Dissemination of IncI plasmid encoding bla CTX-M-1 is not hampered by its fitness cost in the pig's gut.

Multiresistance plasmids belonging to the IncI incompatibility group have become one of the most pervasive plasmid types in extended-spectrum beta-lactamase-producing Escherichia coli of animal origin. The extent of the burden imposed on the bacterial cell by these plasmids seems to modulate the emergence of "epidemic" plasmids. However, in vivo data in the natural environment of the strains are scarce. Here, we investigated the cost of a bla CTX-M-1-IncI1 epidemic plasmid in a commensal E. coli animal strain, UB12-RC, before and after oral inoculation of 15 6- to 8-week- old specific-pathogen-free pigs. Growth rate in rich medium was determined on (i) UB12-RC and derivatives, with or without plasmid, in vivo and/or in vitro evolved, and (ii) strains that acquired the plasmid in the gut during the experiment. Although bla CTX-M-1-IncI1 plasmid imposed no measurable burden on the recipient strain after conjugation and during the longitudinal carriage in the pig's gut, we observed a significant difference in the bacterial growth rate between IncI1 plasmid-carrying and plasmid-free isolates collected during in vivo carriage. Only a few mutations on the chromosome of the UB12-RC derivatives were detected by whole-genome sequencing. RNA-Seq analysis of a selected set of these strains showed that transcriptional responses to the bla CTX-M-1-IncI1 acquisition were limited, affecting metabolism, stress response, and motility functions. Our data suggest that the effect of IncI plasmid on host cells is limited, fitness cost being insufficient to act as a barrier to IncI plasmid spread among natural population of E. coli in the gut niche.

Azithromycin Resistance in Shiga Toxin-Producing Escherichia coli in France between 2004 and 2020 and Detection of mef(C)-mph(G) Genes.

We described and characterized Shiga-toxin-producing Escherichia coli (STEC) strains with high levels of resistance to azithromycin isolated in France between 2004 and 2020. Nine of 1,715 (0.52%) STEC strains were resistant to azithromycin, with an increase since 2017. One isolate carried a plasmid-borne mef(C)-mph(G) gene combination, described here for the first time for E. coli. Azithromycin resistance, although rare, needs consideration, as this treatment may be useful in cases of STEC infection.

KPC Beta-Lactamases Are Permissive to Insertions and Deletions Conferring Substrate Spectrum Modifications and Resistance to Ceftazidime-Avibactam.

To explore the mutational possibilities of insertions and deletions (indels) in the Klebsiella pneumoniae carbapenemase (KPC) beta-lactamase, we selected for ceftazidime-avibactam-resistant mutants. Of 96 screened mutants, we obtained 19 indels (2 to 15 amino acids), all located in the loops surrounding the active site. Three antibiotic susceptibility phenotypes emerged: an extended-spectrum-beta-lactamase-like phenotype, an activity restricted to ceftazidime, and a carbapenem-susceptible KPC-like phenotype. Tolerance for indels reflects the evolvability of KPC beta-lactamase, which could challenge the therapeutic management of patients.

Independent Host Factors and Bacterial Genetic Determinants of the Emergence and Dominance of Escherichia coli Sequence Type 131 CTX-M-27 in a Community Pediatric Cohort Study.

The recent emergence and diffusion in the community of Escherichia coli isolates belonging to the multidrug-resistant and CTX-M-27-producing sequence type 131 (ST131) C1-M27 cluster makes this cluster potentially as epidemic as the worldwide E. coli ST131 subclade C2 composed of multidrug-resistant isolates producing CTX-M-15. Thirty-five extended-spectrum beta-lactamase (ESBL)-producing ST131 isolates were identified in a cohort of 1,885 French children over a 5-year period. They were sequenced to characterize the ST131 E. coli isolates producing CTX-M-27 recently emerging in France. ST131 isolates producing CTX-M-27 (n = 17), and particularly those belonging to the C1-M27 cluster (n = 14), carried many resistance-encoding genes and predominantly an F1:A2:B20 plasmid type. In multivariate analysis, having been hospitalized since birth (odds ratio [OR], 10.9; 95% confidence interval [CI], 2.4 to 48.8; P = 0.002) and being cared for in a day care center (OR, 9.4; 95% CI, 1.5 to 59.0; P = 0.017) were independent risk factors for ST131 CTX-M-27 fecal carriage compared with ESBL-producing non-ST131 isolates. No independent risk factor was found when comparing CTX-M-15 (n = 11)- and CTX-M-1/14 (n = 7)-producing ST131 isolates with ESBL-producing non-ST131 isolates or with non-ESBL-producing isolates. Several factors may contribute to the increase in fecal carriage of CTX-M-27-producing E. coli isolates, namely, resistance to multiple antibiotics, capacity of the CTX-M-27 enzyme to hydrolyze both cefotaxime and ceftazidime, carriage of a peculiar F-type plasmid, and/or capacity to colonize children who have been hospitalized since birth or who attend day care centers.

Advantage of the F2:A1:B- IncF Pandemic Plasmid over IncC Plasmids in In Vitro Acquisition and Evolution of blaCTX-M Gene-Bearing Plasmids in Escherichia coli.

Despite a fitness cost imposed on bacterial hosts, large conjugative plasmids play a key role in the diffusion of resistance determinants, such as CTX-M extended-spectrum ß-lactamases. Among the large conjugative plasmids, IncF plasmids are the most predominant group, and an F2:A1:B- IncF-type plasmid encoding a CTX-M-15 variant was recently described as being strongly associated with the emerging worldwide Escherichia coli sequence type 131 (ST131)-O25b:H4 H30Rx/C2 sublineage. In this context, we investigated the fitness cost of narrow-range F-type plasmids, including the F2:A1:B- IncF-type CTX-M-15 plasmid, and of broad-range C-type plasmids in the K-12-like J53-2 E. coli strain. Although all plasmids imposed a significant fitness cost to the bacterial host immediately after conjugation, we show, using an experimental-evolution approach, that a negative impact on the fitness of the host strain was maintained throughout 1,120 generations with the IncC-IncR plasmid, regardless of the presence or absence of cefotaxime, in contrast to the F2:A1:B- IncF plasmid, whose cost was alleviated. Many chromosomal and plasmid rearrangements were detected after conjugation in transconjugants carrying the IncC plasmids but not in transconjugants carrying the F2:A1:B- IncF plasmid, except for insertion sequence (IS) mobilization from the fliM gene leading to the restoration of motility of the recipient strains. Only a few mutations occurred on the chromosome of each transconjugant throughout the experimental-evolution assay. Our findings indicate that the F2:A1:B- IncF CTX-M-15 plasmid is well adapted to the E. coli strain studied, contrary to the IncC-IncR CTX-M-15 plasmid, and that such plasmid-host adaptation could participate in the evolutionary success of the CTX-M-15-producing pandemic E. coli ST131-O25b:H4 lineage.

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.).

Genome sequencing of strains of the most prevalent clonal group of O1:K1:H7 Escherichia coli that causes neonatal meningitis in France.

BACKGROUND: To describe the temporal dynamics, molecular characterization, clinical and ex vivo virulence of emerging O1:K1 neonatal meningitis Escherichia coli (NMEC) strains of Sequence Type complex (STc) 95 in France. The national reference center collected NMEC strains and performed whole genome sequencing (WGS) of O1:K1 STc95 NMEC strains for phylogenetic and virulence genes content analysis. Data on the clinical and biological features of patients were also collected. Ex vivo virulence was assessed using the Dictyostelium discoideum amoeba model. RESULTS: Among 250 NMEC strains collected between 1998 and 2015, 38 belonged to O1:K1 STc95. This clonal complex was the most frequently collected after 2004, representing up to 25% of NMEC strains in France. Phylogenetic analysis demonstrated that most (74%) belonged to a cluster designated D-1, characterized by the adhesin FimH30. There is no clinical data to suggest that this cluster is more pathogenic than its counterparts, although it is highly predominant and harbors a large repertoire of extraintestinal virulence factors, including a pS88-like plasmid. Ex vivo virulence model showed that this cluster was generally less virulent than STc95 reference strains of O45S88:H7 and O18:H7 serotypes. However, the model showed differences between several subclones, although they harbor the same known virulence determinants. CONCLUSIONS: The emerging clonal group O1:K1 STc95 of NMEC strains is mainly composed of a cluster with many virulence factors but of only moderate virulence. Whether its emergence is due to its ability to colonize the gut thanks to FimH30 or pS88-like plasmid remains to be determined.

Long-term evolution of the natural isolate of Escherichia coli 536 in the mouse gut colonized after maternal transmission reveals convergence in the constitutive expression of the lactose operon.

In vitro experimental evolution has taught us many lessons on the molecular bases of adaptation. To move towards more natural settings, evolution in the mice gut has been successfully performed. Yet, these experiments suffered from the use of laboratory strains as well as the use of axenic or streptomycin-treated mice to maintain the inoculated strains. To circumvent these limitations, we conducted a one-year experimental evolution in vivo using a natural isolate of E. coli, strain 536, in conditions mimicking as much as possible natural environment with mother-to-offspring microbiota transmission. Mice were then distributed in 24 independent cages and separated into two different diets: a regular one (chow diet, CD) and high-fat and high-sugar one (Western Diet, WD). Genome sequences revealed an early and rapid selection during the breastfeeding period that selected the constitutive expression of the well-characterized lactose operon. E. coli was lost significantly more in CD than WD; however, we could not detect any genomic signature of selection, nor any diet specificities during the later part of the experiments. The apparently neutral evolution presumably due to low population size maintained nevertheless at high frequency the early selected mutations affecting lactose regulation. The rapid loss of lactose operon regulation challenges the idea that plastic gene expression is both optimal and stable in the wild.

Human skeletal muscle fibroblasts stimulate in vitro myogenesis and in vivo muscle regeneration.

KEY POINTS: Accumulation of skeletal muscle extracellular matrix is an unfavourable characteristic of many muscle diseases, muscle injury and sarcopenia. The extent of cross-talk between fibroblasts, as the source of matrix protein, and satellite cells in humans is unknown. We studied this in human muscle biopsies and cell-culture studies. We observed a strong stimulation of myogenesis by human fibroblasts in cell culture. In biopsies collected 30 days after a muscle injury protocol, fibroblast number increased to four times control levels, where fibroblasts were found to be preferentially located immediately surrounding regenerating muscle fibres. These novel findings indicate an important role for fibroblasts in supporting the regeneration of muscle fibres, potentially through direct stimulation of satellite cell differentiation and fusion, and contribute to understanding of cell-cell cross-talk during physiological and pathological muscle remodelling. ABSTRACT: Accumulation of skeletal muscle extracellular matrix is an unfavourable characteristic of many muscle diseases, muscle injury and sarcopenia. In addition to the indispensable role satellite cells play in muscle regeneration, there is emerging evidence in rodents for a regulatory influence on fibroblast activity. However, the influence of fibroblasts on satellite cells and muscle regeneration in humans is unknown. The purpose of this study was to investigate this in vitro and during in vivo regeneration in humans. Following a muscle injury protocol in young healthy men (n = 7), the number of fibroblasts (TCF7L2+), satellite cells (Pax7+), differentiating myogenic cells (myogenin+) and regenerating fibres (neonatal/embryonic myosin+) was determined from biopsy cross-sections. Fibroblasts and myogenic precursor cells (MPCs) were also isolated from human skeletal muscle (n = 4) and co-cultured using different cell ratios, with the two cell populations either in direct contact with each other or separated by a permeable membrane. MPC proliferation, differentiation and fusion were assessed from cells stained for BrdU, desmin and myogenin. On biopsy cross-sections, fibroblast number was seen to increase, along with myogenic cell number, by d7 and increase further by d30, where fibroblasts were observed to be preferentially located immediately surrounding regenerating muscle fibres. In vitro, the presence of fibroblasts in direct contact with MPCs was found to moderately stimulate MPC proliferation and strongly stimulate both MPC differentiation and MPC fusion. It thus appears, in humans, that fibroblasts exert a strong positive regulatory influence on MPC activity, in line with observations during in vivo skeletal muscle regeneration.

A combination of mecillinam and amoxicillin/clavulanate can restore susceptibility of high-level TEM-1-producing Escherichia coli to mecillinam.

Objectives: Mecillinam is recommended in France as a first-line treatment for lower urinary tract infections, due to the large increase in resistance of Escherichia coli to other oral treatments, such as co-trimoxazole or fluoroquinolones, its limited impact on faecal microbiota and its stability in the presence of numerous ß-lactamases. However, we recently identified several mecillinam-resistant E. coli isolates with a high-level expression penicillinase (HEP) phenotype that merit further study. Patients and methods: We studied two isogenic clinical isolates from one patient (one susceptible to mecillinam and one resistant to mecillinam) by WGS to determine the mechanism of mecillinam resistance and compared it with other mecillinam-resistant E. coli . We evaluated the synergistic combination of amoxicillin/clavulanate and mecillinam using a simple test, suitable for daily laboratory practice, to determine the MIC of this combination. Results: We showed that the presence of an SNP in the promoter of the plasmidic TEM-1 ß-lactamase gene is sufficient to confer resistance to mecillinam. This mechanism was present in 67% of HEP-phenotype E. coli tested. Combining mecillinam with amoxicillin/clavulanate abolished resistance, with an MIC compatible with clinical use. This association was not sensitive to the inoculum effect, in contrast to mecillinam alone. Conclusions: An HEP phenotype can confer mecillinam resistance in vitro . This resistance is abolished, regardless of the inoculum, by combining mecillinam with amoxicillin/clavulanate, and can be easily tested in the laboratory. This combination may be used as an oral relay treatment of non-complicated pyelonephritis due to multiresistant E. coli strains.

Using long-term experimental evolution to uncover the patterns and determinants of molecular evolution of an Escherichia coli natural isolate in the streptomycin-treated mouse gut.

Although microbial ecology of the gut is now a major focus of interest, little is known about the molecular determinants of microbial adaptation in the gut. Experimental evolution coupled with whole-genome sequencing can provide insights of the adaptive process. In vitro experiments have revealed some conserved patterns: intermediate convergence, and epistatic interactions between beneficial mutations and mutations in global regulators. To test the relevance of these patterns and to identify the selective pressures acting in vivo, we have performed a long-term adaptation of an E. coli natural isolate, the streptomycin-resistant strain 536, in the digestive tract of streptomycin-treated mice. After a year of evolution, a clone from 15 replicates was sequenced. Consistently with in vitro observations, the identified mutations revealed a strong pattern of convergence at the mutation, gene, operon and functional levels. Yet, the rate of molecular evolution was lower than in in vitro, and no mutations in global regulators were recovered. More specific targets were observed: the dgo operon, involved in the galactonate pathway that improved growth on D-galactonate, and rluD and gidB, implicated in the maturation of the ribosomes, which mutations improved growth only in the presence of streptomycin. As in vitro, the nonrandom associations of mutations within the same pathways suggested a role of epistasis in shaping the adaptive landscape. Overall, we show that 'evolve and sequence' approach coupled with an analysis of convergence, when applied to a natural isolate, can be used to study adaptation in vivo and uncover the specific selective pressures of that environment.

Differentially activated macrophages orchestrate myogenic precursor cell fate during human skeletal muscle regeneration.

Macrophages (MPs) exert either beneficial or deleterious effects on tissue repair, depending on their activation/polarization state. They are crucial for adult skeletal muscle repair, notably by acting on myogenic precursor cells. However, these interactions have not been fully characterized. Here, we explored both in vitro and in vivo, in human, the interactions of differentially activated MPs with myogenic precursor cells (MPCs) during adult myogenesis and skeletal muscle regeneration. We showed in vitro that through the differential secretion of cytokines and growth factors, proinflammatory MPs inhibited MPC fusion while anti-inflammatory MPs strongly promoted MPC differentiation by increasing their commitment into differentiated myocytes and the formation of mature myotubes. Furthermore, the in vivo time course of expression of myogenic and MP markers was studied in regenerating human healthy muscle after damage. We observed that regenerating areas containing proliferating MPCs were preferentially associated with MPs expressing proinflammatory markers. In the same muscle, regenerating areas containing differentiating myogenin-positive MPCs were preferentially coupled to MPs harboring anti-inflammatory markers. These data demonstrate for the first time in human that MPs sequentially orchestrate adult myogenesis during regeneration of damaged skeletal muscle. These results support the emerging concept that inflammation, through MP activation, controls stem cell fate and coordinates tissue repair.

Genomic evidence of Escherichia coli gut population diversity translocation in leukemia patients.

Escherichia coli, a commensal species of the human gut, is an opportunistic pathogen that can reach extra-intestinal compartments, including the bloodstream and the bladder, among others. In non-immunosuppressed patients, purifying or neutral evolution of E. coli populations has been reported in the gut. Conversely, it has been suggested that when migrating to extra-intestinal compartments, E. coli genomes undergo diversifying selection as supported by strong evidence for adaptation. The level of genomic polymorphism and the size of the populations translocating from gut to extra-intestinal compartments is largely unknown. To gain insights into the pathophysiology of these translocations, we investigated the level of polymorphism and the evolutionary forces acting on the genomes of 77 E. coli isolated from various compartments in three immunosuppressed patients. Each patient had a unique strain, which was a mutator in one case. In all instances, we observed that translocation encompasses much of the genomic diversity present in the gut. The same signature of selection, whether purifying or diversifying, and as anticipated, neutral for mutator isolates, was observed in both the gut and bloodstream. Additionally, we found a limited number of non-specific mutations among compartments for non-mutator isolates. In all cases, urine isolates were dominated by neutral selection. These findings indicate that substantial proportions of populations are undergoing translocation and that they present a complex compartment-specific pattern of selection at the patient level.IMPORTANCEIt has been suggested that intra and extra-intestinal compartments differentially constrain the evolution of E. coli strains. Whether host particular conditions, such as immunosuppression, could affect the strain evolutionary trajectories remains understudied. We found that, in immunosuppressed patients, large fractions of E. coli gut populations are translocating with variable modifications of the signature of selection for commensal and pathogenic isolates according to the compartment and/or the patient. Such multiple site sampling should be performed in large cohorts of patients to gain a better understanding of E. coli extra-intestinal diseases.

Changing fitness effects of mutations through long-term bacterial evolution.

The distribution of fitness effects of new mutations shapes evolution, but it is challenging to observe how it changes as organisms adapt. Using Escherichia coli lineages spanning 50,000 generations of evolution, we quantify the fitness effects of insertion mutations in every gene. Macroscopically, the fraction of deleterious mutations changed little over time whereas the beneficial tail declined sharply, approaching an exponential distribution. Microscopically, changes in individual gene essentiality and deleterious effects often occurred in parallel; altered essentiality is only partly explained by structural variation. The identity and effect sizes of beneficial mutations changed rapidly over time, but many targets of selection remained predictable because of the importance of loss-of-function mutations. Taken together, these results reveal the dynamic-but statistically predictable-nature of mutational fitness effects.

Variability in cell division among anatomical sites shapes Escherichia coli antibiotic survival in a urinary tract infection mouse model.

Urinary tract infection (UTI), mainly caused by Escherichia coli, are frequent and have a recurrent nature even after antibiotic treatment. Potential bacterial escape mechanisms include growth defects, but probing bacterial division in vivo and establishing its relation to the antibiotic response remain challenging. Using a synthetic reporter of cell division, we follow the temporal dynamics of cell division for different E. coli clinical strains in a UTI mouse model with and without antibiotics. We show that more bacteria are actively dividing in the kidneys and urine compared with the bladder. Bacteria that survive antibiotic treatment are consistently non-dividing in three sites of infection. Additionally, we demonstrate how both the strain in vitro persistence profile and the microenvironment impact infection and treatment dynamics. Understanding the relative contribution of the host environment, growth heterogeneity, non-dividing bacteria, and antibiotic persistence is crucial to improve therapies for recurrent infections.

Perturbation and resilience of the gut microbiome up to 3 months after ß-lactams exposure in healthy volunteers suggest an important role of microbial ß-lactamases.

BACKGROUND: Antibiotics notoriously perturb the gut microbiota. We treated healthy volunteers either with cefotaxime or ceftriaxone for 3 days, and collected in each subject 12 faecal samples up to day 90. Using untargeted and targeted phenotypic and genotypic approaches, we studied the changes in the bacterial, phage and fungal components of the microbiota as well as the metabolome and the ß-lactamase activity of the stools. This allowed assessing their degrees of perturbation and resilience. RESULTS: While only two subjects had detectable concentrations of antibiotics in their faeces, suggesting important antibiotic degradation in the gut, the intravenous treatment perturbed very significantly the bacterial and phage microbiota, as well as the composition of the metabolome. In contrast, treatment impact was relatively low on the fungal microbiota. At the end of the surveillance period, we found evidence of resilience across the gut system since most components returned to a state like the initial one, even if the structure of the bacterial microbiota changed and the dynamics of the different components over time were rarely correlated. The observed richness of the antibiotic resistance genes repertoire was significantly reduced up to day 30, while a significant increase in the relative abundance of ß-lactamase encoding genes was observed up to day 10, consistent with a concomitant increase in the ß-lactamase activity of the microbiota. The level of ß-lactamase activity at baseline was positively associated with the resilience of the metabolome content of the stools. CONCLUSIONS: In healthy adults, antibiotics perturb many components of the microbiota, which return close to the baseline state within 30 days. These data suggest an important role of endogenous ß-lactamase-producing anaerobes in protecting the functions of the microbiota by de-activating the antibiotics reaching the colon. Video Abstract.

Faecal carriage of extended-spectrum ß-lactamase-producing Escherichia coli in a remote region of Niger.

OBJECTIVE: Whole genome sequencing (WGS) of extended-spectrum ß-lactamase-producing Escherichia coli (ESBL-E. coli) in developing countries is lacking. Here we describe the population structure and molecular characteristics of ESBL-E. coli faecal isolates in rural Southern Niger. METHODS: Stools of 383 healthy participants were collected among which 92.4% were ESBL-Enterobacterales carriers. A subset of 90 ESBL-E. coli containing stools (109 ESBL-E. coli isolates) were further analysed by WGS, using short- and long-reads. RESULTS: Most isolates belonged to the commensalism-adapted phylogroup A (83.5%), with high clonal diversity. The blaCTX-M-15 gene was the major ESBL determinant (98.1%), chromosome-integrated in approximately 50% of cases, in multiple integration sites. When plasmid-borne, blaCTX-M-15 was found in IncF (57.4%) and IncY plasmids (26.2%). Closely related plasmids were found in different genetic backgrounds. Genomic environment analysis of blaCTX-M-15 in closely related strains argued for mobilisation between plasmids or from plasmid to chromosome. CONCLUSIONS: Massive prevalence of community faecal carriage of CTX-M-15-producing E. coli was observed in a rural region of Niger due to the spread of highly diverse A phylogroup commensalism-adapted clones, with frequent chromosomal integration of blaCTX-M-15. Plasmid spread was also observed. These data suggest a risk of sustainable implementation of ESBL in community faecal carriage.

A Microbiota-Dependent Response to Anticancer Treatment in an In Vitro Human Microbiota Model: A Pilot Study With Hydroxycarbamide and Daunorubicin.

Background: Anticancer drug efficacy is linked to the gut microbiota's composition, and there is a dire need to better understand these interactions for personalized medicine. In vitro microbiota models are promising tools for studies requiring controlled and repeatable conditions. We evaluated the impact of two anticancer drugs on human feces in the MiniBioReactor Array (MBRA) in vitro microbiota system. Methods: The MBRA is a single-stage continuous-flow culture model, hosted in an anaerobic chamber. We evaluated the effect of a 5-day treatment with hydroxycarbamide or daunorubicine on the fecal bacterial communities of two healthy donors. 16S microbiome profiling allowed analysis of microbial richness, diversity, and taxonomic changes. Results: In this host-free setting, anticancer drugs diversely affect gut microbiota composition. Daunorubicin was associated with significant changes in alpha- and beta-diversity as well as in the ratio of Firmicutes/Bacteroidetes in a donor-dependent manner. The impact of hydroxycarbamide on microbiota composition was not significant. Conclusion: We demonstrated, for the first time, the impact of anticancer drugs on human microbiota composition, in a donor- and molecule-dependent manner in an in vitro human microbiota model. We confirm the importance of personalized studies to better predict drug-associated-dysbiosis in vivo, linked to the host's response to treatment.

Local and Global Protein Interactions Contribute to Residue Entrenchment in Beta-Lactamase TEM-1.

Due to their rapid evolution and their impact on healthcare, beta-lactamases, protein degrading beta-lactam antibiotics, are used as generic models of protein evolution. Therefore, we investigated the mutation effects in two distant beta-lactamases, TEM-1 and CTX-M-15. Interestingly, we found a site with a complex pattern of genetic interactions. Mutation G251W in TEM-1 inactivates the protein's function, just as the reciprocal mutation, W251G, does in CTX-M-15. The phylogenetic analysis revealed that mutation G has been entrenched in TEM-1's background: while rarely observed throughout the phylogeny, it is essential in TEM-1. Using a rescue experiment, in the TEM-1 G251W mutant, we identified sites that alleviate the deviation from G to W. While few of these mutations could potentially involve local interactions, most of them were found on distant residues in the 3D structure. Many well-known mutations that have an impact on protein stability, such as M182T, were recovered. Our results therefore suggest that entrenchment of an amino acid may rely on diffuse interactions among multiple sites, with a major impact on protein stability.

Modeling the bacterial dynamics in the gut microbiota following an antibiotic-induced perturbation.

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.