The bacterial genetic determinants of Escherichia coli capacity to cause bloodstream infections in humans.

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.

Variable fitness effects of bacteriophage resistance mutations in Escherichia coli: implications for phage therapy.

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.

Genome wide association study of Escherichia coli bloodstream infection isolates identifies genetic determinants for the portal of entry but not fatal outcome.

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.

Clinical and Bacteriological Specificities of Escherichia coli Bloodstream Infections From Biliary Portal of Entries.

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.

Inter-phylum circulation of a beta-lactamase-encoding gene: a rare but observable event.

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 (blaMUN-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 blaMUN-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 blaMUN-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. blaMUN-1 encoded for an extended-spectrum Ambler subclass A2 beta-lactamase with 43.0% amino acid identity to TLA-1. blaMUN-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.

Major role of iron uptake systems in the intrinsic extra-intestinal virulence of the genus Escherichia revealed by a genome-wide association study.

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.

Reduced Chlorhexidine Susceptibility Is Associated with Tetracycline Resistance tet Genes in Clinical Isolates of Escherichia coli.

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 Tn10 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.

The Population Genomics of Increased Virulence and Antibiotic Resistance in Human Commensal Escherichia coli over 30 Years in France.

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.

The E phylogroup of Escherichia coli is highly diverse and mimics the whole E. coli species population structure.

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.

Interplay between Bacterial Clones and Plasmids in the Spread of Antibiotic Resistance Genes in the Gut: Lessons from a Temporal Study in Veal Calves.

Intestinal carriage of extended spectrum ß-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 blaESBL genes with a combination of genotyping methods, whole genome sequencing, and conjugation assays. One hundred and seventy-three ESBL-producing E. coli isolates [blaCTX-M-1 (64.7%), blaCTX-M-14 (33.5%), or blaCTX-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 blaCTX-M-carrying plasmids, few blaCTX-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 ß-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 blaCTX-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.

Success of Escherichia coli O25b:H4 Sequence Type 131 Clade C Associated with a Decrease in Virulence.

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 H30-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.

Genomic characterization of 16S rRNA methyltransferase-producing Escherichia coli isolates from the Parisian area, France.

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.

Increased risk of acquisition and transmission of ESBL-producing Enterobacteriaceae in malnourished children exposed to amoxicillin.

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.

Pneumonia-Specific Escherichia coli with Distinct Phylogenetic and Virulence Profiles, France, 2012-2014.

In a prospective, nationwide study in France of Escherichia coli responsible for pneumonia in patients receiving mechanical ventilation, we determined E. coli antimicrobial susceptibility, phylotype, O-type, and virulence factor gene content. We compared 260 isolates with those of 2 published collections containing commensal and bacteremia isolates. The preponderant phylogenetic group was B2 (59.6%), and the predominant sequence type complex (STc) was STc73. STc127 and STc141 were overrepresented and STc95 underrepresented in pneumonia isolates compared with bacteremia isolates. Pneumonia isolates carried higher proportions of virulence genes sfa/foc, papGIII, hlyC, cnf1, and iroN compared with bacteremia isolates. Virulence factor gene content and antimicrobial drug resistance were higher in pneumonia than in commensal isolates. Genomic and phylogenetic characteristics of E. coli pneumonia isolates from critically ill patients indicate that they belong to the extraintestinal pathogenic E. coli pathovar but have distinguishable lung-specific traits.

Ceftriaxone and Cefotaxime Have Similar Effects on the Intestinal Microbiota in Human Volunteers Treated by Standard-Dose Regimens.

Ceftriaxone has a higher biliary elimination than cefotaxime (40% versus 10%), which may result in a more pronounced impact on the intestinal microbiota. We performed a monocenter, randomized open-label clinical trial in 22 healthy volunteers treated by intravenous ceftriaxone (1 g/24 h) or cefotaxime (1 g/8 h) for 3 days. We collected fecal samples for phenotypic analyses, 16S rRNA gene profiling, and measurement of the antibiotic concentration and compared the groups for the evolution of microbial counts and indices of bacterial diversity over time. Plasma samples were drawn at day 3 for pharmacokinetic analysis. The emergence of 3rd-generation-cephalosporin-resistant Gram-negative enteric bacilli (Enterobacterales), Enterococcus spp., or noncommensal microorganisms was not significantly different between the groups. Both antibiotics reduced the counts of total Gram-negative enteric bacilli and decreased the bacterial diversity, but the differences between the groups were not significant. All but one volunteer from each group exhibited undetectable levels of antibiotic in feces. Plasma pharmacokinetic endpoints were not correlated to alteration of the bacterial diversity of the gut. Both antibiotics markedly impacted the intestinal microbiota, but no significant differences were detected when standard clinical doses were administered for 3 days. This might be related to the similar daily amounts of antibiotics excreted through the bile using a clinical regimen. (This study has been registered at ClinicalTrials.gov under identifier NCT02659033.).

The Arginine Deiminase Operon Is Responsible for a Fitness Trade-Off in Extended-Spectrum-ß-Lactamase-Producing Strains of Escherichia coli.

We previously identified an operon involved in an arginine deiminase (ADI) pathway (arc operon) on a CTX-M-producing plasmid from an O102-ST405 strain of Escherichia coli As the ADI pathway was shown to be involved in the virulence of various Gram-positive bacteria, we tested whether the ADI pathway could be involved in the epidemiological success of extended-spectrum-ß-lactamase (ESBL)-producing E. coli strains. We studied two collections of human E. coli isolated in France (n = 493) and England (n = 1,509) and show that the prevalence of the arc operon (i) is higher in ESBL-producing strains (12.1%) than in nonproducers (2.5%), (ii) is higher in CTX-M-producing strains (16%) than in other ESBL producers (3.5%), and (iii) increased over time in ESBL-producing strains from 0% before 2000 to 43.3% in 2011 to 2012. The arc operon, found in strains from various phylogenetic backgrounds, is carried by IncF plasmids (85%) or chromosomes (15%) in regions framed by numerous insertion sequences, indicating multiple arrivals. Competition experiments showed that the arc operon enhances fitness of the strain in vitro in lysogeny broth with arginine. In vivo competition experiments showed that the arc operon is advantageous for the strain in a mouse model of urinary tract infection (UTI), whereas it is a burden in a mouse model of intestinal colonization. In summary, we have identified a trait linked to CTX-M-producing strains that is responsible for a trade-off between two main E. coli lifestyles, UTI and gut commensalism. This trait alone cannot explain the wide spread of ESBLs in E. coli but merits epidemiological surveillance.

Characterization and rapid identification of phylogroup G in Escherichia coli, a lineage with high virulence and antibiotic resistance potential.

The phylogeny of the Escherichia coli species, with the identification of seven phylogroups (A, B1, B2, C, D, E and F), is linked to the lifestyle of the strains. With the accumulation of whole genome sequence data, it became clear that some strains belong to a group intermediate between the F and B2 phylogroups, designated as phylogroup G. Here, we studied the complete sequences of 112 strains representative of the G phylogroup diversity and showed that it is composed of one main sequence type complex (STc)117 and four other STcs (STc657, STc454, STc738 and STc174). STc117, which phylogeny is characterized by very short internal branches, exhibits extensive O diversity, but little H-type and fimH allele diversity, whereas the other STcs are characterized by a main O, H and fimH type. STc117 strains possess many traits associated with extra-intestinal virulence, are virulent in a mouse sepsis model and exhibit multi-drug resistance such as CTX-M production. Epidemiologic data on 4,524 Australian and French strains suggest that STc117 is a poultry-associated lineage that can also establish in humans and cause extra-intestinal diseases. We propose an easy identification method that will help to trace this potentially virulent and resistant phylogroup in epidemiologic studies.

Colistin resistance in Parisian inpatient faecal Escherichia coli as the result of two distinct evolutionary pathways.

BACKGROUND: Beyond plasmid-encoded resistance (mcr genes) prevalence in strain collections, large epidemiological studies to estimate the human burden of colistin-resistant Escherichia coli gut carriage are lacking. OBJECTIVES: To evaluate the prevalence of colistin-resistant E. coli carriage in inpatients and decipher the molecular support of resistance and the genetic background of the strains. METHODS: During a 3 month period in 2017, we prospectively screened patients in six Parisian hospitals for rectal carriage of colistin-resistant E. coli using a selective medium, a biochemical confirmatory test and MIC determination. WGS of the resistant strains and their corresponding plasmids was performed. RESULTS: Among the 1217 screened patients, 153 colistin-resistant E. coli strains were isolated from 152 patients (12.5%). The mcr-1 gene was identified in only seven isolates (4.6%) on different plasmid scaffolds. The genetic background of these MCR-1 producers argued for an animal origin. Conversely, the remaining 146 colistin-resistant E. coli exhibited a phylogenetic distribution corresponding to human gut commensal/clinical population structure (B2 and D phylogroup predominance); 72.6% of those isolates harboured convergent mutations in the PmrA and PmrB proteins, constituting a two-component system shown to be associated with colistin resistance. CONCLUSIONS: We showed that the occurrence at a high rate of colistin resistance in human faecal E. coli is the result of two distinct evolutionary pathways, i.e. the occurrence of chromosomal mutations in an endogenous E. coli population and the rare acquisition of exogenous mcr-1-bearing strains probably of animal origin. The involved selective pressures need to be identified in order to develop preventative strategies.

Escherichia coli B2 Phylogenetic Subgroups in the Infant Gut Microbiota: Predominance of Uropathogenic Lineages in Swedish Infants and Enteropathogenic Lineages in Pakistani Infants.

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.

Evolution of a Dominant Natural Isolate of Escherichia coli in the Human Gut over the Course of a Year Suggests a Neutral Evolution with Reduced Effective Population Size.

In vitro and in vivo evolution experiments on Escherichia coli revealed several principles of bacterial adaptation. However, few data are available in the literature describing the behavior of E. coli in its natural environment. We attempted here to study the evolution in the human gut of a commensal dominant E. coli clone, ED1a belonging to the B2 phylogroup, through a longitudinal genomic study. We sequenced 24 isolates sampled at three different time points within a healthy individual over almost a year. We computed a mutation rate of 6.90 × 10-7 mutations per base per year of the chromosome for E. coli ED1a in healthy human gut. We observed very limited genomic diversity and could not detect any evidence of selection, in contrast to what is observed in experimental evolution over a similar length of time. We therefore suggest that ED1a, being well adapted to the healthy human gut, evolves mostly neutrally with a low effective population size (Ne of ≈500 to 1,700).IMPORTANCE In this study, we follow the genomic fate of a dominant clone of Escherichia coli in the human gut of a healthy individual over about a year. We could compute a low annual mutation rate that supports low diversity, and we could not retrieve any clear signature of selection. These observations support a neutral evolution of E. coli in the human gut, compatible with a very limited effective population size that deviates drastically with the observations made previously in experimental evolution.