Florence Doucet-Populaire

Inducible transfer of conjugative transposon Tn1545 from Enterococcus faecalis to Listeria monocytogenes in the digestive tracts of gnotobiotic mice.

Transfer of conjugative transposon Tn1545 from Enterococcus faecalis to Listeria monocytogenes was studied in vitro and in vivo. Tn1545 transferred following filter mating at a frequency of 2.5 x 10(-7) transconjugants per donor colony. A 20-fold increase in transfer frequency was observed when matings were performed in the presence of a subinhibitory concentration of tetracycline. The frequency of in vivo transfer of Tn1545, expressed as the number of transconjugants per donor cell extracted from the intestines of the gnotobiotic mice after 35 days of experiment, was 1.1 x 10(-8). Presence of a low concentration of tetracycline in the drinking water increased this frequency 10-fold.

Countrywide Spread of Community- and Hospital-Acquired Extended-Spectrum β-Lactamase (CTX-M-15)-Producing Enterobacteriaceae in Lebanon

ABSTRACT A prospective study was carried out to assess the extent of carriage of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae at both hospital and community levels in Lebanon. A total of 1,442 fecal samples were collected from hospital-based patients and 58 from health care workers of six Lebanese tertiary care general hospitals located in different areas of Lebanon between January and March 2003. A total of 382 fecal samples were also collected from healthy subjects between April and June 2003. The samples analysis led to the identification of 118 strains as ESBL producers based on the synergistic effects between clavulanate and selected β-lactams (ceftazidime and cefotaxime). These strains were isolated from 72 subjects: 61 patients, 2 health care workers, and 9 healthy subjects. One representative strain per subject was selected, and a total of 72 nonduplicate ESBL producers, including a high majority of Escherichia coli (n = 56), Klebsiella pneumoniae (n = 9), Enterobacter cloacae (n = 6), and Citrobacter freundii (n = 1), were characterized. The molecular analysis revealed that the majority of the strains (83%) express CTX-M-15 ESBL (pI 8.6). SHV-5a ESBL (pI 8.2) was produced by 18% of the strains. DNA macrorestriction analysis of ESBL-producing E. coli presented 38 different genotypes, revealing the absence of clonal link among these strains. In addition to the fact that the present study highlights the emergence and the countrywide dissemination of CTX-M-15-producing E. coli in Lebanon, it represents the first report of an SHV-5a-producing C. freundii.

Campylobacter Bacteremia: Clinical Features and Factors Associated with Fatal Outcome

BACKGROUND Campylobacter bacteremia is uncommon. The influence of underlying conditions and of the impact of antibiotics on infection outcome are not known. METHODS From January 2000 through December 2004, 183 episodes of Campylobacter bacteremia were identified in 23 hospitals in the Paris, France, area. The medical records were reviewed. Characteristics of bacteremia due to Campylobacter fetus and to other Campylobacter species were compared. Logistic regression analysis was performed to identify risk factors for fatal outcome within 30 days. RESULTS Most affected patients were elderly or immunocompromised. C. fetus was the most commonly identified species (in 53% of patients). The main underlying conditions were liver disease (39%) and cancer (38%). The main clinical manifestations were diarrhea (33%) and skin infection (16%). Twenty-seven patients (15%) died within 30 days. Compared with patients with bacteremia due to other Campylobacter species, patients with C. fetus bacteremia were older (mean age, 69.5 years vs. 55.6 years; P = .001) and were more likely to have cellulitis (19% vs. 7%; P = .03), endovascular infection (13% vs. 1%; P = .007), or infection associated with a medical device (7% vs. 0%; P = .02). Independent risk factors for death were cancer (odds ratio [OR], 5.1; 95% confidence interval [CI], 1.2-20.8) and asymptomatic infection (OR, 6.7; 95% CI, 1.5-29.4) for C. fetus bacteremia, the absence of prescription of appropriate antibiotics (OR, 12.2; 95% CI, 0.9-157.5), and prescription of third-generation cephalosporins (OR, 10.2; 95% CI, 1.9-53.7) for bacteremia caused by other species. CONCLUSIONS Campylobacter bacteremia occurs mainly in immunocompromised patients. Clinical features and risk factors of death differ by infection species.

Multiple Antibiotic Resistance Gene Transfer from Animal to Human Enterococci in the Digestive Tract of Gnotobiotic Mice

ABSTRACT It has been proposed that food animals represent the source of glycopeptide resistance genes present in enterococci from humans. We demonstrated the transfer of vanA and of other resistance genes from porcine to human Enterococcus faecium at high frequency in the digestive tract of gnotobiotic mice. Tylosin in the drinking water favored colonization by transconjugants.

The SrtA Sortase of Streptococcus agalactiae Is Required for Cell Wall Anchoring of Proteins Containing the LPXTG Motif, for Adhesion to Epithelial Cells, and for Colonization of the Mouse Intestine

ABSTRACT Streptococcus agalactiae (group B streptococcus [GBS]) is the leading cause of neonatal pneumonia, sepsis, and meningitis. An in silico genome analysis indicated that GBS strain NEM316 encodes 35 proteins containing an LPXTG motif which are thought to be covalently linked to the peptidoglycan by an enzyme called sortase. The role of these cell wall-anchored proteins in GBS pathogenesis was evaluated on a global level by inactivating the srtA gene. This gene encodes the major sortase SrtA that anchors most of the LPXTG-containing proteins. We chose the C5a peptidase (ScpB) and Alp2, an abundant immunogenic protein, as prototypical LPXTG-containing proteins. As expected, the SrtA knockout mutant was unable to anchor the C5a peptidase (ScpB) and Alp2 to the cell wall. Complementation with plasmid-borne srtA inserted into the chromosome restored the correct surface localization of both ScpB and Alp2. Interestingly, the SrtA mutant was impaired for binding to the major extracellular matrix components fibronectin and fibrinogen and displayed a significant reduction in adherence to human (A549, HeLa, and Caco-2) and murine (L2) epithelial cells compared to the parental wild-type strain. Surprisingly, the inactivation of srtA had no effect on the virulence of the type III strain of GBS in a neonatal rat model (measured by the 50% lethal dose and lung colonization) but strongly impaired the capacity of the strain to colonize the intestines of gnotobiotic mice in a competition assay. These results demonstrate that LPXTG-containing proteins are involved in cell adhesion and GBS persistence in vivo.

Conjugal transfer of plasmid DNA from Enterococcus faecalis to Escherichia coli in digestive tracts of gnotobiotic mice.

We have studied the transfer of the conjugative shuttle plasmid pAT191, which confers resistance to kanamycin, from Enterococcus faecalis to Escherichia coli in the digestive tracts of six gnotobiotic mice. Colonies of E. coli resistant to kanamycin were isolated from the feces of two mice, respectively, on days 25 and 35 after the beginning of the experiment and never thereafter. The transfer frequency of pAT191, expressed as the number of transconjugants per donor cell isolated from intestines of sacrificed mice, was 3 x 10(-9). These results indicate that conjugation is a mechanism that could account for the resistance gene flux from gram-positive to gram-negative bacteria observed in nature. Images

Dose Dependence of Emergence of Resistance to Linezolid in Enterococcus faecalis In Vivo

BACKGROUND The emergence of resistance to antibiotics in vivo, particularly in commensal, potentially pathogenic bacteria, is a factor that is key to the future of antibiotics. To better document the circumstances favoring the emergence of resistance to linezolid (the first of a new class of antibiotics, the oxazolidinones), we modeled the effect of different regimens of linezolid on Enterococcus faecalis in gnotobiotic mice. METHODS We studied the rate of emergence of linezolid-resistant E. faecalis mutants in the digestive tract of gnotobiotic mice monoassociated with linezolid-susceptible E. faecalis and fed with water containing linezolid (0.5, 0.05, or 0.005 g/L). 23S Ribosomal RNA (rRNA) mutations were characterized by sequencing each of the 4 copies of the rRNA genes individually. RESULTS Mutants were readily obtained in vivo, but the frequencies, persistence, and type of mutants were all dependent on the linezolid regimen. Mutations conferring resistance, either the G2505A or G2576U mutation, were present in domain V of the 23S rRNA gene of all resistant isolates. Levels of resistance increased with the number of mutated copies of the 23S rRNA gene and with duration of exposure. CONCLUSION The antibiotic dose appears to be critical in the dynamics and molecular basis of resistance.

Mise en place de la flore intestinale du nouveau-né

Resume Le microbiote intestinal est un ecosysteme extremement complexe, comportant environ 1014 micro-organismes recouvrant pres de 400 especes bacteriennes. C’est un element actif de la physiologie intestinale, avec des fonctions metaboliques, de flore de barriere et de stimulation du systeme immunitaire intestinal. La plupart des etudes de ce microbiote sont basees sur des techniques de culture, mais des techniques de biologie moleculaire permettent de completer ces donnees. La formation de cet ecosysteme debute tres rapidement apres la naissance. Le nouveau-ne, sterile a la naissance, se colonise avec une flore resultant du contact avec sa mere et son environnement. Si les facteurs d’implantation de la flore sont peu connus, de nombreux facteurs vont influencer l’etablissement de ce microbiote : mode d’accouchement, environnement, mode d’alimentation, âge gestationnel et antibiotherapie. Des donnees recentes font etat de modifications dans l’etablissement de cette flore, avec un retard d’implantation des bacteries enteriques d’origine maternelle, dues entre autres aux conditions d’hygiene strictes entourant les accouchements. Les consequences cliniques de ces modifications sont mal connues mais pourraient etre responsables d’une absence de flore de barriere ou d’une mauvaise stimulation du systeme immunitaire intestinal. La question de l’interet de la modulation de la flore du nouveau-ne par l’ajout dans les formules infantiles de probiotiques, prebiotiques, bacteries non viables et leurs metabolites, ou nucleotides est posee et discutee.

Prevalence of resistance to antiseptics and mupirocin among invasive coagulase-negative staphylococci from very preterm neonates in NICU: the creeping threat?

In neonatal intensive care units, topical agents represent an increasing part of the infection control armamentarium. Fifty-one coagulase-negative staphylococci (CNS) isolated from catheter-associated bloodstream infections in very preterm neonates were investigated in this study: 41.2% exhibited decreased susceptibility to at least one antiseptic (chlorhexidine 12%, benzalkonium 24%, acriflavine 33%) and 61% were resistant to mupirocin. QacA/B, mupA and both genes were detected by polymerase chain reaction in 59%, 63% and 49% of CNS, respectively. Seventy-six percent of Staphylococcus epidermidis (5/5 pulsed-field-gel electrophoresis subgroups) and 11% of Staphylococcus capitis (1/3 subgroups) were multi-resistant. Skin antisepsis using low-concentration aqueous formulations and off-label mupirocin indications should benefit from a stewardship programme.

Proficiency Program for Real-Time PCR Diagnosis of Bordetella pertussis Infections in French Hospital Laboratories and at the French National Reference Center for Whooping Cough and other Bordetelloses

ABSTRACT With the support of a ministerial program for innovative and expensive technologies, dedicated to the economic evaluation of laboratory diagnosis of pertussis by real-time PCR, external quality assessment for real-time IS481 PCR was carried out. Coordinated by the National Centre of Reference of Pertussis and other Bordetelloses (NCR), this study aimed to harmonize and to assess the performances of eight participating microbiology hospital laboratories throughout the French territory. Between January 2006 and February 2007, 10 proficiency panels were sent by the NCR (ascending proficiency program), representing a total of 49 samples and including eight panels to analyze and evaluate the global sensitivity and specificity of real-time PCR, one to assess the limit of detection, and one to evaluate nucleic acid extraction methods. As part of the descending proficiency program, extracted DNA from clinical samples was sent by the eight participating laboratories in different panels and analyzed by the NCR. In the ascending proficiency analysis, the sensitivity and specificity of the real-time PCR methods were 92.2% and 94.3%, respectively. The limit of detection of the different methods ranged between 0.1 and 1 fg/μl (0.2 to 2 CFU/μl). The nucleic acid extraction methods showed similar performances. During the descending proficiency analysis, performed with 126 samples, the result of the NCR for 15 samples (11.9%) was discordant with the result obtained by the source laboratory. Despite several initial differences, harmonization was easy and performances were homogeneous. However, the risk of false-positive results remains quite high, and we strongly recommend establishment of uniform quality control procedures performed regularly.