Evolution of fluoroquinolone-resistant Escherichia coli in the gut after ciprofloxacin treatment.

BACKGROUND: Three healthy volunteers carried similar quinolone-resistant E. coli (QREC) (pulsed field gel electrophoresis profiles) in their gut before and after 14 days ciprofloxacin treatment. Given the intensity of the selective pressure and the mutagenic properties of quinolones, we determined whether these strains had evolved at the phenotypic and/or genomic levels. MATERIAL AND METHODS: Commensal QREC from before day-0 (D0), and a month after 14 days of ciprofloxacin (D42) were compared in 3 volunteers. Growth experiments were performed; acetate levels, mutation frequencies, quinolone MICs and antibiotic tolerance were measured at D0 and D42. Genomes were sequenced and single nucleotide polymorphisms (SNPs) between D0 and D42 were analyzed using DiscoSNP and breseq methods. Cytoplasmic proteins were extracted, HPLC performed and proteins identified using X!tandem software; abundances were measured by mass spectrometry using the Spectral Counting (SC) and eXtraction Ion Chromatograms (XIC) integration methods. RESULTS: No difference was found in MICs, growth characteristics, acetate concentrations, mutation frequencies, tolerance profiles, phylogroups, O-and H-types, fimH alleles and sequence types between D0 and D42. No SNP variation was evidenced between D0 and D42 isolates for 2/3 subjects; 2 SNP variations were evidenced in one. At the protein level, very few significant protein abundance differences were identified between D0 and D42. CONCLUSION: No fitness, tolerance, metabolic or genomic evolution of commensal QREC was observed overtime, despite massive exposure to ciprofloxacin in the gut. The three strains behaved as if they had been unaffected by ciprofloxacin, suggesting that gut may act as a sanctuary where bacteria would be protected from the effect of antibiotics and survive without any detrimental effect of stress.

Mortality in Escherichia coli bloodstream infections: antibiotic resistance still does not make it.

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 ±âŸ16.5 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.).

Two levels of specialization in bacteraemic Escherichia coli strains revealed by their comparison with commensal strains.

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.

Escherichia coli bacteraemia in adults: age-related differences in clinical and bacteriological characteristics, and outcome.

To explore the specificities of Escherichia coli bacteraemia in the elderly, the demographic, clinical and bacteriological characteristics and in-hospital mortality rate of 'young' (18-64 years, n = 395), 'old' (65-79 years, n = 372) and 'very old' (⩾80 years, n = 284) adult patients of the multicentre COLIBAFI cohort study were compared. Clinical and bacteriological risk factors for death were jointly identified by logistic regression and multivariate analysis within each group. 'Young' and 'old' patients had more comorbidities than 'very old' patients (comorbidity score: 1·5 ± 1·3 and 1·6 ± 1·2 vs. 1·2 ± 1·2, respectively; P < 0·001), and were more frequently nosocomially infected (22·3% and 23·8% vs. 8·8%, respectively; P < 0·001). 'Old' patients had the poorest prognosis (death rate: 16·4% vs.10·4% for 'young' and 12·0% for 'very old' patients, respectively; P = 0·039). Risk factors for death were age group-specific, suggesting a host-pathogen relationship evolving with age.

Evidence for coexistence of distinct Escherichia coli populations in various aquatic environments and their survival in estuary water.

Escherichia coli, a commensal bacterium from the intestinal tracts of humans and vertebrate animals, has been used as one of two bacterial indicators of fecal contamination, along with intestinal enterococci, to monitor the microbiological quality of water. However, water environments are now recognized as a secondary habitat where some strains can survive. We investigated the survival of E. coli isolates collected from bodies of water in France exhibiting distinct profiles of contamination, defined according to the following criteria: vicinity of the point sources of contamination, land use, hydrology, and physicochemical characteristics of the receiving water. We selected 88 E. coli strains among a collection of 352 strains to carry out a microcosm experiment in filtered estuarine water for 14 days at 10°C. The relationship between the survival of E. coli strains and genotypic and phenotypic characteristics was analyzed. This work showed that distinct E. coli survival types, able to survive from between 7 and 14 days to less than 2 days, coexisted in the water. E. coli isolates that rapidly lost their culturability were more frequently isolated in water recently contaminated by fecal bacteria of human origin, and most were multiresistant to antibiotics and harbored several virulence factors. In contrast, persistent strains able to survive from 4 to 14 days were more often found in water with low levels of fecal bacteria, belonged mainly to the B1 phylogroup, often harbored only one virulence factor, kspE or ompT, and were able to grow at 7°C.

Correlation between severity and SMN protein level in spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a common autosomal recessive neuromuscular disorder characterized by degeneration of motor neurons of the spinal cord. Three different forms of childhood SMA have been recognized on the basis of age at onset and clinical course: Werdnig-Hoffmann disease (type-1), the intermediate form (type-II) and Kugelberg-Welander disease (type-III). A gene termed 'survival of motor neuron' (SMN) has been recognized as the disease-causing gene in SMA. SMN encodes a protein located within a novel nuclear structure and interacts with RNA-binding proteins. To elucidate the molecular mechanism underlying the pathogenesis of the disease, we examined the expression of the SMN gene in both controls and SMA patients by western blot and immunohistochemical analyses using antibodies raised against the SMN protein. The present study shows a marked deficiency of the SMN protein in SMA.

A frame-shift deletion in the survival motor neuron gene in Spanish spinal muscular atrophy patients.

Spinal muscular atrophy (SMA) is a frequent autosomal recessive disease characterized by degeneration of the motor neurons of the spinal cord causing proximal paralysis with muscle atrophy. The region on chromosome 5q13 encompassing the disease gene is particularly unstable and prone to large-scale deletions whose characterization recently led to the identification of the survival motor neuron (SMN) gene. We now present a genetic analysis of 54 unrelated Spanish SMA families that has revealed a 4-basepair (bp) deletion (AGAG) in exon 3 of SMN in four unrelated patients. This deletion, which results in a frameshift and a premature stop codon, occurs on the same haplotype background, suggesting that a single mutational event is involved in the four families. The other patients showed either deletions of the SMN gene (49/54) or a gene conversion event changing SMN exon 7 into its highly homologous copy (cBCD541, 1/54). This observation gives strong support to the view that mutations of the SMN gene are responsible for the SMA phenotype as it is the first frameshift mutation reported in SMA.

The decoupling between genetic structure and metabolic phenotypes in Escherichia coli leads to continuous phenotypic diversity.

To assess the extent of intra-species diversity and the links between phylogeny, lifestyle (habitat and pathogenicity) and phenotype, we assayed the growth yield on 95 carbon sources of 168 Escherichia strains. We also correlated the growth capacities of 14 E. coli strains with the presence/absence of enzyme-coding genes. Globally, we found that the genetic distance, based on multilocus sequence typing data, was a weak indicator of the metabolic phenotypic distance. Besides, lifestyle and phylogroup had almost no impact on the growth yield of non-Shigella E. coli strains. In these strains, the presence/absence of the metabolic pathways, which was linked to the phylogeny, explained most of the growth capacities. However, few discrepancies blurred the link between metabolic phenotypic distance and metabolic pathway distance. This study shows that a prokaryotic species structured into well-defined genetic and lifestyle groups can yet exhibit continuous phenotypic diversity, possibly caused by gene regulatory effects.

De novo and inherited deletions of the 5q13 region in spinal muscular atrophies.

Spinal muscular atrophies (SMAs) represent the second most common fatal autosomal recessive disorder after cystic fibrosis. Childhood spinal muscular atrophies are divided into severe (type I) and mild forms (types II and III). By a combination of genetic and physical mapping, a yeast artificial chromosome contig of the 5q13 region spanning the disease locus was constructed that showed the presence of low copy repeats in this region. Allele segregation was analyzed at the closest genetic loci detected by markers C212 and C272 in 201 SMA families. Inherited and de novo deletions were observed in nine unrelated SMA patients. Moreover, deletions were strongly suggested in at least 18 percent of SMA type I patients by the observation of marked heterozygosity deficiency for the loci studied. These results indicate that deletion events are statistically associated with the severe form of spinal muscular atrophy.

Determination of Escherichia coli phylogroups in elderly patients with urinary tract infection or asymptomatic bacteriuria.

OBJECTIVES: Distinguishing between urinary tract infection (UTI) and asymptomatic bacteriuria (ABU) is difficult in the geriatric population since specific symptoms are often lacking. Escherichia coli is the most frequent UTI pathogen in this population but also a common urine colonizer. We hypothesized that detecting E. coli phylogroups B2 or D, which were previously associated with virulent strains responsible for extra-intestinal infections outside elderly patients, could help in distinguishing UTI from ABU. METHODS: Consecutive cases of E. coli bacteriuria diagnosed in hospitalized patients >75 years old during 3 months were investigated for E. coli phylogroups. Multiplex PCR was used to search for several virulence genes as previously described. Characteristics of UTI and ABU cases, assessed retrospectively according to definitions and geriatric expertise, were compared. RESULTS: Out of 233 bacteriuria cases, 60 were assessed to be UTI and 163 to be ABU, with 10 cases unclassified. E. coli strains belonging to the phylogroups B2 and D were significantly more frequent in UTI (48/60, 80%) than in ABU (101/163, 62%) by univariate and multivariate analyses (OR 3.05, 1.44-6.86, p 0.005). Out of all the host and bacterial characteristics studied, falls (p 0.032), comorbidities (p 0.041), and altered autonomy evaluated by a low activity of daily living score (p 0.027) were also associated with UTI using univariate and multivariate analysis. CONCLUSIONS: Determination of the E. coli phylogroup, in addition to some host characteristics, can help to distinguish UTI from ABU in elderly patients with bacteriuria. If this hypothesis is confirmed by prospective studies, then inappropriate use of antibiotics may be reduced in ABU cases.

The Odyssey of the Ancestral Escherich Strain through Culture Collections: an Example of Allopatric Diversification.

More than a century ago, Theodor Escherich isolated the bacterium that was to become Escherichia coli, one of the most studied organisms. Not long after, the strain began an odyssey and landed in many laboratories across the world. As laboratory culture conditions could be responsible for major changes in bacterial strains, we conducted a genome analysis of isolates of this emblematic strain from different culture collections (England, France, the United States, Germany). Strikingly, many discrepancies between the isolates were observed, as revealed by multilocus sequence typing (MLST), the presence of virulence-associated genes, core genome MLST, and single nucleotide polymorphism/indel analyses. These differences are correlated with the phylogeographic history of the strain and were due to an unprecedented number of mutations in coding DNA repair functions such as mismatch repair (MutL) and oxidized guanine nucleotide pool cleaning (MutT), conferring a specific mutational spectrum and leading to a mutator phenotype. The mutator phenotype was probably acquired during subculturing and corresponded to second-order selection. Furthermore, all of the isolates exhibited hypersusceptibility to antibiotics due to mutations in efflux pump- and porin-encoding genes, as well as a specific mutation in the sigma factor-encoding gene rpoS. These defects reflect a self-preservation and nutritional competence tradeoff allowing survival under the starvation conditions imposed by storage. From a clinical point of view, dealing with such mutator strains can lead microbiologists to draw false conclusions about isolate relatedness and may impact therapeutic effectiveness. IMPORTANCE Mutator phenotypes have been described in laboratory-evolved bacteria, as well as in natural isolates. Several genes can be impacted, each of them being associated with a typical mutational spectrum. By studying one of the oldest strains available, the ancestral Escherich strain, we were able to identify its mutator status leading to tremendous genetic diversity among the isolates from various collections and allowing us to reconstruct the phylogeographic history of the strain. This mutator phenotype was probably acquired during the storage of the strain, promoting adaptation to a specific environment. Other mutations in rpoS and efflux pump- and porin-encoding genes highlight the acclimatization of the strain through self-preservation and nutritional competence regulation. This strain history can be viewed as unintentional experimental evolution in culture collections all over the word since 1885, mimicking the long-term experimental evolution of E. coli of Lenski et al. (O. Tenaillon, J. E. Barrick, N. Ribeck, D. E. Deatherage, J. L. Blanchard, A. Dasgupta, G. C. Wu, S. Wielgoss, S. Cruveiller, C. Médigue, D. Schneider, and R. E. Lenski, Nature 536:165-170, 2016, https://doi.org/10.1038/nature18959) that shares numerous molecular features.

Survival motor neuron gene deletion in the arthrogryposis multiplex congenita-spinal muscular atrophy association.

The survival motor neuron (SMN) gene was lacking in 6/12 patients with arthrogryposis multiplex congenita (AMC) associated with spinal muscular atrophy (SMA). Neither point mutation in the SMN gene nor evidence for linkage to chromosome 5q13 were found in the other patients. Hitherto, arthrogryposis was regarded as an exclusion criterion in SMA. Our data strongly suggest that AMC of neurogenic origin is genetically heterogeneous, with a subgroup being allelic to SMA. Absence or interruption of the SMN gene in the AMC-SMA association will make the diagnosis easier and genetic counselling will now become feasible.

The Yersinia high-pathogenicity island is highly predominant in virulence-associated phylogenetic groups of Escherichia coli.

The high-pathogenicity island (HPI) present in pathogenic Yersinia and encoding the siderophore yersiniabactin, has recently been identified in the asnT tRNA region of various Escherichia coli pathotypes, especially those responsible for bacteremia and urosepsis. Most E. coli strains causing such extra-intestinal infections belong to phylogenetic groups B2 and D. In this study we investigated (i) the distribution and localization of HPI among the different E. coli phylogenetic groups, using the ECOR reference collection; and (ii) the prevalence of HPI among a set of 124 phylogenetically characterized E. coli strains responsible for neonatal meningitis. Ninety-three percent of the ECOR strains belonging to groups B2 and D harbored HPI. In contrast, the island was present in 32% and 25% of strains belonging to groups A and B1, respectively, which are considered to be non-pathogenic. HPI was found in 100% of the neonatal meningitis strains, 13 of which belonged to groups A and B1, suggesting that HPI might contain virulent factors required for the development of neonatal meningitis. Moreover, we observed for the first time that HPI can be inserted in a site different from the asnT tRNA region.

Escherichia coli bacteraemia in pregnant women is life-threatening for foetuses.

In order to improve knowledge on Escherichia coli bacteraemia during pregnancy, we studied clinical data and performed molecular characterization of strains for 29 E. coli bacteraemia occurring in pregnant women. Bacteraemia mostly occurred in the third trimester of pregnancy (45%) and was community-acquired (79%). Portals of entry were urinary (55%) and genital (45%). E. coli strains belonged mainly to phylogroups B2 (72%) and D (17%). Four clonal lineages (i.e. sequence type complex (STc) 73, STc95, STc12 and STc69) represented 65% of the strains. The strains exhibited a high number of virulence factor coding genes (10 (3-16)). Six foetuses died (27%), five of them due to bacteraemia of genital origin (83%). Foetal deaths occurred despite adequate antibiotic regimens. Strains associated with foetal mortality had fewer virulence factors (8 (6-10)) than strains involved in no foetal mortality (11 (4-12)) (p 0.02). When comparing E. coli strains involved in bacteraemia with a urinary portal of entry in non-immunocompromised pregnant vs. non-immunocompromised non-pregnant women from the COLIBAFI study, there was no significant difference of phylogroups and virulence factor coding genes. These results show that E. coli bacteraemia in pregnant women involve few highly virulent clones but that severity, represented by foetal death, is mainly related to bacteraemia of genital origin.

Severity of Escherichia coli bacteraemia is independent of the intrinsic virulence of the strains assessed in a mouse model.

Extraintestinal pathogenic Escherichia coli (ExPEC) strains, a major cause of bacteraemia, typically belong to phylogenetic group B2 and express diverse accessory traits that contribute to virulence in mouse infection models. However, their high genomic diversity obscures the relationship between virulence factors and severity of infection in patients. In this study, we analysed concomitantly the strain's expression of virulence in a mouse model, genomic determinants and the clinical severity of infection in 60 bacteraemic patients. We show that bacterial virulence based on an animal model study and virulence factor determination is not correlated with pejorative outcome of E. coli human blood infections.

Bacteraemia caused by third-generation cephalosporin-resistant Escherichia coli in France: prevalence, molecular epidemiology and clinical features.

Escherichia coli is one of the major pathogens responsible for bactaeremia. Empirical antibiotherapy of these infections usually relies on third-generation cephalosporins (3GCs). Thus, the occurrence and epidemiology of 3GC-resistant strains have to be monitored. The French prospective multicentre study COLIBAFI collected 1081 strains of E. coli responsible for bacteraemia in 2005. In the present work, the prevalence of resistance to 3GCs was evaluated, and the implicated molecular mechanisms were characterized by specific PCR and sequencing. Phylogenetic grouping, O-typing, pulsed-field gel electrophoresis and virulence factor analysis were used to investigate the genetic background of the 3GC-resistant (3GC-R) strains. Clinical features of the patients with documented data (n = 1051) were analysed. Decreased susceptibility to 3GCs was observed in 41 strains (3.8%): 19, 18 and four had extended-spectrum ß-lactamase (ESBL), AmpC cephalosporinase and OXA-type penicillinase phenotypes, respectively. Pulsed-field gel electrophoresis revealed that the 3GC-R strains constitute a diverse population. All but one of the strains with an ESBL phenotype produced a CTX-M-type enzyme, and six of them belonged to the widespread intercontinental clone O25b:H4-ST131. AmpC phenotype strains harboured various chromosomal ampC promoter and coding region mutations and/or the bla(CMY-2) plasmidic gene. 3GC-R strains carried fewer virulence factors and were more co-resistant to other antibiotics than 3GC-susceptible (3GC-S) strains. Infections with 3GC-R strains were mostly community-acquired and, as compared with those caused by their 3GC-S counterparts, were more severe. Underlying chronic disease and prior use of antibiotics were independent risk factors for development of a 3GC-R strain bacteraemia. The fact that the molecular support of 3GC resistance is mainly plasmid-mediated represents a potentially epidemic threat.

Rapid and simple determination of the Escherichia coli phylogenetic group.

Phylogenetic analysis has shown that Escherichia coli is composed of four main phylogenetic groups (A, B1, B2, and D) and that virulent extra-intestinal strains mainly belong to groups B2 and D. Actually, phylogenetic groups can be determined by multilocus enzyme electrophoresis or ribotyping, both of which are complex, time-consuming techniques. We describe a simple and rapid phylogenetic grouping technique based on triplex PCR. The method, which uses a combination of two genes (chuA and yjaA) and an anonymous DNA fragment, was tested with 230 strains and showed excellent correlation with reference methods.

Automated ribotyping provides rapid phylogenetic subgroup affiliation of clinical extraintestinal pathogenic Escherichia coli strains.

Using the automated Riboprinter system, we have initiated the construction of an electronic Riboprint database composed of 72 ECOR reference strains and 15 archetypal virulent strains in order to provide a new simple molecular characterization method. More than 90% of the ECOR strains clustered in their original phylogenetic group. All but one of the archetypal virulent strains had a profile identical to that of one of the ECOR strains and could be easily affiliated with a phylogenetic group. This method appears to be an accurate and practical tool especially for investigating the genetic relationship between clinical extraintestinal pathogenic strains and B2 subgroup ECOR strains or archetypal pathotype strains.

In vivo selection of a target/efflux double mutant of Pseudomonas aeruginosa by ciprofloxacin therapy.

We report the emergence after 4 days of ciprofloxacin monotherapy of a double mutant of Pseudomonas aeruginosa overexpressing the multidrug efflux system MexAB-OprM and harbouring a mutation in the gyrB gene. Compared with its initial susceptible counterpart, this mutant exhibited a significant increase in resistance to most of the beta-lactam antibiotics tested (16 x MIC of ticarcillin) and to ciprofloxacin (128 x MIC). Combined ceftazidime and amikacin therapy finally eradicated the resistant isolate and cured the patient of his infection. This case illustrates how strains of P. aeruginosa may develop high levels of fluoroquinolone resistance by combining efflux mechanisms and target alterations.