 8 ) discovered that two were actually E. marmotae ( 5 ). Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) has become a frequently used tool for identifying bacterial isolates ( 9 ); however, its use for identification of E. marmotae has been problematic as the software for a long time contained no E. marmotae spectra. The first E. marmotae spectrum in the Bruker database was included in 2021 (database version L2020 9607MSP), but did not reliably distinguish E. marmotae from E. coli , rarely achieving an identity score >2.0, as required for the most confident identification in the Bruker system. Later versions have included additional spectra that align better with typical E. marmotae, often achieving identity scores >2.2 (Sivertsen et al., 2022). In another laboratory ( 6 ), an isolate was identified as E. marmotae by 16S RNA gene sequencing was initially classified as E. coli with 99% probability by the colorimetric and substrate-specific tests with the VITEK 2 XL GNI ID card system (BioMérieux, Australia). A subsequent test on a Bruker MALDI-TOF-MS using a database that included E. marmotae reported a confidence score of 2.39 for E. marmotae ( 6 ). In this paper, we describe two methods, potentially applicable in a clinical laboratory, for reliably distinguishing E. marmotae from E. coli. The first is a Taqman PCR method based on sequence differences between E. marmotae and E. coli . In the second method, we identify reliable spectral differences between E. marmotae and E. coli on a Biomerieux VITEK MALDI-TOF-MS system, as all previous studies have reported analysis of E. marmotae using the Bruker system. In preliminary studies to the results shown in this paper, we found that these VITEK systems classify most isolates of E. marmotae as E. coli (i.e., they report In Vitro Diagnostic (IVD) confidence scores for E. coli greater than 99%) or, for a minority of E. marmotae isolates, fail to identify them at all. In this paper, we identify a specific peak in the MALDI-TOF-MS spectra that is exclusively and reliably associated with E. marmotae that enabled the discovery of the first E. marmotae strain isolated in North America. MATERIALS AND METHODS Bacterial Isolates The Ram laboratory previously isolated six strains of E. marmotae from aquatic environments and raccoons ( 4 ) identified by whole genome sequencing. These isolates were archived at −80 ± 2°C in glycerol stocks (in Colilert 18 (IDEXX US) media with 15% glycerol) and have reliably yielded viable subcultures. Seventeen additional E. marmotae isolates, designated with the “TW” prefix, were retrieved from the Thomas Whittam strain collection at Michigan State University. Some of the TW strains were originally derived from the same E. marmotae isolates produced by the Ram lab and other environmental sources ( 2 ). Therefore, comparisons between Ram lab and TW strains serve as internal replicates, enabling assessment of reproducibility in measurements from independently maintained stocks of the same strain. In addition to these isolates, the present study also investigated other strains i) E. marmotae isolated by others from human clinical sources (Sivertsen et al., 2022), and additional isolates of E. marmotae : ii) Representative isolates of other cryptic clades and Escherichia species from the microbial archives of Michigan State University, described originally by ( 2 , 10 ), and iii) 175 strains of putative E. coli that were originally identified as such by MALDI-TOF-MS at Henry Ford Health. Table 1 shows the metadata of the strains of E. marmotae and E.coli that are analyzed in the present study, except for the strains from Henry Ford Health, which are described in further detail in the methods section on MALDI-TOF-MS. View this table: View inline View popup Download powerpoint Table 1. Sample labels (Sample ID), species, sources of isolates, and previous publications (if any) in which the isolate was previously reported or used Primers and TaqMan probe design Sequence data of Ram lab strains in another study in our lab ( 11 ) were used to find suitable assay targets. The genes uidA and uidB , which encode for beta-glucuronidase and the glucuronide carrier protein, respectively, and have both conserved and variable regions among various Escherichia species, were chosen for designing primers and Taqman probes for detecting E. marmotae and E. coli . Studies of the variability of sequences of uidA among environmental and fecal isolates ( 4 , 12 ) had previously led to the discovery of phenotypically similar strains of E. coli and the strain subsequently identified as E. marmotae . The genes exhibit >8% mismatches in nucleotide identity between E. marmotae and E.coli . Geneious Prime software (version 2024.0, Dotmatics) was used to design forward and reverse primers, and a TaqMan probe targeting the mismatch region specific to E. marmotae , following best practices for specificity and efficiency in amplification ( 13 , 14 ). We also tested E.coli specific lipB primers, as previously described by ( 15 ) across all 176 clinical isolates.In addition, we used universal primers targeting the adenylate kinase ( adk ) gene ( 2 ) and designed a probe conserved in all Escherichia species as a normalization standard, enabling amplification of both E. marmotae and E.coli . Sequences of the primers and probes are provided in Table 2 . Specificity of the E. marmotae -primers and probes in Table 2 was studied by in silico analysis using PubMLST, to compare them against E. coli strains and other Escherichia genomes available in GenBank. Primers and probes were synthesized by Integrated DNA Technologies (IDT). View this table: View inline View popup Download powerpoint Table 2: Primers and probes used in the analysis Quantitative PCR Procedure Single colonies were picked using 200 µL pipet tips and suspended in 300 µL of nuclease-free water. The qPCR was performed on a Bio-Rad CFX 96 Touch Real-time PCR detection system with Bio-Rad CFX manager software version 3.1. The optimal annealing temperature was determined by performing a temperature gradient (55° - 65°C) within a single run using DNA samples of E. marmotae and E.coli . The optimized cycling parameters were 95°C for 5 min; 30 cycles of 95°C for 30 s, annealing temperature as described in table 2 , and 72°C for 40 s; then hold at 4°C. The amplification was performed in duplicate in 22 µL, containing 5.5 µL of SsoAdvanced Universal Probes Supermix (Bio-Rad), 3.3 µL each of uidA, uidB or adk probe and primers stock solution (250 nM final concentration for each probe and 900 nM final concentration for each primer), 1 µL of sample, and nuclease free water to a total volume of 22 µL. The reaction was run in duplex for adk and uidA targets, while the amplification of uidB was performed in a separate reaction. MALDI-TOF MS MALDI-TOF-MS spectra were obtained for the 39 Strains of E. marmotae and 12 other Escherichia isolates listed in Table 1 . These spectra were also compared to MALDI-TOF-MS spectra of 176 anonymized clinical isolates that had been identified as E. coli in routine tests in the microbiology laboratory at Henry Ford Health. Cultures were incubated on Trypticase Soy Agar + 5% sheep blood (Remel, USA) at 35°C ± 2°C for 16-24hr. Colonies were picked and placed in a single well of a barcode-labeled VITEK MS-DS target slide (bioMérieux, France) using a VITEK Pick-Me pen and nibs (bioMérieux, France). Then the colony was covered with 1 µL of VITEK MS-CHCA Alpha-cyano-4- hydroxy-cinnamic acid (bioMérieux, France) and air-dried. All of the Henry Ford Health isolates used in this study were identified as E. coli by MALDI-TOF MS using the Vitek MS instrument (BioMérieux VITEK MS Software version 3.2.0) by the in vitro diagnostic (IVD) database with confidence scores of 99% and above (usually >99.9%); in rare instances, the IVD result for an E. marmotae strains was reported as “no identification.” We also recorded the percent confidence scores provided by Vitek’s Research Use Only (RUO) database. Images of the spectra, including the m/z values of several of the peaks identified by the Vitek software, were captured as an image for each of these isolates, as illustrated in the results. Statistical Analysis The m/z values of the peaks of each strain of E. marmotae and E. coli were analyzed to compare the statistical significance of their median or average positions and ranges. Statistical analysis was performed using GraphPad Prism, employing descriptive statistics (medians, averages, variance, ranges), parametric and non-parametric and mixed methods comparisons where data were not normally distributed as determined by a Shapiro-Wilks test, and other statistical tests (e.g., outlier tests), as described with the results. Alpha levels for statistical significance were 0.05, except where Bonferroni-corrected alpha levels were used where indicated in Results. Whole Genome Sequencing and Data analyses As will be described, one isolate among 176 clinical E. coli samples from Henry Hord Health was reclassified as a potential E. marmotae based on MALDI-TOF MS findings, and this identification was further confirmed by qPCR and whole genome sequencing (WGS). For WGS, E. marmotae HFH1 was streaked on LB (Luria Bertani) Agar (Fisher bioreagents) and revived overnight at 37 °C. One colony was picked and grown in LB Broth (37 °C, overnight). Genomic DNA extraction utilized a DNeasy Blood & Tissue kit (Qiagen) according to the manufacturer’s instructions. Sequencing was performed on an Illumina NovaSeq 6000 Sequencer by SeqCenter in Pittsburgh, PA USA. De novo assembly of short reads was performed using Unicycler 0.5.0 ( 17 ). Assembly statistics were recorded with QUAST 5.2.0 ( 18 ), which measures the quality and completeness of the genome assembly. Genome annotation was performed using Prokka v1.14.6, a prokaryotic genome annotation software tool ( 19 ). Genome analysis was performed with ABRIcate version 1.01. ( https://github.com/tseeman/abricate ) identified antimicrobial resistance genes using the CARD database ( 20 ), virulence genes using the E.coli Virulence Factor Database ( 21 ), serotype using SerotypeFinder ( 22 ) and the presence of plasmids using PlasmidFinder ( 23 ). Motility Assay Bacterial motility was measured in semi-solid (0.25% agar) plates containing LB broth. The strain was grown in LB broth overnight at 37 °C with shaking at 180 rpm. Cultures were adjusted to OD 600 = 0.06-0.1. A sterile stab was used to inoculate the culture into the center of the semi-solid agar plate (100 mm x 15 mm). Plates were incubated at 37 °C for 18-24 hours. If a bacterial spread diameter was <0.5 cm, the strain was categorized as “non-motile.” RESULTS PCR IDENTIFICATION In silico analysis to compare the primers and probes in Table 2 against E. coli strains and other Escherichia genomes available in GenBank showed that the forward primer differed from all E.coli and other cryptic Escherichia clades by four bases, the reverse primer differed by two bases, and the probe differed by 3 bases, suggesting that the design would be completely specific to E. marmotae . Representative qPCR results illustrated in Figure 1 demonstrate the specificity of the uidA and uidB primers and probes targeting E. marmotae , and the efficacy of the adk primers and probe as a positive control amplification of all Escherichia . Table S1 summarizes this experimental validation as tested on 27 E. marmotae strains, 4 environmental E. coli strains, and 3 strains from other cryptic clades. Amplification of uidA and uidB was also negative on all clinical E. coli isolates tested (n = 175). The uidA and uidB primers and probes amplified target DNA exclusively from E. marmotae with 100% specificity, while the adk primers and probe amplified DNA from all Escherichia strains tested. Sanger DNA sequencing of the PCR products was done from a subset of these amplifications confirmed the correct amplification targets. Download figure Open in new tab Figure 1: Taqman PCR identification of E. marmotae by species-specific primers and probes for genes uidA (blue), uidB (red), lipB (purple) and positive control amplification by an all Escherichia adk- targeted primer-probe. The “X” symbol indicates that no amplification was detected within 30 cycles. Bars show the average Cq threshold cycle number with error bars indicating the standard deviation of triplicate assays for a representative set of isolates of E. marmotae (C4B, C5, RAM 3024,RAM 3032 & HFH1), E. coli , other Escherichia clades and no template control. MALDI-TOF-MS IVD and RUO MALDI-TOF-MS scores were determined for 176 putative E. coli clinical isolates from Henry Ford Health and for isolates of E. marmotae and other Escherichia listed in Table 1 that were submitted to the Henry Ford Health microbiology laboratory. The Henry Ford Health E. coli isolates had average IVD confidence scores of 99.9% (no variation was reported) and RUO scores in the range of 0% to 99.9% (median = 87.4%). All those that were tested (175 isolates) by our E. marmotae/E. coli qPCR test and an E. coli- specific test by ( 15 ) were confirmed as E. coli, except one isolate which had an IVD Score of 99.1% and RUO =0% . Similarly, as shown in Table S2, four environmental E. coli isolates had IVD E. coli confidence scores of 99.9% and RUO scores ranging from 93.9% to 99.9% (median = 98.7%). By comparison, out of 39 E. marmotae strains, 36 isolates had MALDI-TOF-MS IVD scores for “ E. coli ” ranging from 98.3% - 99.9% (median = 99.9%) while 3 isolates were reported as “NO ID”. The RUO scores for the 39 E. marmotae ranged from 0% - 84.2% (median = 0%), significantly different from the RUO scores of E. coli (p <0.0001; Mann-Whitney U test). Other Escherichia cryptic clades had IVD scores of 99.9% and RUO scores overlapping both the E. coli and E. marmotae scores (range 0% - 99.9%; median: 82.3%, p<0.0001, significantly higher than for E. marmotae ). Although the RUO scores of E. marmotae isolates indicate that significant differences exist between the spectra from E. coli , the RUO scores alone do not provide an absolute identification of E. marmotae since these scores overlap other Escherichia clades and even some rare variants of E. coli . However, by analyzing details of the mass spectra peaks provided by the “plus” version of the VITEK software, we determined that a peak between 7260 and 7268 daltons m/z was specific to E. marmotae ; the comparable peak in E. coli was usually located between 7270 and 7285 daltons, and never in the range of 7260 to 7268. This is shown in representative spectra from E. marmotae and E. coli , illustrated in Figure 2 , in which E. marmotae has a peak at m/z of 7263.837 daltons in a spectrum from a clinical sample and 7260.751 daltons in a spectrum from an environmental isolate. The comparable peak in E. coli has an m/z peak at approximately 7274 daltons in both environmental and clinical isolates. Download figure Open in new tab Figure 2: MALDI-TOF-MS spectra for E. marmotae and E. coli in the range of 5200 to 8800 daltons, highlighting the species-specific peak that usually occurs in the range of 7260 – 7268 for E. marmotae and between 7268 and 7285 in E. coli . The specific peak is highlighted in larger font for each spectrum for clarity. These representative spectra have m/z for the species-specific peak close to the median peak locations as summarized in Figure 3 , for clinical and environmental strains. Figure 3 summarizes these data statistically, comparing the m/z values both by species ( E. marmotae vs. E. coli ) and by whether the isolate was from a clinical or an environmental source. Since there was no overlap between E. marmotae and E. coli in the m/z values for the peak occurring in the range of 7250 – 7280 daltons, it is not surprising that the m/z for all E. marmotae differs significantly from all E. coli (unpaired t-test, p<0.0001). Environmental and clinical strains among each species had overlapping ranges for this peak within species but nevertheless differed significantly from each other for E. marmotae (means: environmental 7260.949; clinical 7264.137; Bonferroni-corrected unpaired t-test, p<0.0001); the E. coli clinical v. environmental comparison was not significant. See the supplement for additional statistical details on the comparisons in Figure 3 . The statistical analysis in Figure 3 confirmed that the peak range of 7260–7268 m/z for E. marmotae was distinct and non-overlapping with the 7268–7285 m/z range observed for E. coli . Download figure Open in new tab Figure 3: Comparative analysis of MALDI- TOF MS peaks for E. marmotae and E. coli in the m/z range of 7250 - 7280. The violin plots, with a dashed line representing the median, illustrate values of the m/z peak for 17 environmental E. marmotae (blue), 18 clinical E. marmotae (red), 4 environmental E. coli (green), and 169 clinical E. coli (purple). Groups with different letters differed from one another on statistical tests at p<0.0001 (multiple t-tests and Mann-Whitney tests, Bonferroni corrected for 6 comparisons; see supplement). The two groups with the same letter were not different (p = 0.034, not significant with the Bonferroni correction). While most strains of Escherichia fit this described paradigm, a small proportion of both E. marmotae and E. coli lacked the peak in the described range, but rather had a nearby lower m/z peak. Thus, 3 of 39 E. marmotae isolates had a peak at 7221daltons, and one isolate had a peak at 7200 daltons. In E. coli , all of the environmental strains and all of the other Escherichia cryptic clades showed a peak in the 7270–7280 daltons m/z range (Table S1); however, among the 175 clinical E. coli tested from Henry Ford Health, 3 isolates had their nearby peak in the range of 7200 – 7221, as listed in Table S3 and 3 strains had a nearby peak in the range of 7280 - 7286. The idea that these low and high m/z peaks are distinctively different is supported by statistical outlier tests (GraphPad, Outlier test ROUT with Q set at 1%) on the complete data sets. Clinical Case Identification and Characterization of E. marmotae HFH1 Among the 176 clinical Escherichia coli isolates tested by MALDI-TOF-MS, one strain—designated HFH1—was flagged for further analysis due to its unique spectral profile. Specifically, this isolate displayed a peak at 7261.439 m/z ( Figure 4 ), which falls within the E. marmotae -specific range (7260–7268 m/z) identified in our spectral analysis ( Figure 2 – 3 ). Despite being identified by the IVD MALDI-TOF system as E. coli with a high confidence score of 99.1%, the RUO confidence score was 0%, consistent with other E. marmotae isolates. To further investigate, we tested the isolate with our species-specific qPCR assay targeting E. marmotae uidA and uidB genes. The assay had a positive result, and sequencing of the amplicons and the whole genome (HFH1 WGS assembly, completeness, and annotation statistics are given in Table S4) further confirmed its identity as E. marmotae . WGS of HFH1 showed that this isolate is more than 99.0% identical to other E. marmotae genomes in GenBank and differs from E. coli by 10%. The sequence of HFH1 has been submitted to GenBank as accession ID JBOZGX000000000. Download figure Open in new tab Figure 4: MALDI-TOF-MS spectra for E. marmotae HFH1 highlighting the range of E. marmotae -specific peak which is 7261.439. Strain HFH1 was isolated from a blood culture of a patient with clinical signs of sepsis, indicating its pathogenic potential. Motility tests revealed that HFH1 is non-motile and its serotype is O21:H56. Virulence factor profiling identified genes involved in adhesion and fimbrial assembly, such as fimA-I, ompA, papD-H, hofB/C/Q, sfpC/H, eaeH, nada/b, fanD-H, ppdA-D , and the curli operon csgA/B/D/E/F/G ( 24 , 25 ). The genome of HFH1 encodes genes related to flagellar biosynthesis and chemotaxis, including flgA-N , flhA-E , fliA-Z , flk , motA/B , and cheA/B/R/W/Y/Z ( 26 ). Toxin- and secretion-related genes included cdtABC , encoding cytolethal distending toxins that induce DNA damage in host cells ( 27 ) and astA , which encodes the enteroaggregative heat-stable enterotoxin EAST1. Additional virulence elements included genes for Type II ( gspC-M ), Type III (T3SS), and Type VI (T6SS) secretion systems, as well as invasion-associated loci such as kpsD , kpsM , and ibeB/C . The complete virulence profile is listed in Table S5 and is consistent with previously reported clinical and environmental E. marmotae strains ( 28 – 30 ) Antimicrobial resistance (AMR) gene analysis revealed several genes capable of conferring resistance to various antibiotic classes. Genes associated with resistance to antimicrobial peptides included eptA , bacA , ugd , yojI , and pmrF . Nitroimidazole resistance is mediated by msbA , while emrE conferred resistance to macrolides. Multidrug efflux systems were represented by acrA/B/E/F , marA , gadX , mdtE/F , and tolC . Additionally, β-lactam resistance genes ampH and ampC were present, suggesting potential resistance to penams and cephalosporins. A full list of resistance genes along with sequence coverage and identity based on the CARD database in isolate HFH1 is presented in Table S6. Plasmid analysis using PlasmidFinder revealed the presence of three plasmid replicon types in the E. marmotae HFH1 genome: IncX4_2, IncFIB(AP001918)_1, and IncFII(pHN7A8)_1_pHN7A8. The IncX4 replicon has been frequently associated with the global dissemination of mcr-1, a plasmid-mediated colistin resistance gene among Enterobacteriaceae ( 31 , 32 ) although no mcr genes were detected in HFH1. The IncFIB(AP001918) and IncFII(pHN7A8) plasmid types are commonly found in extraintestinal pathogenic E. coli (ExPEC) and are often linked to virulence factors and antimicrobial resistance genes ( 33 ). The phenotypic and genotypic data confirm HFH1 as E. marmotae and demonstrate its potential to cause invasive human infection. DISCUSSION Accuracy in identifying the organisms infecting an ill person may be a necessary pre-requisite to designing proper treatment. For an emerging pathogen, such as E. marmotae , which is frequently mistaken for E. coli because of its similar phenotype on standard tests, the lack of accurate identification means that until now the relative efficacy of different treatments for E. marmotae than for E. coli infections cannot be determined. Since the genomic sequence of E. marmotae differs on average from E coli by about 10%, sequence-based tests can easily be applied; however, to our knowledge, no FDA-approved PCR test to do so is commercially available. We have demonstrated here two Taqman PCR targets that specifically identify E. marmotae and could be used in such tests. MALDI-TOF-MS is a frequently used clinical laboratory method for fast identification of bacterial isolates; however, MALDI-TOF-MS databases do not universally include E. marmotae . The Bruker reference database includes several E. marmotae spectra, but publications reported that the confidence scores with the current database are relatively low ( 6 , 7 ). The Biomérieux VITEK MALDI- TOF-MS database does not include E. marmotae at all, and therefore, as reported here, either mistakenly identifies verified E. marmotae as E. coli most of the time or doesn’t identify the isolate at all. However, we report here a specific spectral peak observed on the VITEK MALDI-TOF-MS that unambiguously identifies most strains of E. marmotae , differentiating them, with no overlap, from isolates of E. coli. The species-specific spectral peak in E. marmotae occurs at m/z of 7260 – 7268, while the comparable peak in E. coli is at 7268 – 7286. A small percentage (<10%) of both species lacks the peak at these m/z values, but seem to have a peak that is otherwise absent in the m/z range of 7200 – 7225. If the isolate has a peak in the range of 7260 – 7268, this study unambiguously identifies it as E. marmotae . Until now, the prevalence of E. marmotae in clinical cases appears to be low compared to E. coli and have previously only been reported in publications from Norway, France, and Australia ( 5 – 7 ); This study provides the first documented clinical identification of E. marmotae in the United States, highlighting the need for updated diagnostic protocols capable of detecting this pathogen. The prevalence of E. marmotae compared to E. coli is below 1% (0.2% in France ( 5 ); and 0.4% in Norway ( 7 ). In the present study, in which almost 200 clinical isolates putatively identified as E. coli were tested for being E. marmotae, one strain, HFH1, proved to be E. marmotae ; the rest were confirmed as E. coli . With only one isolate out of 176, we cannot calculate an accurate prevalence, but this frequency is consistent with a prevalence of less than 1%, as reported elsewhere. The isolate was recovered from a patient with sepsis, indicating its potential to cause invasive disease. Motility analysis revealed that HFH1 is not motile at 37 °C and therefore may cause a different range of infections than E. coli , which is typically motile at this temperature. The temperature dependence of motility and motility gene expression of E. marmotae , in comparison to E. coli , is the subject of another study from our laboratory ( 11 ). Genetic analysis of HFH1 also reveals numerous non-synonymous sequence differences from E. coli in genes that are important for extraintestinal pathogenic E. coli (ExPEC) infections, including genes for fimbrial adhesion, toxin production, and multiple secretion systems. E. marmotae is known to occur in the environment in the USA, where it was originally identified as a “cryptic clade” of Escherichia ( 2 ). Although the identifying MALDI TOF MS peak in the environmental strains of E marmotae in the present study occurs at a lower m/z than most clinical strains we have tested, our HFH1 isolate is closer to the average environmental isolate (all of which were from Michigan) than the average clinical isolate of E. marmotae , most of which were from Norway. The significant difference may therefore be one of geographical origin and not environment vs. clinic. Future studies may investigate these differences in the identifying peak. Clinical tests based on these observations may assist in therapeutic decisions. Moreover, discovering the identity of the structure underlying the shift in the MALDI-TOF-MS peak may help determine if it has a special role in infection; however, until now the molecule(s) that is detected by this peak are unknown. Although currently rare among clinical isolates of Escherichia , it seems likely that additional clinical isolates will be detected globally. We wonder whether E. marmotae is an emerging pathogen or whether it has always been rare and will remain so. Future research should explore the host immune response to E. marmotae , the functional role of its unique proteomic features, and comparative infection models to elucidate how its pathogenesis diverges from that of E. coli . Data Availability All data produced in the present work are contained in the manuscript DECLARATIONS Ethics approval and consent to participate The E. coli and E. marmotae isolates used in this research were collected from Henry Ford Health. All patient data were anonymized, prior to research use. E. marmotae cultures from Norway were collected in a study approved by the Regional Ethical Committee of Western Norway (REK 322324) ( 7 ). This research adhered to the Declaration of Helsinki. Availability of data and materials The raw sequencing data and genome assemblies of the RAM lab environmental and clinical isolates have been published under the BioProject PRJNA1261436 and PRJNA1268432, and accession numbers are provided in Table 1 . Consent for publication All authors contributed to writing – review & editing and approved the final version of the manuscript. Competing interests I declare that the authors have no competing interests as defined by BMC, or other interests that might be perceived to influence the results and/or discussion reported in this paper. Funding No Funding Authors’ contributions P.M.O. conceived the study and led conceptualization, data curation, investigation, software, formal analysis, and writing – original draft. R.J.T., J.M.B., M.S., and M.Z. contributed resources, methodology and supported data acquisition from Henry Ford Health. A.S. and T.S.B. contributed to data validation and investigation. A.F. supported investigation and laboratory analyses. J.L.R. contributed to data curation, formal analysis, methodology, project administration, software, resources, writing—editing of original draft, supervision and validation. Acknowledgements Not Applicable Abbreviations MALDI-TOF MS Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry IVD in vitro diagnostic RUO Research Use Only AMR Antimicrobial resistance LB Luria Bertani WGS Whole Genome Sequencing CARD Comprehensive Antibiotic Resistance Database ExPEC Extraintestinal Pathogenic Escherichia coli FDA Food and Drug Administration qPCR Quantitative Polymerase Chain Reaction PCR Polymerase Chain Reaction m/z Mass-to-charge ratio T3SS Type III Secretion System T6SS Type VI Secretion System IDT Integrated DNA Technologies References 1. ↵ Walk ST. The “Cryptic” Escherichia . Rasko DA , editor. EcoSal Plus [Internet] . 2015 Oct 23 [cited 2024 Nov 1]; 6 ( 2 ) : doi: 10.1128/ecosalplus.ESP-0002-2015 . Available from: https://journals.asm.org/doi/10.1128/ecosalplus.esp-0002-2015 OpenUrl CrossRef 2. ↵ Walk ST , Alm EW , Gordon DM , Ram JL , Toranzos GA , Tiedje JM , et al. Cryptic Lineages of the Genus Escherichia . Appl Environ Microbiol [Internet] . 2009 Oct 15 [cited 2024 Oct 30]; 75 ( 20 ): 6534 – 44 . Available from: https://journals.asm.org/doi/10.1128/AEM.01262-09 OpenUrl 3. ↵ Liu S , Jin D , Lan R , Wang Y , Meng Q , Dai H , et al. Escherichia marmotae sp. nov . , isolated from faeces of Marmota himalayana. International Journal of Systematic and Evolutionary Microbiology [Internet] . 2015 Jul 1 [cited 2024 Nov 1]; 65 ( Pt_7 ): 2130 – 4 . Available from: https://www.microbiologyresearch.org/content/journal/ijsem/10.1099/ijs.0.000228 OpenUrl 4. ↵ Ram JL , Thompson B , Turner C , Nechvatal JM , Sheehan H , Bobrin J . Identification of pets and raccoons as sources of bacterial contamination of urban storm sewers using a sequence-based bacterial source tracking method . Water Research [Internet] . 2007 Aug [cited 2024 Oct 30]; 41 ( 16 ): 3605 – 14 . Available from: https://linkinghub.elsevier.com/retrieve/pii/S0043135407002527 OpenUrl 5. ↵ Clermont O , Gordon DM , Brisse S , Walk ST , Denamur E . Characterization of the cryptic Escherichia lineages: rapid identification and prevalence . Environmental Microbiology [Internet] . 2011 Sep [cited 2024 Nov 1]; 13 ( 9 ): 2468 – 77 . Available from: https://sfamjournals.onlinelibrary.wiley.com/doi/10.1111/j.1462-2920.2011.02519.x OpenUrl 6. ↵ Sinha T , Merlino J , Rizzo S , Gatley A , Siarakas S , Gray T . Unrecognised: isolation of Escherichia marmotae from clinical urine sample, phenotypically similar to Escherichia coli . Pathology [Internet] . 2024 Jun [cited 2024 Nov 1]; 56 ( 4 ): 577 – 8 . Available from: https://linkinghub.elsevier.com/retrieve/pii/S0031302523002611 OpenUrl 7. ↵ Sivertsen A , Dyrhovden R , Tellevik MG , Bruvold TS , Nybakken E , Skutlaberg DH , et al. Escherichia marmotae—a Human Pathogen Easily Misidentified as Escherichia coli . Theis KR , editor. Microbiol Spectr [Internet] . 2022 Apr 27 [cited 2024 Nov 1]; 10 ( 2 ): e02035 – 21 . Available from: https://journals.asm.org/doi/10.1128/spectrum.02035-21 OpenUrl 8. ↵ Lefort A , Panhard X , Clermont O , Woerther P , Branger C , Mentré F , et al. Host Factors and Portal of Entry Outweigh Bacterial Determinants To Predict the Severity of Escherichia coli Bacteremia . JOURNAL OF CLINICAL MICROBIOLOGY . 2011 Mar ; 49 ( 3 ): 777 – 83 . OpenUrl Abstract / FREE Full Text 9. ↵ Florio W , Tavanti A , Barnini S , Ghelardi E , Lupetti A . Recent Advances and Ongoing Challenges in the Diagnosis of Microbial Infections by MALDI-TOF Mass Spectrometry . Front Microbiol [Internet] . 2018 May 29 [cited 2024 Nov 1]; 9 : 1097 . Available from: https://www.frontiersin.org/article/10.3389/fmicb.2018.01097/full OpenUrl 10. ↵ Mire M , Kim C , Baffaut C , Liu F , Wuliji T , Zheng G . Escherichia cryptic clade II through clade VIII: Rapid detection and prevalence in feces and surface water . Science of The Total Environment . 2022 Nov ; 848 : 157741 . OpenUrl PubMed 11. ↵ Oladipo P , Withey JH , Ram JL . Not so cryptic Anymore: Genetic and Functional Differences of Escherichia marmotae from E. coli . Abstract and poster presented at: ASM Microbe 2024 ; 2024 Jun ; Atlanta, GA . 12. ↵ Ram JL , Ritchie RP , Fang J , Gonzales FS , Selegean JP . Sequence-Based Source Tracking of Escherichia coli Based on Genetic Diversity of β-Glucuronidase . J of Env Quality [Internet] . 2004 May [cited 2024 Oct 30]; 33 ( 3 ): 1024 – 32 . Available from: https://acsess.onlinelibrary.wiley.com/doi/10.2134/jeq2004.1024 OpenUrl 13. ↵ Untergasser A , Cutcutache I , Koressaar T , Ye J , Faircloth BC , Remm M , et al. Primer3—new capabilities and interfaces . Nucleic Acids Research . 2012 Aug ; 40 ( 15 ): e115 – e115 . OpenUrl CrossRef PubMed 14. ↵ Ye J , Coulouris G , Zaretskaya I , Cutcutache I , Rozen S , Madden TL . Primer-BLAST: A tool to design target-specific primers for polymerase chain reaction . BMC Bioinformatics . 2012 Dec ; 13 ( 1 ): 134 . OpenUrl CrossRef PubMed 15. ↵ Moinet M , Collis RM , Rogers L , Devane ML , Biggs PJ , Stott R , et al. Development of a multiplex droplet digital PCR assay for simultaneous detection and quantification of Escherichia coli, E. marmotae, and E. ruysiae in water samples . Journal of Microbiological Methods [Internet] . 2024 May [cited 2024 Nov 1]; 220 : 106909 . Available from: https://linkinghub.elsevier.com/retrieve/pii/S0167701224000216 OpenUrl 16. Mire M , Kim C , Baffaut C , Liu F , Wuliji T , Zheng G . Escherichia cryptic clade II through clade VIII: Rapid detection and prevalence in feces and surface water . SCIENCE OF THE TOTAL ENVIRONMENT . 2022 Nov 20; 848 . 17. ↵ Wick RR , Judd LM , Gorrie CL , Holt KE. Unicycler: Resolving bacterial genome assemblies from short and long sequencing reads . Phillippy AM , editor. PLoS Comput Biol . 2017 Jun 8; 13 ( 6 ): e1005595 . OpenUrl CrossRef PubMed 18. ↵ Gurevich A , Saveliev V , Vyahhi N , Tesler G . QUAST: quality assessment tool for genome assemblies . Bioinformatics . 2013 Apr 15; 29 ( 8 ): 1072 – 5 . OpenUrl CrossRef PubMed Web of Science 19. ↵ Seemann T . Prokka: rapid prokaryotic genome annotation . Bioinformatics . 2014 Jul 15; 30 ( 14 ): 2068 – 9 . OpenUrl CrossRef PubMed Web of Science 20. ↵ Alcock BP , Raphenya AR , Lau TTY , Tsang KK , Bouchard M , Edalatmand A , et al. CARD 2020: antibiotic resistome surveillance with the comprehensive antibiotic resistance database . Nucleic Acids Research . 2019 Oct 29 ; gkz935 . OpenUrl 21. ↵ Chen L . VFDB: a reference database for bacterial virulence factors . Nucleic Acids Research . 2004 Dec 17; 33 ( Database issue ): D325 – 8 . OpenUrl CrossRef 22. ↵ Joensen KG , Tetzschner AMM , Iguchi A , Aarestrup FM , Scheutz F. Rapid and Easy In Silico Serotyping of Escherichia coli Isolates by Use of Whole-Genome Sequencing Data . Carroll KC , editor. J Clin Microbiol . 2015 Aug; 53 ( 8 ): 2410 – 26 . OpenUrl Abstract / FREE Full Text 23. ↵ Carattoli A , Zankari E , García-Fernández A , Voldby Larsen M , Lund O , Villa L , et al. In silico detection and typing of plasmids using PlasmidFinder and plasmid multilocus sequence typing . Antimicrob Agents Chemother . 2014 Jul ; 58 ( 7 ): 3895 – 903 . OpenUrl Abstract / FREE Full Text 24. ↵ Crabbé A , Ledesma MA , Nickerson CA . Mimicking the host and its microenvironment in vitro for studying mucosal infections by Pseudomonas aeruginosa . Pathogens Disease . 2014 Jun ; 71 ( 1 ): 1 – 19 . OpenUrl PubMed 25. ↵ Snyder JA , Haugen BJ , Lockatell CV , Maroncle N , Hagan EC , Johnson DE , et al. Coordinate Expression of Fimbriae in Uropathogenic Escherichia coli . Infect Immun . 2005 Nov ; 73 ( 11 ): 7588 – 96 . OpenUrl Abstract / FREE Full Text 26. ↵ Girgis HS , Liu Y , Ryu WS , Tavazoie S . A comprehensive genetic characterization of bacterial motility . PLoS Genet . 2007 Sep ; 3 ( 9 ): 1644 – 60 . OpenUrl CrossRef PubMed Web of Science 27. ↵ Huerta-Cantillo J , Chavez-Dueñas L , Zaidi MB , Estrada-García T , Navarro-Garcia F . Cytolethal distending toxin-producing Escherichia coli clinical isolates from Mexican children harbor different cdt types causing CDT-induced epithelial pathological phenotypes . Med Microbiol Immunol . 2025 Dec ; 214 ( 1 ): 7 . OpenUrl PubMed 28. ↵ Binsker U , Deneke C , Hamid HM , Gadicherla AK , Göhler A , Käsbohrer A , et al. Genomic dissection of Escherichia marmotae provides insights into diversity and pathogenic potential . ISME Communications . 2024 Jan 8; 4 ( 1 ): ycae126 . OpenUrl 29. Liu S , Feng J , Pu J , Xu X , Lu S , Yang J , et al. Genomic and molecular characterisation of Escherichia marmotae from wild rodents in Qinghai-Tibet plateau as a potential pathogen . Sci Rep . 2019 Jul 23; 9 ( 1 ): 10619 . OpenUrl CrossRef PubMed 30. ↵ Sivertsen A , Dyrhovden R , Tellevik MG , Bruvold TS , Nybakken E , Skutlaberg DH , et al. Escherichia marmotae—a Human Pathogen Easily Misidentified as Escherichia coli . Theis KR , editor. Microbiol Spectr . 2022 Apr 27; 10 ( 2 ): e02035 – 21 . OpenUrl 31. ↵ Matamoros S , Van Hattem JM , Arcilla MS , Willemse N , Melles DC , Penders J , et al. Global phylogenetic analysis of Escherichia coli and plasmids carrying the mcr-1 gene indicates bacterial diversity but plasmid restriction . Sci Rep . 2017 Nov 10; 7 ( 1 ): 15364 . OpenUrl CrossRef PubMed 32. ↵ Sun J , Fang LX , Wu Z , Deng H , Yang RS , Li XP , et al. Genetic Analysis of the IncX4 Plasmids: Implications for a Unique Pattern in the mcr-1 Acquisition . Sci Rep . 2017 Mar 24; 7 ( 1 ): 424 . OpenUrl CrossRef PubMed 33. ↵ Johnson TJ , Nolan LK . Pathogenomics of the Virulence Plasmids of Escherichia coli . Microbiol Mol Biol Rev . 2009 Dec ; 73 ( 4 ): 750 – 74 . OpenUrl Abstract / FREE Full Text View the discussion thread. Back to top Previous Next Posted July 14, 2025. Download PDF Supplementary Material Data/Code Email Thank you for your interest in spreading the word about medRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following New Diagnostic Methods for Escherichia marmotae and the First Report of its Identification in Clinical Isolates in North America Message Subject (Your Name) has forwarded a page to you from medRxiv Message Body (Your Name) thought you would like to see this page from the medRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share New Diagnostic Methods for Escherichia marmotae and the First Report of its Identification in Clinical Isolates in North America Pelumi M. Oladipo , Robert J. Tibbetts , Audun Sivertsen , Justin M. Barger , Torbjørn S. Bruvold , Alemu Fite , Matthew Sims , Marcus Zervos , Ali Jomaa , Jeffrey L. Ram medRxiv 2025.07.10.25331261; doi: https://doi.org/10.1101/2025.07.10.25331261 Share This Article: Copy Citation Tools New Diagnostic Methods for Escherichia marmotae and the First Report of its Identification in Clinical Isolates in North America Pelumi M. Oladipo , Robert J. Tibbetts , Audun Sivertsen , Justin M. Barger , Torbjørn S. Bruvold , Alemu Fite , Matthew Sims , Marcus Zervos , Ali Jomaa , Jeffrey L. Ram medRxiv 2025.07.10.25331261; doi: https://doi.org/10.1101/2025.07.10.25331261 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Infectious Diseases (except HIV/AIDS) Subject Areas All Articles Addiction Medicine (569) Allergy and Immunology (863) Anesthesia (300) Cardiovascular Medicine (4442) Dentistry and Oral Medicine (444) Dermatology (383) Emergency Medicine (609) Endocrinology (including Diabetes Mellitus and Metabolic Disease) (1511) Epidemiology (15230) Forensic Medicine (30) Gastroenterology (1126) Genetic and Genomic Medicine (6610) Geriatric Medicine (668) Health Economics (998) Health Informatics (4542) Health Policy (1370) Health Systems and Quality Improvement (1613) Hematology (543) HIV/AIDS (1266) Infectious Diseases (except HIV/AIDS) (15923) Intensive Care and Critical Care Medicine (1103) Medical Education (623) Medical Ethics (147) Nephrology (668) Neurology (6607) Nursing (346) Nutrition (999) Obstetrics and Gynecology (1146) Occupational and Environmental Health (957) Oncology (3337) Ophthalmology (974) Orthopedics (369) Otolaryngology (420) Pain Medicine (436) Palliative Medicine (130) Pathology (664) Pediatrics (1693) Pharmacology and Therapeutics (692) Primary Care Research (712) Psychiatry and Clinical Psychology (5448) Public and Global Health (9238) Radiology and Imaging (2202) Rehabilitation Medicine and Physical Therapy (1370) Respiratory Medicine (1196) Rheumatology (596) Sexual and Reproductive Health (714) Sports Medicine (530) Surgery (712) Toxicology (99) Transplantation (289) Urology (265) (function(){function c(){var b=a.contentDocument||a.contentWindow.document;if(b){var d=b.createElement('script');d.innerHTML="window.__CF$cv$params={r:'a01ca5dd3e1f8739',t:'MTc3OTc5NzEyNA=='};var a=document.createElement('script');a.src='/cdn-cgi/challenge-platform/scripts/jsd/main.js';document.getElementsByTagName('head')[0].appendChild(a);";b.getElementsByTagName('head')[0].appendChild(d)}}if(document.body){var a=document.createElement('iframe');a.height=1;a.width=1;a.style.position='absolute';a.style.top=0;a.style.left=0;a.style.border='none';a.style.visibility='hidden';document.body.appendChild(a);if('loading'!==document.readyState)c();else if(window.addEventListener)document.addEventListener('DOMContentLoaded',c);else{var e=document.onreadystatechange||function(){};document.onreadystatechange=function(b){e(b);'loading'!==document.readyState&&(document.onreadystatechange=e,c())}}}})();