Catherine Branger

The CTX-M-15-producing Escherichia coli diffusing clone belongs to a highly virulent B2 phylogenetic subgroup

OBJECTIVESnA clone of CTX-M-15-producing Escherichia coli has recently been reported to be spreading through Europe and Africa. The aim of this work was to thoroughly characterize this clone.nnnMATERIALS AND METHODSnRepresentative isolates of this clone were subjected to multilocus sequence typing, O typing, virulence gene detection, adhesion assay on human cells, biofilm production assay and mouse lethality assay.nnnRESULTSnThe clone: (i) belongs to a unique B2 phylogenetic subgroup encompassing the pyelonephritogenic diffusely adhering EC7372 strain; (ii) exhibits a specific O25b molecular subtype; (iii) is identical to the E. coli clone O25:H4-ST131 producing CTX-M-15; (iv) produces biofilm; and (v) is highly virulent in mice despite lacking classical extraintestinal pathogenicity islands (except for high pathogenicity island) and the afa/dra gene.nnnCONCLUSIONSnThe CTX-M-15-producing E. coli diffusing clone is associated with a high level of antibiotic resistance and with high virulence, showing that, under certain selective pressures, the previously observed trade-off between resistance and virulence may not apply.

Replicon typing of plasmids in Escherichia coli producing extended-spectrum β-lactamases

OBJECTIVESnEscherichia coli producing CTX-M-15 and CTX-M-14 extended-spectrum beta-lactamases (ESBLs) are spreading worldwide. The aim of this work was to investigate the replicons involved in the emergence and spread of ESBLs in relation to ESBL type.nnnMETHODSnA collection of 125 TEM, SHV and CTX-M ESBL-producing E. coli strains was analysed. The replicons carrying the ESBLs and the total plasmid content of the strains have been characterized by PCR replicon typing in relation to the type of ESBL. The ESBL replicons were then compared with the replicon content of E. coli strains carrying TEM-1 or inhibitor-resistant TEM (IRT) beta-lactamases.nnnRESULTSnIncF plasmids were the most frequently carried replicons in our collection, but none carried TEM ESBL. Of TEM ESBLs, 67% were carried on IncA/C replicons except for TEM-52 genes, which were carried preferentially on IncI1 replicons. Although CTX-M enzymes can be carried by various replicons, the great majority of genes encoding CTX-M-14 and CTX-M-15 ESBLs were carried by IncF replicons, as were TEM-1 and IRT beta-lactamases.nnnCONCLUSIONSnResistance genes borne by the narrow host-range IncF replicon spread readily as this replicon is well adapted to E. coli. This is observed for blaTEM-1 and blaCTX-M-15 and, to a lesser extent, for blaCTX-M-14. Transposition immunity seems to play an important role in the diffusion process.

Host Factors and Portal of Entry Outweigh Bacterial Determinants To Predict the Severity of Escherichia coli Bacteremia

ABSTRACT Escherichia coli ranks among the organisms most frequently isolated from cases of bacteremia. The relative contribution of the host and bacteria to E. coli bacteremia severity remains unknown. We conducted a prospective multicenter cohort study to identify host and bacterial factors associated with E. coli bacteremia severity. The primary endpoint was in-hospital death, up to 28 days after the first positive blood culture. Among 1,051 patients included, 136 (12.9%) died. Overall, 604 (57.5%) patients were female. The median age was 70 years, and 202 (19.2%) episodes were nosocomial. The most frequent comorbidities were immunocompromised status (37.9%), tobacco addiction (21.5%), and diabetes mellitus (20.1%). The most common portal of entry was the urinary tract (56.9%). Most E. coli isolates belonged to phylogenetic group B2 (52.0%). The multivariate analysis retained the following factors as predictive of death: older age (odds ratio [OR] = 1.25 [95% confidence interval {CI}, 1.09 to 1.43] for each 10-year increment), cirrhosis (OR = 4.85 [95% CI, 2.49 to 9.45]), hospitalization before bacteremia (OR = 4.13 [95% CI, 2.49 to 6.82]), being an immunocompromised patient not hospitalized before bacteremia (OR = 3.73 [95% CI, 2.25 to 6.18]), and a cutaneous portal of entry (OR = 6.45 [95% CI, 1.68 to 24.79]); a urinary tract portal of entry and the presence of the ireA virulence gene were negatively correlated with death (OR = 0.46 [95% CI, 0.30 to 0.70] and OR = 0.53 [95% CI, 0.30 to 0.91], respectively). In summary, host factors and the portal of entry outweigh bacterial determinants for predicting E. coli bacteremia severity.

Molecular characterization of addiction systems of plasmids encoding extended-spectrum β-lactamases in Escherichia coli

OBJECTIVESnEscherichia coli producing CTX-M-type extended-spectrum beta-lactamases (ESBLs) are spreading worldwide. The aim of this work was to investigate the addiction systems carried by the replicons involved in the emergence and spread of ESBLs in relation to ESBL and replicon types.nnnMETHODSnA collection of 125 TEM, SHV and CTX-M ESBL-producing E. coli isolates and their 125 transconjugants or transformants was analysed. Five plasmid protein antitoxin-regulated systems and three plasmid antisense RNA-regulated systems were sought by PCR.nnnRESULTSnTwo hundred and ninety-eight plasmid addiction systems were detected in the parental strains (mean 2.38, range 0-6 per strain) and 86 were detected in the recipient strains (mean 0.69, range 0-5 per strain). PemKI, CcdAB, Hok-Sok and VagCD were the most frequently represented systems in both recipient and parental strains. The parental SHV and CTX-M ESBL-producing strains had more addiction systems than the TEM ESBL producers. In the recipient strains, the frequency of addiction systems was significantly higher in IncF plasmids. Among the IncF replicons carrying CTX-M-type enzymes, the frequency of addiction systems was significantly higher in IncF plasmids carrying CTX-M-15 (mean 3.5) or CTX-M-9 (mean 4) than in those carrying CTX-M-14 (mean 0.6).nnnCONCLUSIONSnIn E. coli producing CTX-M-15 or CTX-M-9 ESBLs, plasmids bearing the bla(CTX-M) gene have multiple addiction systems that could contribute to their maintenance in host strains.

Multiple acquisitions of CTX-M plasmids in the rare D2 genotype of Escherichia coli provide evidence for convergent evolution.

Over the last decade, CTX-M enzymes have become the most prevalent extended-spectrum beta-lactamases (ESBLs) worldwide, mostly in Escherichia coli, causing a major health problem. An epidemiological relationship has been established between a rare genotype of E. coli, the D(2) genotype, and the presence of CTX-M genes. We investigated this striking association by exploring the genetic backgrounds of 18 D(2) genotype CTX-M-producing strains and of the plasmids encoding CTX-M enzymes. The 18 strains had different genetic backgrounds, as assessed by multilocus sequence and O typing, and were associated with various plasmids bearing diverse CTX-M genes. The region encompassing the genetic marker of the D(2) genotype (TSPE4.C2) was not correlated with the presence of CTX-M genes. CTX-M-producing D(2) strains had far fewer virulence factors than a control group of 8 non-ESBL-producing D(2) strains, and an inverse relationship was found between the number of co-resistances associated with the CTX-M gene and the number of virulence factors found in the strain. These findings provide evidence for multiple acquisitions of plasmids carrying CTX-M genes in different D(2) genotype strains. They strongly suggest that convergent evolution has occurred, and indicate that there has been selection for the association of a specific genetic background of the strain and the CTX-M gene. This fine-tuning of the relationship between the D(2) genotype and CTX-M genes presumably increases the fitness of the strain, indicating a role for the host cell in the acquisition and dissemination of CTX-M genes.

Probable intrafamily transmission of a highly virulent CTX-M-3-producing Escherichia coli belonging to the emerging phylogenetic subgroup D2 O102-ST405 clone

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Targeting relaxase genes for classification of the predominant plasmids in Enterobacteriaceae

Plasmids are the main vectors of antimicrobial drug resistance and virulence genes, especially in Enterobacteriaceae. Identification and classification of plasmids is essential for analysis of their distribution. The most widely used typing method is PCR-based replicon typing (PBRT). A new classification scheme based on relaxase gene typing has been described recently. We propose a practical application of this method, with the development of a multiplex PCR set targeting relaxase genes found on plasmids most frequently encountered in Enterobacteriaceae. This method, here called plasmid relaxase gene typing (PRaseT), was validated with 60 transconjugants and transformants harboring various replicon types. The method was tested with 39 multidrug-resistant clinical isolates including Escherichia coli, Klebsiella pneumoniae and Salmonella enterica subsp. enterica carrying 1-7 replicons as well as with 17 plasmids non-typeable using PBRT; all replicons were tested in parallel with PBRT for comparison. Six multiplex PCRs and one simplex PCR, including 24 pairs of primers, recognized plasmids of groups A/C, B/O, colE, FIA, FIB, FIC, FV, FIIk, HI1, HI2, I1, K, L/M, N, P1α, Q1, U, W, X1, X2, X3 and X4. There was perfect correlation between PRaseT and PBRT results in 31/39 (79.5%) clinical isolates. Moreover, 11/17 (64.7%) plasmids non-typeable by PBRT could be typed by PRaseT. Our set of multiplex PCRs showed high sensitivity and specificity for the classification of resistance plasmids. It has proved complementary to the widely used PBRT and will improve the monitoring of plasmid distribution in every-day practice.

Risk factors for Clostridium difficile infection in a hepatology ward.

During 2001, Clostridium difficile infection was observed in 23 patients hospitalized in a hepatology ward (attack rate, 0.9%). Since strain typing ruled out a clonal dissemination, we performed a case-control study. In addition to antibiotic use as a risk factor, the C. difficile infection rate was higher among patients with autoimmune hepatitis (P<.01).

Complexity of Escherichia coli bacteremia pathophysiology evidenced by comparison of isolates from blood and portal of entry within single patients

The portal of entry of Escherichia coli bacteremia, a frequent and severe disease, is most commonly the urinary tract followed by the digestive tract. Recent reports have evidenced the presence of several distinct E. coli clones within a single patient suffering of extra-intestinal infection. To explore the relationships between the blood and portal of entry strains, we thoroughly studied 98 bacteremic patients from the French prospective COLIBAFI cohort. In these patients, we compared genotypically and phenotypically E. coli strains isolated from the blood and the suspected portal of entry [non-urinary pus (n=52) and urine (n=52)]. We found genetically distinct strains exhibiting distinct antibiotypes in the blood and pus samples (8 patients; 15%) and the blood and urine samples (2 patients; 3.8%) (p=0.09). These data highlight the complexity of pathophysiology of E. coli bacteremia and should be taken into consideration when strain antibiotic susceptibility is tested, especially in bacteremia of pus origin.

Extended-spectrum β-lactamase-encoding genes are spreading on a wide range of Escherichia coli plasmids existing prior to the use of third-generation cephalosporins.

To understand the evolutionary dynamics of extended-spectrum β-lactamase (ESBL)-encoding genes in Escherichia coli, we undertook a comparative genomic analysis of 116 whole plasmid sequences of human or animal origin isolated over a period spanning before and after the use of third-generation cephalosporins (3GCs) using a gene-sharing network approach. The plasmids included 82 conjugative, 22 mobilizable and 9 non-transferable plasmids and 3 P-like bacteriophages. ESBL-encoding genes were found on 64 conjugative, 6 mobilizable, 2 non-transferable plasmids and 2 P1-like bacteriophages, indicating that these last three types of mobile elements also play a role, albeit modest, in the diffusion of the ESBLs. The network analysis showed that the plasmids clustered according to their genome backbone type, but not by origin or period of isolation or by antibiotic-resistance type, including type of ESBL-encoding gene. There was no association between the type of plasmid and the phylogenetic history of the parental strains. Finer scale analysis of the more abundant clusters IncF and IncI1 showed that ESBL-encoding plasmids and plasmids isolated before the use of 3GCs had the same diversity and phylogenetic history, and that acquisition of ESBL-encoding genes had occurred during multiple independent events. Moreover, the blaCTX-M-15 gene, unlike other CTX-M genes, was inserted at a hot spot in a blaTEM-1-Tn2 transposon. These findings showed that ESBL-encoding genes have arrived on wide range of pre-existing plasmids and that the successful spread of blaCTX-M-15 seems to be favoured by the presence of well-adapted IncF plasmids that carry a Tn2-blaTEM-1 transposon.