Abstract: Enterobacteriaceae are opportunistic pathogens responsible for local or systemic infections in both human and veterinary medicine. To monitor circulating strains in stud farms in Normandy (France), we investigated a collection of Enterobacteriaceae isolated from necropsied equids performed in the region between 1997 and 2020. These strains were initially identified using MALDI-TOF; however, as this method failed to identify some isolates, whole genome sequencing followed by rMLST analysis was subsequently performed. Different genera were identified: Enterobacter spp., Huaxiibacter spp., Lelliottia spp., Rahnella spp.. MALDI-TOF and rMLST identifications were concordant for only 26.5% of the strains studied, leading us to conclude that rMLST is a more reliable method for both genus- and species-level identification, particularly for less-studied genera such as Huaxiibacter spp. and Rahnella spp.. The genus Enterobacter spp. (E. hormaechei and E. ludwigii) accounted for 53% of the strains with a high degree of sequence type (ST) diversity. These include E. hormaechei ST114 and ST171, known as high-risk clone in human clinical medicine. These clones, containing plasmids and acquired resistance genes such as blaOXA-1, blaSHV-12 or blaTEM-1B, are resistant to at least four classes of antibiotics. The presence of genes encoding the enteroaggregative heat-stable enterotoxin 1 or the bacteriocin colicin, probably carried by plasmids, implies that Enterobacter spp. form a reservoir of antibiotic resistance and virulence factors. Conversely, strains of the genera Huaxiibacter spp., Lelliottia spp. and Rahnella spp. naturally found in the environment, showed a lean resistome and virulome. This analysis shows that genomic studies are essential to obtain precise species identification, monitor and detect high-risk clones, and to highlight the circulation of resistance and virulence genes through mobile genetic elements.
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.
Overview
This study analyzed bacterial strains isolated from horse necropsy samples in Normandy, France, originally classified as Enterobacter spp.
Using whole genome sequencing and ribosomal multilocus sequence typing (rMLST), the research reclassified these strains, assessed antibiotic resistance, and identified virulence factors, highlighting the importance of genomic tools in veterinary pathogen surveillance.
Background and Purpose
Enterobacteriaceae are a family of bacteria that can cause opportunistic infections in humans and animals.
Monitoring bacterial strains from stud farms is crucial for understanding infectious risks and managing treatment strategies in veterinary settings.
MALDI-TOF, a common bacterial identification tool, sometimes fails to accurately identify environmental or less-studied strains.
The study aimed to improve identification accuracy for Enterobacteriaceae isolates from equine necropsy samples collected over more than two decades (1997–2020) in Normandy, France.
Methods
Initial bacterial identification was performed using MALDI-TOF mass spectrometry.
For strains that MALDI-TOF failed to identify confidently, whole genome sequencing (WGS) was performed.
rMLST analysis, a genetic sequencing approach targeting ribosomal gene loci, was used for genus and species determination after WGS.
Comparisons between MALDI-TOF and rMLST identifications were made to evaluate accuracy and concordance.
Genomic analysis included screening for antibiotic resistance genes, virulence factors like enterotoxins and bacteriocins, and plasmids (mobile genetic elements facilitating gene transfer).
Key Findings
The strains originally classified as Enterobacter spp. were actually from multiple genera:
Enterobacter spp. (including E. hormaechei and E. ludwigii)
Huaxiibacter spp.
Lelliottia spp.
Rahnella spp.
Only about 26.5% of strain identifications matched between MALDI-TOF and rMLST, indicating MALDI-TOF’s limitations for some genera.
rMLST was considered more reliable for accurate genus and species classification, especially for under-studied genera like Huaxiibacter and Rahnella.
Genetic Diversity and Resistance
53% of strains were confirmed as Enterobacter spp., showing high sequence type diversity.
Notably, high-risk Enterobacter clones known from human medicine were detected:
E. hormaechei ST114
E. hormaechei ST171
These clones carried plasmids with acquired antibiotic resistance genes such as:
blaOXA-1
blaSHV-12
blaTEM-1B
These genes confer resistance to at least four antibiotic classes, highlighting multidrug resistance.
Virulence Factors
Some Enterobacter strains contained genes encoding:
Enteroaggregative heat-stable enterotoxin 1 (a toxin linked with pathogenicity)
Bacteriocin colicin (a protein that can kill competing bacteria)
These virulence factors are probably plasmid-encoded, meaning they can spread between bacteria.
This suggests Enterobacter spp. in horses may act as reservoirs for both antibiotic resistance and virulence genes.
Environmental Genera Characteristics
Strains from Huaxiibacter, Lelliottia, and Rahnella genera were found to be naturally environmental bacteria.
These genera showed minimal antibiotic resistance genes (lean resistome) and few virulence factors (lean virulome).
This supports their lesser role as opportunistic pathogens compared to Enterobacter spp.
Conclusions and Implications
Genomic studies like WGS and rMLST are essential for:
Precise bacterial species identification beyond traditional methods like MALDI-TOF.
Monitoring emerging and high-risk clones that pose risks in veterinary and potentially human contexts.
Tracking dissemination of antibiotic resistance and virulence genes, particularly via plasmids and mobile genetic elements.
This study highlights a potential reservoir of multidrug-resistant and virulent Enterobacter strains in equine populations.
Veterinary microbiology should adopt genomic surveillance to inform infection control, antibiotic stewardship, and disease management strategies in animal farms.
Cite This Article
APA
Harel B, Sévin C, Le Hello S, Moreau P, Giard JC, Petry S, Gravey F.
(2025).
Genomic epidemiology of strains currently and formerly classified as Enterobacter spp. recovered from equine necropsy samples.
PLoS One, 20(11), e0333701.
https://doi.org/10.1371/journal.pone.0333701
Sutton GG, Brinkac LM, Clarke TH, Fouts DE. Enterobacter hormaechei subsp. hoffmannii subsp. nov., Enterobacter hormaechei subsp. xiangfangensis comb. nov., Enterobacter roggenkampii sp. nov., and Enterobacter muelleri is a later heterotypic synonym of Enterobacter asburiae based on computational analysis of sequenced Enterobacter genomes.. F1000Research 2018;7:521.
Brady C, Cleenwerck I, Venter S, Coutinho T, De Vos P. Taxonomic evaluation of the genus Enterobacter based on multilocus sequence analysis (MLSA): Proposal to reclassify E. nimipressuralis and E. amnigenus into Lelliottia gen. nov. as Lelliottia nimipressuralis comb. nov. and Lelliottia amnigena comb. nov., respectively, E. gergoviae and E. pyrinus into Pluralibacter gen. nov. as Pluralibacter gergoviae comb. nov. and Pluralibacter pyrinus comb. nov., respectively, E. cowanii, E. radicincitans, E. oryzae and E. arachidis into Kosakonia gen. nov. as Kosakonia cowanii comb. nov., Kosakonia radicincitans comb. nov., Kosakonia oryzae comb. nov. and Kosakonia arachidis comb. nov., respectively, and E. turicensis, E. helveticus and E. pulveris into Cronobacter as Cronobacter zurichensis nom. nov., Cronobacter helveticus comb. nov. and Cronobacter pulveris comb. nov., respectively, and emended description of the genera Enterobacter and Cronobacter.. Syst Appl Microbiol 2013;36(5):309–19.
Huang S, Dai W, Sun S, Zhang X, Zhang L. Prevalence of plasmid-mediated quinolone resistance and aminoglycoside resistance determinants among carbapeneme non-susceptible Enterobacter cloacae.. PLoS One 2012;7(10):e47636.
Davin-Regli A, PagÃs JM. and ; versatile bacterial pathogens confronting antibiotic treatment. Front Microbiol. 2015. Available from: http://www.frontiersin.org/Antimicrobials%2c_Resistance_and_Chemotherapy/10.3389/fmicb.2015.00392/abstract
Kaneko K, Okamoto R, Nakano R, Kawakami S, Inoue M. Gene mutations responsible for overexpression of AmpC beta-lactamase in some clinical isolates of Enterobacter cloacae.. J Clin Microbiol 2005;43(6):2955–8.
Liu S, Chen L, Wang L, Zhou B, Ye D, Zheng X. Cluster Differences in Antibiotic Resistance, Biofilm Formation, Mobility, and Virulence of Clinical Enterobacter cloacae Complex.. Front Microbiol 2022;13:814831.
Brust FR, Boff L, da Silva Trentin D, Pedrotti Rozales F, Barth AL, Macedo AJ. Macrocolony of NDM-1 Producing Enterobacterhormaechei subsp. oharae Generates Subpopulations with Different Features Regarding the Response of Antimicrobial Agents and Biofilm Formation.. Pathogens 2019;8(2):49.
Soria-Bustos J, Ares MA, Gómez-Aldapa CA, González-Y-Merchand JA, Girón JA, De la Cruz MA. Two Type VI Secretion Systems of Enterobacter cloacae Are Required for Bacterial Competition, Cell Adherence, and Intestinal Colonization.. Front Microbiol 2020;11:560488.
Ewels P, Magnusson M, Lundin S, Käller M. MultiQC: summarize analysis results for multiple tools and samples in a single report.. Bioinformatics 2016;32(19):3047–8.
Miyoshi-Akiyama T, Hayakawa K, Ohmagari N, Shimojima M, Kirikae T. Multilocus sequence typing (MLST) for characterization of Enterobacter cloacae.. PLoS One 2013;8(6):e66358.
Florensa AF, Kaas RS, Clausen PTLC, Aytan-Aktug D, Aarestrup FM. ResFinder - an open online resource for identification of antimicrobial resistance genes in next-generation sequencing data and prediction of phenotypes from genotypes.. Microb Genom 2022;8(1):000748.
Liu B, Zheng D, Zhou S, Chen L, Yang J. VFDB 2022: a general classification scheme for bacterial virulence factors.. Nucleic Acids Res 2022;50(D1):D912–7.
Carattoli A, Zankari E, García-Fernández A, Voldby Larsen M, Lund O, Villa L. In silico detection and typing of plasmids using PlasmidFinder and plasmid multilocus sequence typing.. Antimicrob Agents Chemother 2014;58(7):3895–903.
Molano L-AG, Hirsch P, Hannig M, Müller R, Keller A. The PLSDB 2025 update: enhanced annotations and improved functionality for comprehensive plasmid research.. Nucleic Acids Res 2025;53(D1):D189–96.
Rebelo AR, Ibfelt T, Bortolaia V, Leekitcharoenphon P, Hansen DS, Nielsen HL. One Day in Denmark: Nationwide point-prevalence survey of human bacterial isolates and comparison of classical and whole-genome sequence-based species identification methods.. PLoS One 2022;17(2):e0261999.
He Y, Wu S, Feng Y, Zong Z. Huaxiibacter chinensis gen. nov., sp. nov., recovered from human sputum.. Int J Syst Evol Microbiol 2022;72(8):10.1099/ijsem.0.005484.
Gomez-Simmonds A, Hu Y, Sullivan SB, Wang Z, Whittier S, Uhlemann AC. Evidence from a New York City hospital of rising incidence of genetically diverse carbapenem-resistant Enterobacter cloacae and dominance of ST171, 2007–14.. J Antimicrob Chemother 2016;71(8):2351–3.
Hu S, Xie W, Cheng Q, Zhang X, Dong X, Jing H. Molecular eidemiology of carbapenem-resistant Enterobacter cloacae complex in a tertiary hospital in Shandong, China.. BMC Microbiol 2023;23(1):177.
Ballash GA, Mathys DA, Feicht SM, Mollenkopf DF, Albers AL, Adams RJ. Antimicrobial-Resistant Enterobacterales Recovered from the Environment of Two Zoological Institutions Include Enterobacter cloacae Complex ST171 Producing KPC-4 Carbapenemase.. Appl Environ Microbiol 2023;89(5):e0025723.
de Mendieta JM, Argüello A, Menocal MA, Rapoport M, Albornoz E, Más J. Emergence of NDM-producing Enterobacterales infections in companion animals from Argentina.. BMC Vet Res 2024;20(1):174.
Pot M, Guyomard-Rabenirina S, Couvin D, Ducat C, Enouf V, Ferdinand S. Dissemination of Extended-Spectrum-β-Lactamase-Producing Enterobacter cloacae Complex from a Hospital to the Nearby Environment in Guadeloupe (French West Indies): ST114 Lineage Coding for a Successful IncHI2/ST1 Plasmid.. Antimicrob Agents Chemother 2021;65(3):e02146-20.
Gravey F, Michel A, Langlois B, Gérard M, Galopin S, Gakuba C. Central role of the ramAR locus in the multidrug resistance in ESBL-Enterobacterales.. Microbiol Spectr 2024;12(8):e0354823.
Pelludat C, Rakin A, Jacobi CA, Schubert S, Heesemann J. The yersiniabactin biosynthetic gene cluster of Yersinia enterocolitica: organization and siderophore-dependent regulation.. J Bacteriol 1998;180(3):538–46.
Diamant I, Adani B, Sylman M, Rahav G, Gal-Mor O. The transcriptional regulation of the horizontally acquired iron uptake system, yersiniabactin and its contribution to oxidative stress tolerance and pathogenicity of globally emerging salmonella strains.. Gut Microbes 2024;16(1):2369339.