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Bacteria exposed to bactericidal treatment, such as antibiotics or bacteriophages (phages), often develop resistance. While phage therapy is proposed as a solution to the antibiotic resistance crisis, the bacterial resistance emerging during phage therapy remains poorly characterized. In this study, we examined a large population of phage-resistant extra-intestinal pathogenic Escherichia coli 536 clones that emerged from both in vitro (non-limited liquid medium) and in vivo (murine pneumonia) conditions. Genome sequencing uncovered a convergent mutational pattern in phage resistance mechanisms under both conditions, particularly targeting two cell-wall components, the K15 capsule and the lipopolysaccharide (LPS). This suggests that their identification in vivo could be predicted from in vitro assays. Phage-resistant clones exhibited a wide range of fitness according to in vitro tests, growth rate, and resistance to amoeba grazing, which could not distinguish between the K15 capsule and LPS mutants. In contrast, K15 capsule mutants retained virulence comparable to the wild-type strain, whereas LPS mutants showed significant attenuation in the murine pneumonia model. Additionally, we observed that resistance to the therapeutic phage through a nonspecific mechanism, such as capsule overproduction, did not systematically lead to co-resistance to other phages that were initially capable or incapable of infecting the wild-type strain. Our findings highlight the importance of incorporating a diverse range of phages in the design of therapeutic cocktails to target potential future phage-resistant clones effectively., Importance: This study isolated more than 50 phage-resistant mutants from both in vitro and in vivo conditions, exposing an extra-intestinal pathogenic Escherichia coli strain to a single virulent phage. The characterization of these clones revealed several key findings: (1) mutations occurring during phage treatment affect the same pathways as those identified in vitro ; (2) the resistance mechanisms are associated with the modification of two cell-wall components, with one involving receptor deletion (phage-specific mechanism) and the other, less frequent, involving receptor masking (phage-nonspecific mechanism); (3) an in vivo virulence assay demonstrated that the absence of the receptor abolishes virulence while masking the receptor preserves it; and (4) clones with a resistance mechanism nonspecific to a particular phage can remain susceptible to other phages. This supports the idea of incorporating diverse phages into therapeutic cocktails designed to collectively target both wild-type and phage-resistant strains, including those with resistance mechanisms nonspecific to a phage.

Background: Antimicrobial resistance (AMR) is a major public health threat, affecting not only people but also animals and the environment. The One Health dimension of AMR is well known; however, data are lacking on the circulation of resistance-conferring genes, particularly in low-income countries. In 2017, WHO proposed a protocol called Tricycle, focusing on extended-spectrum β-lactamase (ESBL)-Escherichia coli surveillance in the three sectors (humans, animals, and the environment). We implemented Tricycle in Madagascar to assess ESBL-E coli prevalence and describe intrasector and intersector circulation of ESBL-E coli and plasmids., Methods: In this prospective study, we collected blood culture data from hospitalised patients with a suspected bloodstream infection processed from May 1, 2018, to April 30, 2019, and rectal swabs from healthy pregnant women from July 30, 2018, to April 27, 2019, both from three hospitals in Antananarivo, Madagascar; and caeca from farm chickens and surface waters from the Ikopa river, wastewater, and slaughterhouse effluents in the Antananarivo area, Madagascar, from April 9, 2018, to April 30, 2019. All samples were tested for ESBL-E coli. The genomes of all isolates were sequenced using a short-read method on NextSeq 500 and NovaSeq 6000 platforms (Illumina, San Diego, CA, USA) and those carrying plasmid replicons using an additional long-read method on a MinION platform (Oxford Nanopore Technologies, Oxford, UK). We characterised genomes of isolated strains (sequence type, resistance and virulence gene content, and plasmid replicons). We then compared isolates using the variant calling method (single-nucleotide polymorphism)., Findings: Data from 1056 blood cultures were collected and 289 pregnant women, 246 chickens, and 28 surface waters were sampled. Of the blood cultures, 18 contained E coli, of which seven (39%) were ESBL. ESBL-E coli was present in samples from 86 (30%) of 289 pregnant women, 140 (57%) of 246 chickens, and 28 (100%) of 28 surface water samples. The wet season (November to April) was associated with higher rates of carriage in humans (odds ratio 3·08 [1·81-5·27]) and chickens (2·79 [1·65-4·81]). Sequencing of 277 non-duplicated isolates (82 from pregnant women, 118 from chickens, and 77 from environmental samples) showed high genetic diversity (90 sequence types identified) with sector-specific genomic features. Single nucleotide polymorphism (SNP) analysis revealed that 169 (61%) of 277 isolates grouped into 44 clusters (two or more isolates) of closely related isolates (<40 SNPs), of which 24 clusters contained isolates from two sectors and five contained isolates from all three sectors. ESBL genes were all bla CTX-M variants (215 [78%] of 277 being bla CTX-M-15 ) and were located on a plasmid in 113 (41%) of 277 isolates. These ESBL-carrying plasmids were mainly IncF (63 [55%] of 114; one strain carried two plasmids) and IncY (42 [37%] of 114). The F31/36:A4:B1 (n=13) and F-:A-:B53 (n=8) pMLST subtypes, and the IncY plasmids, which were all highly conserved, were observed in isolates of differing genetic backgrounds from all sectors and were transferable in vitro by conjugation., Interpretation: Despite sector-specific population structures, both ESBL-E coli strains and plasmids are circulating among humans, chickens, and the environment in Antananarivo, Madagascar. The Tricycle protocol can be implemented in a low-income country and represents a powerful tool for investigating dissemination of AMR from a One Health perspective., Funding: Fondation Mérieux and INSERM, Université Paris Cité., Competing Interests: Declaration of interests We declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: Escherichia coli is frequently responsible for bloodstream infections (BSIs). Among digestive BSIs, biliary infections appear to be less severe. Respective roles of host factors, bacterial determinants (phylogroups, virulence, and antibiotic resistance), and portal of entry on outcome are unknown., Methods: Clinical characteristics and prognosis of 770 episodes of E coli BSI were analyzed and isolates sequenced (Illumina technology) comparing phylogroups, multilocus sequence type, virulence, and resistance gene content. BSI isolates were compared with 362 commensal E coli from healthy subjects., Results: Among 770 episodes, 135 were biliary, 156 nonbiliary digestive, and 479 urinary. Compared to urinary infections, BSIs of digestive origin occurred significantly more in men, comorbid, and immunocompromised patients. Digestive portal of entry was significantly associated with septic shock and death. Among digestive infections, patients with biliary infections were less likely to die (P = .032), despite comparable initial severity. Biliary E coli resembled commensals (phylogroup distribution, sequence type, and few virulence-associated genes) whereas nonbiliary digestive and urinary strains carried many virulence-associated genes., Conclusions: Escherichia coli strains responsible for biliary infections exhibit commensal characteristics and are associated with lower mortality rates, despite similar initial severity, than other digestive BSIs. Biliary drainage in addition to antibiotics in the management of biliary infections may explain improved outcome., Competing Interests: Potential conflicts of interest . All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Beta-lactamase-mediated degradation of beta-lactams is the most common mechanism of beta-lactam resistance in Gram-negative bacteria. Beta-lactamase-encoding genes can be transferred between closely related bacteria, but spontaneous inter-phylum transfers (between distantly related bacteria) have never been reported. Here, we describe an extended-spectrum beta-lactamase (ESBL)-encoding gene ( bla MUN-1 ) shared between the Pseudomonadota and Bacteroidota phyla. An Escherichia coli strain was isolated from a patient in Münster (Germany). Its genome was sequenced. The ESBL-encoding gene (named bla MUN-1 ) was cloned, and the corresponding enzyme was characterized. The distribution of the gene among bacteria was investigated using the RefSeq Genomes database. The frequency and relative abundance of its closest homolog in the global microbial gene catalog (GMGC) were analyzed. The E. coli strain exhibited two distinct morphotypes. Each morphotype possessed two chromosomal copies of the bla MUN-1 gene, with one morphotype having two additional copies located on a phage-plasmid p0111. Each copy was located within a 7.6-kb genomic island associated with mobility. bla MUN-1 encoded for an extended-spectrum Ambler subclass A2 beta-lactamase with 43.0% amino acid identity to TLA-1. bla MUN-1 was found in species among the Bacteroidales order and in Sutterella wadsworthensis (Pseudomonadota). Its closest homolog in GMGC was detected frequently in human fecal samples. This is, to our knowledge, the first reported instance of inter-phylum transfer of an ESBL-encoding gene, between the Bacteroidota and Pseudomonadota phyla. Although the gene was frequently detected in the human gut, inter-phylum transfer was rare, indicating that inter-phylum barriers are effective in impeding the spread of ESBL-encoding genes, but not entirely impenetrable., Competing Interests: The authors declare no conflict of interest.

Escherichia coli - one of the most characterized bacteria and a major public health concern - remains invisible across the temporal landscape. Here, we present the meticulous reconstruction of the first ancient E. coli genome from a 16 th century gallstone from an Italian mummy with chronic cholecystitis. We isolated ancient DNA and reconstructed the ancient E. coli genome. It consisted of one chromosome of 4446 genes and two putative plasmids with 52 genes. The E. coli strain belonged to the phylogroup A and an exceptionally rare sequence type 4995. The type VI secretion system component genes appears to be horizontally acquired from Klebsiella aerogenes, however we could not identify any pathovar specific genes nor any acquired antibiotic resistances. A sepsis mouse assay showed that a closely related contemporary E. coli strain was avirulent. Our reconstruction of this ancient E. coli helps paint a more complete picture of the burden of opportunistic infections of the past., (© 2022. The Author(s).)

Longitudinal studies of extraintestinal pathogenic Escherichia coli (ExPEC) and epidemic clones of E. coli in association with New Delhi metallo-β-lactamase ( bla NDM ) in septicaemic neonates are rare. This study captured the diversity of 80 E. coli isolates collected from septicaemic neonates in terms of antibiotic susceptibility, resistome, phylogroups, sequence types (ST), virulome, plasmids, and integron types over a decade (2009 to 2019). Most of the isolates were multidrug-resistant and, 44% of them were carbapenem-resistant, primarily due to bla NDM . NDM-1 was the sole NDM-variant present in conjugative IncFIA/FIB/FII replicons until 2013, and it was subsequently replaced by other variants, such as NDM-5/-7 found in IncX3/FII. A core genome analysis for bla NDM +ve isolates showed the heterogeneity of the isolates. Fifty percent of the infections were caused by isolates of phylogroups B2 (34%), D (11.25%), and F (4%), whereas the other half were caused by phylogroups A (25%), B1 (11.25%), and C (14%). The isolates were further distributed in approximately 20 clonal complexes (ST C ), including five epidemic clones (ST131, ST167, ST410, ST648, and ST405). ST167 and ST131 (subclade H30Rx) were dominant, with most of the ST167 being bla NDM +ve and bla CTX-M-15 +ve . In contrast, the majority of ST131 isolates were bla NDM -ve but bla CTX-M-15 +ve , and they possessed more virulence determinants than did ST167. A single nucleotide polymorphism (SNP)-based comparative genome analysis of epidemic clones ST167 and ST131 in a global context revealed that the study isolates were present in close proximity but were distant from global isolates. The presence of antibiotic-resistant epidemic clones causing sepsis calls for a modification of the recommended antibiotics with which to treat neonatal sepsis. IMPORTANCE Multidrug-resistant and virulent ExPEC causing sepsis in neonates is a challenge to neonatal health. The presence of enzymes, such as carbapenemases ( bla NDM ) that hydrolyze most β-lactam antibiotic compounds, result in difficulties when treating neonates. The characterization of ExPECs collected over 10 years showed that 44% of ExPECs were carbapenem-resistant, possessing transmissible bla NDM genes. The isolates belonged to different phylogroups that are considered to be either commensals or virulent. The isolates were distributed in around 20 clonal complexes (ST C ), including two predominant epidemic clones (ST131 and ST167). ST167 possessed few virulence determinants but was bla NDM +ve . In contrast, ST131 harbored several virulence determinants but was bla NDM -ve . A comparison of the genomes of these epidemic clones in a global context revealed that the study isolates were present in close proximity but were distant from global isolates. The presence of epidemic clones in a vulnerable population with contrasting characteristics and the presence of resistance genes call for strict vigilance., Competing Interests: The authors declare no conflict of interest.

Escherichia coli is both a highly prevalent commensal and a major opportunistic pathogen causing bloodstream infections (BSI). A systematic analysis characterizing the genomic determinants of extra-intestinal pathogenic vs. commensal isolates in human populations, which could inform mechanisms of pathogenesis, diagnostic, prevention and treatment is still lacking. We used a collection of 912 BSI and 370 commensal E. coli isolates collected in France over a 17-year period (2000-2017). We compared their pangenomes, genetic backgrounds (phylogroups, STs, O groups), presence of virulence-associated genes (VAGs) and antimicrobial resistance genes, finding significant differences in all comparisons between commensal and BSI isolates. A machine learning linear model trained on all the genetic variants derived from the pangenome and controlling for population structure reveals similar differences in VAGs, discovers new variants associated with pathogenicity (capacity to cause BSI), and accurately classifies BSI vs. commensal strains. Pathogenicity is a highly heritable trait, with up to 69% of the variance explained by bacterial genetic variants. Lastly, complementing our commensal collection with an older collection from 1980, we predict that pathogenicity continuously increased through 1980, 2000, to 2010. Together our findings imply that E. coli exhibit substantial genetic variation contributing to the transition between commensalism and pathogenicity and that this species evolved towards higher pathogenicity., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Burgaya et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

To get a global picture of the population structure of the Escherichia coli phylogroup E, encompassing the O157:H7 EHEC lineage, we analysed the whole genome of 144 strains isolated from various continents, hosts and lifestyles and representative of the phylogroup diversity. The strains possess 4331 to 5440 genes with a core genome of 2771 genes and a pangenome of 33 722 genes. The distribution of these genes among the strains shows an asymmetric U-shaped distribution. E phylogenetic strains have the largest genomes of the species, partly explained by the presence of mobile genetic elements. Sixty-eight lineages were delineated, some of them exhibiting extra-intestinal virulence genes and being virulent in the mouse sepsis model. Except for the EHEC lineages and the reference EPEC, EIEC and ETEC strains, very few strains possess intestinal virulence genes. Most of the strains were devoid of acquired resistance genes, but eight strains possessed extended-spectrum beta-lactamase genes. Human strains belong to specific lineages, some of them being virulent and antibiotic-resistant [sequence type complexes (STcs) 350 and 2064]. The E phylogroup mimics all the features of the species as a whole, a phenomenon already observed at the STc level, arguing for a fractal population structure of E. coli., (© 2021 Society for Applied Microbiology and John Wiley & Sons Ltd.)

The intrinsic virulence of extra-intestinal pathogenic Escherichia coli is associated with numerous chromosomal and/or plasmid-borne genes, encoding diverse functions such as adhesins, toxins, and iron capture systems. However, the respective contribution to virulence of those genes seems to depend on the genetic background and is poorly understood. Here, we analyze genomes of 232 strains of sequence type complex STc58 and show that virulence (quantified in a mouse model of sepsis) emerged in a sub-group of STc58 due to the presence of the siderophore-encoding high-pathogenicity island (HPI). When extending our genome-wide association study to 370 Escherichia strains, we show that full virulence is associated with the presence of the aer or sit operons, in addition to the HPI. The prevalence of these operons, their co-occurrence and their genomic location depend on strain phylogeny. Thus, selection of lineage-dependent specific associations of virulence-associated genes argues for strong epistatic interactions shaping the emergence of virulence in E. coli., (© 2023. The Author(s).)

Bacteria from the same species can differ widely in their gene content. In Escherichia coli, the set of genes shared by all strains, known as the core genome, represents about half the number of genes present in any strain. Although recent advances in bacterial genomics have unravelled genes required for fitness in various experimental conditions, most studies have focused on single model strains. As a result, the impact of the species' genetic diversity on core processes of the bacterial cell remains largely under-investigated. Here, we have developed a CRISPR interference platform for high-throughput gene repression that is compatible with most E. coli isolates and closely related species. We have applied it to assess the importance of ~3,400 nearly ubiquitous genes in three growth conditions in 18 representative E. coli strains spanning most common phylogroups and lifestyles of the species. Our screens revealed extensive variations in gene essentiality between strains and conditions. Investigation of the genetic determinants for these variations highlighted the importance of epistatic interactions with mobile genetic elements. In particular, we have shown how prophage-encoded defence systems against phage infection can trigger the essentiality of persistent genes that are usually non-essential. This study provides broad insights into the evolvability of gene essentiality and argues for the importance of studying various isolates from the same species under diverse conditions.

Background: Colistin is an antibiotic of last resort in the management of highly drug-resistant Enterobacterales infections. Travel to some destinations presents a high risk of acquiring multidrug-resistant Enterobacterales, but little data are available on the risk of acquiring colistin-resistant strains. Here, we use the VOYAG-R sample collection (2012-2013) in order to evaluate the rate of acquisition of colistin-resistant Enterobacterales, excluding species with intrinsic resistance (CRE), following travel to tropical regions., Methods: A total of 574 frozen stool samples of travellers returning from tropical regions were screened for colistin-resistant strains using ChromID Colistin R agar (bioMerieux®) after pre-enrichment culture with 1 mg/L of colistin. Genomes were obtained by Illumina sequencing and genetic determinants of colistin resistance (mutational events and mcr genes) were searched., Results: A total of 22 travellers (3.8%) acquired colistin-resistant Enterobacterales carrying an mcr gene. Acquisition rates varied between visited regions: 9.2% (18/195) for Asia (southeast Asia: 17/18), 2.2% (4/184) for Latin America (Peru: 4/4) and 0% from Africa (0/195). Acquired strains were predominantly Escherichia coli (92%) and carried mostly the mcr-1 variant (83%). Escherichia coli strains belonged mainly to commensal phylogroups A and B1, and were genetically highly diverse (5 non-clonal sequence type (ST)10 and 17 ST singletons). Only four non mcr colistin-resistant strains (two E. coli and two Enterobacter cloacae complex) were identified. Among all the strains, two also carried extended-spectrum beta-lactamase genes., Conclusions: Travel to tropical regions, and particularly to Southeast Asia, is a risk factor for the acquisition of mcr-carrying Enterobacterales. This study highlights the community dissemination of mcr in humans as early as 2012, 4 years prior to its first published description., (© International Society of Travel Medicine 2022. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Escherichia coli is a commensal of the vertebrate gut that is increasingly involved in various intestinal and extra-intestinal infections as an opportunistic pathogen. Numerous pathotypes that represent groups of strains with specific pathogenic characteristics have been described based on heterogeneous and complex criteria. The democratization of whole-genome sequencing has led to an accumulation of genomic data that render possible a population phylogenomic approach to the emergence of virulence. Few lineages are responsible for the pathologies compared with the diversity of commensal strains. These lineages emerged multiple times during E. coli evolution, mainly by acquiring virulence genes located on mobile elements, but in a specific chromosomal phylogenetic background. This repeated emergence of stable and cosmopolitan lineages argues for an optimization of strain fitness through epistatic interactions between the virulence determinants and the remaining genome.

Escherichia coli is a commensal species of the lower intestine but is also a major pathogen causing intestinal and extraintestinal infections that is increasingly prevalent and resistant to antibiotics. Most studies on genomic evolution of E. coli used isolates from infections. Here, instead, we whole-genome sequenced a collection of 403 commensal E. coli isolates from fecal samples of healthy adult volunteers in France (1980 to 2010). These isolates were distributed mainly in phylogroups A and B2 (30% each) and belonged to 152 sequence types (STs), the five most frequent being ST10 (phylogroup A; 16.3%), ST73 and ST95 (phylogroup B2; 6.3 and 5.0%, respectively), ST69 (phylogroup D; 4.2%), and ST59 (phylogroup F; 3.9%), and 224 O:H serotypes. ST and serotype diversity increased over time. The O1, O2, O6, and O25 groups used in bioconjugate O-antigen vaccine against extraintestinal infections were found in 23% of the strains of our collection. The increase in frequency of virulence-associated genes and antibiotic resistance was driven by two evolutionary mechanisms. Evolution of virulence gene frequency was driven by both clonal expansion of STs with more virulence genes ("ST-driven") and increases in gene frequency within STs independent of changes in ST frequencies ("gene-driven"). In contrast, the evolution of resistance was dominated by increases in frequency within STs ("gene-driven"). This study provides a unique picture of the phylogenomic evolution of E. coli in its human commensal habitat over 30 years and will have implications for the development of preventive strategies. IMPORTANCE Escherichia coli is an opportunistic pathogen with the greatest burden of antibiotic resistance, one of the main causes of bacterial infections and an increasing concern in an aging population. Deciphering the evolutionary dynamics of virulence and antibiotic resistance in commensal E. coli is important to understand adaptation and anticipate future changes. The gut of vertebrates is the primary habitat of E. coli and probably where selection for virulence and resistance takes place. Unfortunately, most whole-genome-sequenced strains are isolated from pathogenic conditions. Here, we whole-genome sequenced 403 E. coli commensals isolated from healthy French subjects over a 30-year period. Virulence genes increased in frequency by both clonal expansion of clones carrying them and increases in frequency within clones, whereas resistance genes increased by within-clone increased frequency. Prospective studies of E. coli commensals should be performed worldwide to have a broader picture of evolution and adaptation of this species.

Background: Escherichia coli bloodstream infections (BSIs) account for high mortality rates (5%-30%). Determinants of death are unclear, especially since the emergence of ESBL producers.Objectives: To determine the relative weight of host characteristics, bacterial virulence and antibiotic resistance in the outcome of patients suffering from E. coli BSI.Methods: All consecutive patients suffering from E. coli BSI in seven teaching hospitals around Paris were prospectively included for 10 months. E. coli isolates were sequenced using Illumina NextSeq technology to determine the phylogroup, ST/ST complex (STc), virulence and antimicrobial resistance gene content. Risk factors associated with death at discharge or Day 28 were determined.Results: Overall, 545 patients (mean ± SD age 68.5 ± 16.5 years; 52.5% male) were included. Mean Charlson comorbidity index (CCI) was 5.6 (± 3.1); 19.6% and 12.8% presented with sepsis and septic shock, respectively. Portals of entry were mainly urinary (51.9%), digestive (41.9%) and pulmonary (3.5%); 98/545 isolates (18%) were third-generation cephalosporin resistant (3GC-R), including 86 ESBL producers. In-hospital death (or at Day 28) was 52/545 (9.5%). Factors independently associated with death were a pulmonary portal of entry [adjusted OR (aOR) 6.54, 95% CI 2.23-19.2, P = 0.0006], the iha_17 virulence gene (aOR 4.41, 95% CI 1.23-15.74, P = 0.022), the STc88 (aOR 3.62, 95% CI 1.30-10.09, P = 0.014), healthcare-associated infections (aOR 1.98, 95% CI 1.04-3.76, P = 0.036) and high CCI (aOR 1.14, 95% CI 1.04-1.26, P = 0.006), but not ESBL/3GC-R.Conclusions: Host factors, portal of entry and bacterial characteristics remain major determinants associated with mortality in E. coli BSIs. Despite a high prevalence of ESBL producers, antibiotic resistance did not impact mortality. (ClinicalTrials.gov identifier: NCT02890901.). [ABSTRACT FROM AUTHOR]

Recent studies have highlighted the importance of ecological interactions in dysbiosis of gut microbiota, but few focused on their role in antibiotic-induced perturbations. We used the data from the CEREMI trial in which 22 healthy volunteers received a 3-day course of ceftriaxone or cefotaxime antibiotics. Fecal samples were analyzed by 16S rRNA gene profiling, and the total bacterial counts were determined in each sample by flux cytometry. As the gut exposure to antibiotics could not be experimentally measured despite a marked impact on the gut microbiota, it was reconstructed using the counts of susceptible Escherichia coli. The dynamics of absolute counts of bacterial families were analyzed using a generalized Lotka-Volterra equations and nonlinear mixed effect modeling. Bacterial interactions were studied using a stepwise approach. Two negative and three positive interactions were identified. Introducing bacterial interactions in the modeling approach better fitted the data, and provided different estimates of antibiotic effects on each bacterial family than a simple model without interaction. The time to return to 95% of the baseline counts was significantly longer in ceftriaxone-treated individuals than in cefotaxime-treated subjects for two bacterial families: Akkermansiaceae (median [range]: 11.3 days [0; 180.0] vs. 4.2 days [0; 25.6], p = 0.027) and Tannerellaceae (13.7 days [6.1; 180.0] vs. 6.2 days [5.4; 17.3], p = 0.003). Taking bacterial interaction as well as individual antibiotic exposure profile into account improves the analysis of antibiotic-induced dysbiosis., (© 2022 The Authors. CPT: Pharmacometrics & Systems Pharmacology published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

Escherichia coli is an important cause of bloodstream infections (BSI), which is of concern given its high mortality and increasing worldwide prevalence. Finding bacterial genetic variants that might contribute to patient death is of interest to better understand infection progression and implement diagnostic methods that specifically look for those factors. E. coli samples isolated from patients with BSI are an ideal dataset to systematically search for those variants, as long as the influence of host factors such as comorbidities are taken into account. Here we performed a genome-wide association study (GWAS) using data from 912 patients with E. coli BSI from hospitals in Paris, France. We looked for associations between bacterial genetic variants and three patient outcomes (death at 28 days, septic shock and admission to intensive care unit), as well as two portals of entry (urinary and digestive tract), using various clinical variables from each patient to account for host factors. We did not find any association between genetic variants and patient outcomes, potentially confirming the strong influence of host factors in influencing the course of BSI; we however found a strong association between the papGII operon and entrance of E. coli through the urinary tract, which demonstrates the power of bacterial GWAS when applied to actual clinical data. Despite the lack of associations between E. coli genetic variants and patient outcomes, we estimate that increasing the sample size by one order of magnitude could lead to the discovery of some putative causal variants. Given the wide adoption of bacterial genome sequencing of clinical isolates, such sample sizes may be soon available., Competing Interests: The authors have declared that no competing interests exist.

Chlorhexidine is a widely used antiseptic in hospital and community health care. Decreased susceptibility to this compound has been recently described in Klebsiella pneumoniae and Pseudomonas aeruginosa, together with cross-resistance to colistin. Surprisingly, few data are available for Escherichia coli, the main species responsible for community and health care-associated infections. In order to decipher chlorhexidine resistance mechanisms in E. coli, we studied both in vitro derived and clinical isolates through whole-genome sequence analysis. Comparison of strains grown in vitro under chlorhexidine pressure identified mutations in the gene mlaA coding for a phospholipid transport system. Phenotypic analyses of single-gene mutants from the Keio collection confirmed the role of this mutation in the decreased susceptibility to chlorhexidine. However, mutations in mlaA were not found in isolates from large clinical collections. In contrast, genome wide association studies (GWAS) showed that, in clinical strains, chlorhexidine reduced susceptibility was associated with the presence of tetA genes of class B coding for efflux pumps and located in a Tn 10 transposon. Construction of recombinant strains in E. coli K-12 confirmed the role of tetA determinant in acquired resistance to both chlorhexidine and tetracycline. Our results reveal that two different evolutionary paths lead to chlorhexidine decreased susceptibility: one restricted to in vitro evolution conditions and involving a retrograde phospholipid transport system; the other observed in clinical isolates associated with efflux pump TetA. None of these mechanisms provide cross-resistance to colistin. This work demonstrates the GWAS power to identify new resistance mechanisms in bacterial species.

Antimicrobial resistance is a major public health concern worldwide affecting humans, animals and the environment. However, data is lacking especially in developing countries. Thus, the World Health Organization developed a One-Health surveillance project called Tricycle focusing on the prevalence of ESBL-producing Escherichia co li in humans, animals, and the environment. Here we present the first results of the human community component of Tricycle in Madagascar. From July 2018 to April 2019, rectal swabs from 492 pregnant women from Antananarivo, Mahajanga, Ambatondrazaka, and Toamasina were tested for ESBL- E. coli carriage. Demographic, sociological and environmental risk factors were investigated, and E. coli isolates were characterized (antibiotic susceptibility, resistance and virulence genes, plasmids, and genomic diversity). ESBL- E. coli prevalence carriage in pregnant women was 34% varying from 12% (Toamasina) to 65% (Ambatondrazaka). The main risk factor associated with ESBL -E. coli carriage was the rainy season (OR = 2.9, 95% CI 1.3-5.6, p = 0.009). Whole genome sequencing was performed on 168 isolates from 144 participants. bla CTX-M-15 was the most frequent ESBL gene (86%). One isolate was resistant to carbapenems and carried the bla NDM- 5 gene. Most isolates belonged to commensalism associated phylogenetic groups A, B1, and C (90%) and marginally to extra-intestinal virulence associated phylogenetic groups B2, D and F (10%). Multi locus sequence typing showed 67 different sequence types gathered in 17 clonal complexes (STc), the most frequent being STc10/phylogroup A (35%), followed distantly by the emerging STc155/phylogroup B1 (7%), STc38/phylogroup D (4%) and STc131/phylogroup B2 (3%). While a wide diversity of clones has been observed, SNP analysis revealed several genetically close isolates ( n = 34/168) which suggests human-to-human transmissions. IncY plasmids were found with an unusual prevalence (23%), all carrying a bla CTX-M-15 . Most of them (85%) showed substantial homology (≥85%) suggesting a dissemination of IncY ESBL plasmids in Madagascar. This large-scale study reveals a high prevalence of ESBL- E. coli among pregnant women in four cities in Madagascar associated with warmth and rainfall. It shows the great diversity of E. coli disseminating throughout the country but also transmission of specific clones and spread of plasmids. This highlights the urgent need of public-health interventions to control antibiotic resistance in the country., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Milenkov, Rasoanandrasana, Rahajamanana, Rakotomalala, Razafindrakoto, Rafalimanana, Ravelomandranto, Ravaoarisaina, Westeel, Petitjean, Mullaert, Clermont, Raskine, Samison, Endtz, Andremont, Denamur, Komurian-Pradel and Armand-Lefevre.)

Intestinal carriage of extended spectrum β-lactamase (ESBL)-producing Escherichia coli is a frequent, increasing, and worrying phenomenon, but little is known about the molecular scenario and the evolutionary forces at play. We screened 45 veal calves, known to have high prevalence of carriage, for ESBL-producing E. coli on 514 rectal swabs (one randomly selected colony per sample) collected over 6 months. We characterized the bacterial clones and plasmids carrying bla ESBL genes with a combination of genotyping methods, whole genome sequencing, and conjugation assays. One hundred and seventy-three ESBL-producing E. coli isolates [ bla CTX-M-1 (64.7%), bla CTX-M-14 (33.5%), or bla CTX-M-15 (1.8%)] were detected, belonging to 32 bacterial clones, mostly of phylogroup A. Calves were colonized successively by different clones with a trend in decreasing carriage. The persistence of a clone in a farm was significantly associated with the number of calves colonized. Despite a high diversity of E. coli clones and bla CTX-M -carrying plasmids, few bla CTX-M gene/plasmid/chromosomal background combinations dominated, due to (i) efficient colonization of bacterial clones and/or (ii) successful plasmid spread in various bacterial clones. The scenario "clone versus plasmid spread" depended on the farm. Thus, epistatic interactions between resistance genes, plasmids, and bacterial clones contribute to optimize fitness in specific environments. IMPORTANCE The gut microbiota is the epicenter of the emergence of resistance. Considerable amount of knowledge on the molecular mechanisms of resistance has been accumulated, but the ecological and evolutionary forces at play in nature are less studied. In this context, we performed a field work on temporal intestinal carriage of extended spectrum β-lactamase (ESBL)-producing Escherichia coli in veal farms. Veal calves are animals with one of the highest levels of ESBL producing E. coli fecal carriage, due to early high antibiotic exposure. We were able to show that calves were colonized successively by different ESBL-producing E. coli clones, and that two main scenarios were at play in the spread of bla CTX-M genes among calves: efficient colonization of several calves by a few bacterial clones and successful plasmid spread in various bacterial clones. Such knowledge should help develop new strategies to fight the emergence of antibiotic-resistance.

Travel to tropical regions is associated with high risk of acquiring extended-spectrum beta-lactamase-producing Enterobacterales (ESBL-E) that are typically cleared in less than 3 months following return. The conditions leading to persistent carriage that exceeds 3 months in some travellers require investigation. Whole-genome sequencing (Illumina MiSeq) was performed on the 82 ESBL-E isolates detected upon return and 1, 2, 3, 6 and 12 months later from the stools of 11 long-term (>3 months) ESBL-E carriers following travel abroad. One to five different ESBL Escherichia coli strains were detected per traveller upon return, and this diminished to one after 3 months. Long-term carriage was due to the presence of the same ESBL E. coli strain, for more than 3 months, in 9 out of 11 travellers, belonging to epidemic sequence type complexes (STc 10, 14, 38, 69, 131 and 648). The mean carriage duration of strains belonging to phylogroups B2/D/F, associated with extra-intestinal virulence, was higher than that for commensal-associated A/B1/E phylogroups (3.5 vs 0.5 months, P =0.021). Genes encoding iron capture systems ( fyuA, irp ), toxins ( senB , sat ), adhesins ( flu, daaF, afa/nfaE , pap , ecpA ) and colicin ( cjrA ) were more often present in persistent strains than in transient ones. Single-nucleotide polymorphism (SNP) analysis in persistent strains showed a maximum divergence of eight SNPs over 12 months without signs of adaptation. Genomic plasticity was observed during the follow-up with the loss or gain of mobile genetic elements such as plasmids, integrons and/or transposons that may contain resistance genes at different points in the follow-up. Long-term colonization of ESBL-E following travel is primarily due to the acquisition of E. coli strains belonging to epidemic clones and harbouring 'virulence genes', allowing good adaptation to the intestinal microbiota.

Background: Escherichia coli is the leading cause of bloodstream infections, associated with a significant mortality. Recent genomic analyses revealed that few clonal lineages are involved in bloodstream infections and captured the emergence of some of them. However, data on within sequence type (ST) population genetic structure evolution are rare., Methods: We compared whole genome sequences of 912 E. coli isolates responsible for bloodstream infections from two multicenter clinical trials that were conducted in the Paris area, France, 12 years apart, in teaching hospitals belonging to the same institution ("Assistance Publique-Hôpitaux de Paris"). We analyzed the strains at different levels of granularity, i.e., the phylogroup, the ST complex (STc), and the within STc clone taking into consideration the evolutionary history, the resistance, and virulence gene content as well as the antigenic diversity of the strains., Results: We found a mix of stability and changes overtime, depending on the level of comparison. Overall, we observed an increase in antibiotic resistance associated to a restricted number of genetic determinants and in strain plasmidic content, whereas phylogroup distribution and virulence gene content remained constant. Focusing on STcs highlighted the pauci-clonality of the populations, with only 11 STcs responsible for more than 73% of the cases, dominated by five STcs (STc73, STc131, STc95, STc69, STc10). However, some STcs underwent dramatic variations, such as the global pandemic STc131, which replaced the previously predominant STc95. Moreover, within STc131, 95 and 69 genomic diversity analysis revealed a highly dynamic pattern, with reshuffling of the population linked to clonal replacement sometimes coupled with independent acquisitions of virulence factors such as the pap gene cluster bearing a papGII allele located on various pathogenicity islands. Additionally, STc10 exhibited huge antigenic diversity evidenced by numerous O:H serotype/fimH allele combinations, whichever the year of isolation., Conclusions: Altogether, these data suggest that the bloodstream niche is occupied by a wide but specific phylogenetic diversity and that highly specialized extra-intestinal clones undergo frequent turnover at the within ST level. Additional worldwide epidemiological studies overtime are needed in different geographical and ecological contexts to assess how generalizable these data are.

Bacteraemia caused by Escherichia coli are particularly frequent and severe, contrasting with the commensal character of the strains found in the digestive tract. A better understanding of the relationships between strains of both origins is needed to unravel the pathogenesis of this disease. Two hundred and forty-three commensal strains were compared to 243 bacteraemic strains isolated from adult hosts matched in terms of gender and age, and from similar location and epoch. Phylogenetic grouping, O-type determination, virulence factor content and antibiotic resistance were compared. Compared to commensal strains, the bacteraemic strains were characterized by a higher proportion of B2, C and D phylogroups, and a lower proportion of A, E and F phylogroups. They also had a lower proportion of the B2 subgroup IV (STc141), a higher proportion of virulence factors, and a higher frequency of antibiotic resistance. These differences were more marked for the bacteraemic strains of urinary tract origin with the presence of specific clones, whereas the bacteraemic strains of digestive origin remained non-significantly different from the commensal strains, except for their antibiotic resistance. Thus, two levels of specialization from commensal strains were demonstrated in the bacteraemic strains: resistance to antibiotics in all cases, and virulence for those of urinary tract origin. [ABSTRACT FROM PUBLISHER]

Adaptive processes in chronic bacterial infections are well described, but much less is known about the processes at play during acute infections. Here, by sequencing seven randomly selected isolates per patient, we analyzed Escherichia coli populations from three acute extraintestinal infections in adults (meningitis, pyelonephritis, and peritonitis), in which a high-mutation-rate isolate or mutator isolate was found. The isolates of single patients displayed between a few dozen and more than 200 independent mutations, with up to half being specific to the mutator isolate. Multiple signs of positive selection were evidenced: a high ratio of nonsynonymous to synonymous mutations ( K a / K s ratio) and strong mutational convergence within and between patients, some of them at loci well known for their adaptive potential, such as rpoS , rbsR , fimH , and fliC For all patients, the mutator isolate was likely due to a large deletion of a methyl-directed mismatch repair gene, and in two instances, the deletion extended to genes involved in some genetic convergence, suggesting potential coselection. Intrinsic extraintestinal virulence assessed in a mouse model of sepsis showed variable patterns of virulence ranging from non-mouse killer to mouse killer for the isolates from single patients. However, genomic signature and gene inactivation experiments did not establish a link between a single gene and the capacity to kill mice, highlighting the complex and multifactorial nature of the virulence. Altogether, these data indicate that E. coli isolates are adapting under strong selective pressure when colonizing an extraintestinal site. IMPORTANCE Little is known about the dynamics of adaptation in acute bacterial infections. By sequencing multiple isolates from monoclonal extraintestinal Escherichia coli infections in several patients, we were able to uncover traces of selection taking place at short time scales compared to chronic infection. High genomic diversity was observed in the patient isolates, with an excess of nonsynonymous mutations, and the comparison within and between different infections showed patterns of convergence at the gene level, both constituting strong signs of adaptation. The genes targeted were coding mostly for proteins involved in global regulation, metabolism, and adhesion/motility. Moreover, virulence assessed in a mouse model of sepsis was variable among the isolates of single patients, but this difference was left unexplained at the molecular level. This work gives us clues about the E. coli lifestyle transition between commensalism and pathogenicity., (Copyright © 2021 Bridier-Nahmias et al.)

Escherichia coli O25b:H4 sequence type 131 (ST131), which is resistant to fluoroquinolones and which is a producer of CTX-M-15, is globally one of the major extraintestinal pathogenic E. coli (ExPEC) lineages. Phylogenetic analyses showed that multidrug-resistant ST131 strains belong to clade C, which recently emerged from clade B by stepwise evolution. It has been hypothesized that features other than multidrug resistance could contribute to this dissemination since other major global ExPEC lineages (ST73 and ST95) are mostly antibiotic susceptible. To test this hypothesis, we compared early biofilm production, presence of ExPEC virulence factors (VFs), and in vivo virulence in a mouse sepsis model in 19 and 20 epidemiologically relevant strains of clades B and C, respectively. Clade B strains were significantly earlier biofilm producers ( P  < 0.001), carriers of more VFs ( P  = 4e-07), and faster killers of mice ( P  = 2e-10) than clade C strains. Gene inactivation experiments showed that the H 30- fimB and ibeART genes were associated with in vivo virulence. Competition assays in sepsis, gut colonization, and urinary tract infection models between the most anciently diverged strain (B1 subclade), one C1 subclade strain, and a B4 subclade recombining strain harboring some clade C-specific genetic events showed that the B1 strain always outcompeted the C1 strain, whereas the B4 strain outcompeted the C1 strain, depending on the mouse niches. All these findings strongly suggest that clade C evolution includes a progressive loss of virulence involving multiple genes, possibly enhancing overall strain fitness by avoiding severe infections, even if it comes at the cost of a lower colonization ability., (Copyright © 2020 Duprilot et al.)

The genus Escherichia is composed of several species and cryptic clades, including E. coli, which behaves as a vertebrate gut commensal, but also as an opportunistic pathogen involved in both diarrheic and extra-intestinal diseases. To characterize the genetic determinants of extra-intestinal virulence within the genus, we carried out an unbiased genome-wide association study (GWAS) on 370 commensal, pathogenic and environmental strains representative of the Escherichia genus phylogenetic diversity and including E. albertii (n = 7), E. fergusonii (n = 5), Escherichia clades (n = 32) and E. coli (n = 326), tested in a mouse model of sepsis. We found that the presence of the high-pathogenicity island (HPI), a ~35 kbp gene island encoding the yersiniabactin siderophore, is highly associated with death in mice, surpassing other associated genetic factors also related to iron uptake, such as the aerobactin and the sitABCD operons. We confirmed the association in vivo by deleting key genes of the HPI in E. coli strains in two phylogenetic backgrounds. We then searched for correlations between virulence, iron capture systems and in vitro growth in a subset of E. coli strains (N = 186) previously phenotyped across growth conditions, including antibiotics and other chemical and physical stressors. We found that virulence and iron capture systems are positively correlated with growth in the presence of numerous antibiotics, probably due to co-selection of virulence and resistance. We also found negative correlations between virulence, iron uptake systems and growth in the presence of specific antibiotics (i.e. cefsulodin and tobramycin), which hints at potential "collateral sensitivities" associated with intrinsic virulence. This study points to the major role of iron capture systems in the extra-intestinal virulence of the genus Escherichia., Competing Interests: The authors have declared that no competing interests exist.

Background: The resistance to all aminoglycosides (AGs) conferred by 16S rRNA methyltransferase enzymes (16S-RMTases) is a major public health concern., Objectives: To characterize the resistance genotype, its genetic environment and plasmid support, and the phylogenetic relatedness of 16S-RMTase-producing Escherichia coli from France., Methods: We screened 137 E. coli isolates resistant to all clinically relevant AGs from nine Parisian hospitals for 16S-RMTases. WGS was performed on clinical isolates with high-level AG resistance (MIC ≥256 mg/L) and their transformants., Results: Thirty of the 137 AG-resistant E. coli produced 16S-RMTases: 11 ArmA, 18 RmtB and 1 RmtC. The 16S-RMTase producers were also resistant to third-generation cephalosporins (90% due to a blaCTX-M gene), co-trimoxazole, fluoroquinolones and carbapenems (blaNDM and blaVIM genes) in 97%, 83%, 70% and 10% of cases, respectively. Phylogenomic diversity was high in ArmA producers, with 10 different STs, but a similar genetic environment, with the Tn1548 transposon carried by a plasmid closely related to pCTX-M-3 in 6/11 isolates. Conversely, RmtB producers belonged to 12 STs, the most frequent being ST405 and ST complex (STc) 10 (four and four isolates, respectively). The rmtB gene was carried by IncF plasmids in 10 isolates and was found in different genetic environments. The rmtC gene was carried by the pNDM-US plasmid., Conclusions: ArmA and RmtB are the predominant 16S-RMTases in France, but their spread follows two different patterns: (i) dissemination of a conserved genetic support carrying armA in E. coli with high levels of genomic diversity; and (ii) various genetic environments surrounding rmtB in clonally related E. coli., (© The Author(s) 2020. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Objectives: Routine amoxicillin for children with uncomplicated severe acute malnutrition raises concerns of increasing antibiotic resistance. We performed an ancillary study nested within a double-blind, placebo-controlled trial in Niger testing the role of routine 7 day amoxicillin therapy in nutritional recovery of children 6 to 59 months of age with uncomplicated severe acute malnutrition., Methods: We screened 472 children for rectal carriage of ESBL-producing Enterobacteriaceae (ESBL-E) as well as their household siblings under 5 years old, at baseline and Week 1 (W1) and Week 4 (W4) after start of therapy, and characterized strains by WGS. ClinicalTrials.gov: NCT01613547., Results: Carriage in index children at baseline was similar in the amoxicillin and the placebo groups (33.8% versus 27.9%, P = 0.17). However, acquisition of ESBL-E in index children at W1 was higher in the amoxicillin group than in the placebo group (53.7% versus 32.2%, adjusted risk ratio = 2.29, P = 0.001). Among 209 index and sibling households possibly exposed to ESBL-E transmission, 16 (7.7%) had paired strains differing by ≤10 SNPs, suggesting a high probability of transmission. This was more frequent in households from the amoxicillin group than from the placebo group [11.5% (12/104) versus 3.8% (4/105), P = 0.04]., Conclusions: Among children exposed to amoxicillin, ESBL-E colonization was more frequent and the risk of transmission to siblings higher. Routine amoxicillin should be carefully balanced with the risks associated with ESBL-E colonization., (© The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Escherichia coli segregates into phylogenetic groups, with group B2 containing both extraintestinal pathogenic E. coli (ExPEC) and enteropathogenic E. coli (EPEC) strains. Ten main B2 subgroups (subgroups I to X)/sequence type complexes (STcs), as well as EPEC lineages, have been identified. In the current study, we characterized ExPEC and EPEC strains of E. coli B2 phylogenetic subgroups/STcs that colonize Swedish and Pakistani infants. Gut commensal E. coli B2 strains, 120 from Swedish infants ( n  = 87) and 19 from Pakistani infants ( n  = 12), were assigned to B2 subgroups. Carriage of the bundle-forming pili and intimin adhesin was examined in the EPEC lineages. The ExPEC virulence markers and the time of persistence of the strains in the microbiota were previously determined. In total, 84% of the Swedish strains and 47% of the Pakistani strains belonged to 1 of the 10 main B2 subgroups ( P  = 0.001). Among the Swedish strains, the most common B2 subgroups were IX/STc95 (19%), II/STc73 (17%), VI/STc12 (13%), and III/STc127 (11%), with each subgroup carrying distinctive sets of ExPEC virulence markers. EPEC lineages with few ExPEC features constituted 47% of the Pakistani B2 strains but only 7% of the Swedish B2 strains ( P  = 0.0001). The subgroup distribution within phylogenetic group B2 strains colonizing the gut differed between Swedish and Pakistani infants. B2 subgroups with uropathogenic characteristics dominated the gut microbiota of Swedish infants, while EPEC lineage 1 strains frequently colonized the intestines of Pakistani infants. Moreover, within the B2 subgroups, ExPEC virulence genes were more prevalent in Swedish strains than in Pakistani strains. Thus, ExPEC traits exemplify the intestinal B2 strains from Western populations. IMPORTANCE The intestinal microbiota is an important reservoir for bacteria that cause extraintestinal infections. Escherichia coli is found ubiquitously in the gut microbiota, and it also causes urinary tract infections, infantile septicemia, and meningitis. Urinary tract infections are usually caused by E. coli strains that originate in the intestinal microbiota. E. coli also causes gastrointestinal infections and is a major cause of diarrhea in infants worldwide. The abilities of certain E. coli strains to cause infections are attributed to their virulence factors, i.e., bacterial components that contribute to the development of different diseases. Our study shows that different subtypes of potentially pathogenic E. coli strains dominate in the gut microbiota of infants in different geographical areas and expands our knowledge of the interplay between bacterial commensalism and pathogenicity., (Copyright © 2019 American Society for Microbiology.)

Background: Oral function tests have been shown to reliably detect impaired bulbar function in adults with spinal muscular atrophy (SMA). Although not routinely recorded, it is known that persons with SMA are affected to varying degrees. Detecting differences in bite and tongue force, endurance, and maximum mouth opening has become particularly promising since the introduction of causal therapy for SMA. This study aimed to compare oral function among adult persons with SMA with different SMA types, walking abilities, and treatment status to a healthy control group., Methods: Data from oral function tests conducted on 58 persons with SMA and 45 healthy individuals were analyzed. Differences in oral function between SMA subgroups were pairwise tested and compared to the healthy control group using Wilcoxon rank sum tests., Results: In an overall comparison, three out of five oral function tests revealed lower values for the SMA group compared to the control group. Subgroup analyses indicated lower scores for most oral function tests in non-ambulatory, untreated patients with SMA type 2 compared to controls. Ambulatory, treated patients with SMA type 3 achieved strength and endurance values comparable to those of healthy individuals., Conclusions: The impairment of oral function varies across persons with SMA. Routine measurement of oral function is warranted to determine individual bulbar involvement stages. Further evaluation should be scheduled if indicators such as restricted maximum mouth opening arise. Trial registration DRKS, DRKS00015842. Registered 30 July 2019, https://drks.de/register/de/trial/DRKS00015842/preview ., (© 2024. The Author(s).)