Raman spectroscopic analysis of the clonal and horizontal spread of CTX-M-15-producing Klebsiella pneumoniae in a neonatal intensive care unit.

Nosocomial outbreaks of extended-spectrum ß-lactamase (ESBL)-producing Klebsiella pneumoniae are an increasing concern in neonatal intensive care units (NICUs). We describe an outbreak of ESBL-producing K. pneumoniae that lasted 5 months and affected 23 neonates in our NICU. Proton pump inhibitor and extended-spectrum cephalosporin exposure were significantly associated with the risk of ESBL-producing K. pneumoniae colonisation and/or infection. Thirty isolates recovered from clinical, screening and environmental samples in the NICU were studied by means of Raman spectroscopy, pulsed-field gel electrophoresis and repetitive extragenic palindromic polymerase chain reaction (rep-PCR). The Raman clustering was in good agreement with the results of the other two molecular methods. Fourteen isolates belonged to the Raman clone 1 and 16 to the Raman clone 3. Molecular analysis showed that all the strains expressed SHV-1 chromosomal resistance, plasmid-encoded TEM-1 and CTX-M-15 ß-lactamases. Incompatibility groups of plasmid content identified by PCR-based replicon typing indicated that resistance dissemination was due to the clonal spread of K. pneumoniae and horizontal CTX-M-15 gene transfer between the two clones.

Salmonella enterica serotype Gambia with CTX-M-3 and armA resistance markers: nosocomial infections with a fatal outcome.

We report two cases of bacteremia caused by the Salmonella enterica serotype Gambia in our children's hospital, with one fatal outcome. The isolates showed indistinguishable genotypes and infrequent resistance markers: CTX-M-3 extended-spectrum ß-lactamase and armA methyltransferase. This is the first report of S. Gambia exhibiting CTX-M-3 and armA markers involved in serious infections.

Selective strategy for urethral catheterization in febrile young girls to confirm urinary tract infection diagnosis.

BACKGROUND: Urinary tract infections (UTIs) are a common source of bacterial infections in childhood. Making a proper diagnosis is important but requires invasive urine collection techniques. We aimed to derive a clinical decision rule to identify non-toilet-trained febrile girls at high risk for UTIs to restrict urethral catheterizations (UCs) to this high-risk group of patients. METHODS: We included all non-toilet-trained girls with a positive microscopic urinalysis from urine collected by sterile bag in a prospective cohort study to derive a model to predict UTI assessed by urine culture from UC. RESULTS: Thirty-seven patients were included. Absence of another source of fever on examination and the child's unusual behaviour were found to be independent predictors of UTI. The corresponding model offered an 85% sensitivity [95% confidence interval (CI): 56-96], with a 59% specificity (95% CI: 30-83) for UTI. The internal cross-validation by bootstrap led to an 85% sensitivity (95% CI: 68-100), and a 59% specificity (95% CI: 35-83). CONCLUSION: We derived a clinical decision model to selectively identify young febrile girls at high risk for UTI with a positive microscopic analysis and propose UC with an 85% sensitivity, which would avoid approximately 60% of unnecessary UCs; although further validation is necessary before daily clinical use.

[Phenotypic and genotypic (randomly amplified polymorphic DNA analysis and multiple-locus variable-number tandem-repeat analysis) charaterization of 96 clinical isolates of Pseudomonas aeruginosa in the F. Bourguiba hospital (Monastir, Tunisia)]. / Caractérisation phénotypique et génotypique (randomly amplified polymorphic DNA analysis et multiple-locus variable-number tandem-repeat analysis) de 96 isolats cliniques de Pseudomonas aeruginosa à l'hôpital F. Bourguiba (Monastir, Tunisie).

AIM OF THE STUDY: Phenotypic and genotypic characterization of 96 clinical isolates of Pseudomonas aeruginosa recovered in a Tunisian teaching hospital during a 16-month period. MATERIALS AND METHODS: All the isolates were characterized by serotyping, antimicrobial susceptibility typing and genotyping with randomly amplified polymorphic DNA (RAPD) analysis and multiple-locus variable-number tandem-repeat analysis (MLVA). RESULTS: Forty-one isolates out of 96 (43%) were recovered from two intensive care units (medical and chirurgical). Most of the isolates (48%) belonged to serotype O:11. Among the 13 antibiotypes, three multidrug resistant ones were mostly observed within the two intensive care units. Genotyping showed 83 RAPD types and 52 MLVA types. Isolates showing the same serotype could show different genotypes. A limited number of clusters was highlighted with MLVA typing, of which an outbreak of nine cases within the surgical intensive care unit. CONCLUSION: Except this outbreak of nine cases, the heterogeneity observed for most of the P. aeruginosa isolates showed that outbreak situations were rare in the F. Bourguiba hospital during the study period. MLVA genotyping is a good tool for genotyping P. aeruginosa clinical isolates.

[Disseminated fusarium infection in two neutropenic children]. / Fusariose disséminée chez deux enfants neutropéniques.

UNLABELLED: Disseminated fusariosis in children is a rare and serious fungal infection, that occurs especially in neutropenic immunosuppressed patients, treated for malignant hemopathy, or bone marrow transplant recipient. Treatment is difficult and mortality is estimated between 50 and 70% in adult patients. CASE REPORT 1: A ten-year-old boy, treated for an acute lymphoblastic leukemia in second relapse, presented a disseminated fusarium spp infection, that occurred during neutropenia. He died due to fusariosis infection in spite of amphotericin B treatment. CASE REPORT 2: A ten-year-old neutropenic girl, treated for an acute myeloïd leukemia, presented disseminated fusariosis, uncontrolled by amphotericin B. Recovery was observed after voriconazole introduction and resolution of neutropenia. Ten months later, she presented a leukemia's relapse, treated by new intensive chemotherapy with secondary prophylaxis by voriconazole, without fusariosis's recurrence. CONCLUSION: Voriconazole, a new triazole agent, seems to be an alternative antifungal agent to amphotericin B for disseminated fusarium infection, either at the acute phase or for secondary prophylaxis.

Mycobacterial lung disease in cystic fibrosis: a prospective study.

BACKGROUND: Patients with cystic fibrosis (CF) may be predisposed to airway infections with unusual organisms, such as mycobacteria. The aim of the study was to determine the incidence and clinical picture of mycobacterial infection in CF children. METHODS: At least 2 acid-fast bacillus (AFB) smears and mycobacterial cultures were performed on a prospective basis on 682 sputum specimens from 106 patients during a 1-year period. RESULTS: Thirty-three percent of the cultures were contaminated with other bacteria. Seven children had at least one sputum culture positive for one mycobacterium. Five children had only one positive AFB culture. Their clinical status and lung function remained stable during follow-up. Two teenagers with severe lung disease had several positive AFB smears and cultures for Mycobacterium chelonae and Mycobacterium abscessus. The isolation of M. chelonae and M. abscessus was associated with a clinical and functional decline. Clarithromycin treatment resulted in temporary improvement with the disappearance of the mycobacteria after 6 months of treatment. This prospective study shows an incidence of 2.3% for positive cultures. The prevalence was 6.6% for mycobacterial colonization but only 1.9% for mycobacterial lung disease in our pediatric population. CONCLUSIONS: We recommend performing AFB smears and cultures in CF children with severe lung disease and/or during a lung exacerbation. In these patients persistence of M. chelonae or M. abscessus in sputum should lead to consideration of treatment with clarithromycin.

Comparative DNA analysis of Bordetella pertussis clinical isolates by pulsed-field gel electrophoresis, randomly amplified polymorphism DNA, and ERIC polymerase chain reaction.

We used DNA fingerprinting by pulsed-field gel electrophoresis (PFGE), randomly amplified polymorphic DNA (RAPD) and PCR amplification of enterobacterial repetitive intergenic consensus sequences (ERIC-PCR) to compare 15 clinical isolates of Bordetella pertussis recovered between August 1993 and September 1995 from 13 infants and two adults, living in the same geographic area. PFGE produced 10 patterns and made it possible to differentiate all the isolates and to indicate an intrafamilial transmission. RAPD and ERIC-PCR generated banding patterns with small differences and had a poor discriminatory power. During the last 2 years, at Armand-Troussau pediatric hospital, 10 distinct clones of clinical B. pertussis isolates, with a predominant clone including seven strains, could be determined by the PFGE method.

[Description and investigation of an outbreak of extended-spectrum beta-lactamase producing Escherichia coli strain in a neonatal unit]. / Description et investigation d'une épidémie nosocomiale de colonisations et d'infections à Escherichia coli producteur d'une bêta-lactamase à spectre étendu dans un service de néonatologie.

An outbreak of colonization and infection with an Escherichia coli strain producing extended-spectrum beta-lactamase (ESBL) occurred in a neonatal unit : a high rate of cases was observed, 27/59 neonates were colonized : one of them developed meningitis with favourable outcome and another baby developed conjunctivitis. Despite intensive efforts to control the outbreak by standard methods of hand hygiene, patients screening and isolation, the spread was uncontrolled and the unit was closed to all admission in order to stop the outbreak. The investigation was not able to identify a single outbreak's source. Emergence and spread of ESBL producing E. coli strains from community and hospital acquired infections are a significant public health problem with difficult choice of treatment for serious infections.

Comparative in vitro activities of caspofungin and micafungin, determined using the method of the European Committee on Antimicrobial Susceptibility Testing, against yeast isolates obtained in France in 2005-2006.

The in vitro activities of caspofungin and micafungin against 1,038 yeast isolates have been determined. The caspofungin and micafungin MICs were lower for Candida albicans, Candida glabrata, and Candida tropicalis than for Candida parapsilosis, Candida guilliermondii, and Candida krusei. A clear correlation was seen between the MICs for the two drugs.

[Use of 16S rRNA gene sequencing for identification of "Pseudomonas-like" isolates from sputum of patients with cystic fibrosis]. / Mucoviscidose: identification des bacilles isolés d'infections bronchiques par séquençage du gène de l'ARN ribosomal 16S.

Since nonfermenting, Gram negative bacilli recovered from patients with cystic fibrosis could be misidentified with phenotypic procedures, we used partial 16S ribosomal RNA gene (16S gene) sequencing to identify these "Pseudomonas-like" isolates. 473 isolates were recovered from 66 patients in 2003. Sequencing was used to identify 29 (from 24 patients) of the 473 isolates, showing unclear results with routine tests. PCR with specific primers was carried out to amplify a 995 bp fragment, which was then sequenced. The sequences were analyzed with GenBank database for species assignment. Phenotypic and genotypic results were concordant for 20/29 isolates (10 Pseudomonas aeruginosa, 5 Burkholderia cepacia, 3 Stenotrophomonas maltophilia, 2 Achromobacter xylosoxidans). However, 3 of the 5 B. cepacia isolates were then identified as Burkholderia multivorans with a PCR-RFLP procedure. Phenotypic misidentification was observed for 9/29 isolates: 4 A. xylosoxidans, 1 P. aeruginosa, 1 Bordetella petrii, 1 Bordetella bronchiseptica, 1 Ralstonia respiraculi and 1 Ralstonia mannitolilytica. Partial 16S gene sequencing improved the identification of "Pseudomonas-like" isolates from cystic fibrosis patients, but the accuracy to distinguish between genomovars of the B. cepacia complex was inadequate.

[Nosocomial infections in immunocompromised children]. / Infections nosocomiales chez l'enfant immunodéprimé.

Immunocompromised children are at high risk for developing nosocomial infections which may cause significant morbidity and mortality in this population. In paediatric oncology, reported prevalence of nosocomial infections varies from 10 to 20%. Major predisposing factors are neutropenia, central venous catheter, corticosteroid therapy and hospital construction or renovation for invasive aspergillosis. The management of patients with febrile neutropenia should take into account the previous history of infection and the microbiologic environment of each department. Nowadays, Gram positives infections are predominant, but fungal infections remain a major threat. In organ transplant recipients, wound infections are the main early problems, followed by viral infections often due to the donor CMV seropositivity. In HIV-infected children, nosocomial infections are difficult to define, and can implicate unusual pathogens. In general, adapted preventive infection control strategy warrants prospective studies.

Molecular epidemiology of Burkholderia cepacia, Stenotrophomonas maltophilia, and Alcaligenes xylosoxidans in a cystic fibrosis center.

Burkholderia cepacia, Stenotrophomonas maltophilia, and Alcaligenes xylosoxidans have been isolated with increasing frequency from the sputum of patients with cystic fibrosis in a pediatric hospital. In 1994-95, 27 of 120 patients were persistently colonized, 17 with Burkholderia cepacia, eight with Alcaligenes xylosoxidans, and five with Stenotrophomonas maltophilia. Genotyping of 220 clinical isolates revealed that most of the Burkholderia cepacia strains were clonally related, suggesting either cross-infection or a common source of exposure. In contrast, neither cross-infection nor a common source of exposure appear to have occurred in the cases of Alcaligenes xylosoxidans or Stenotrophomonas maltophilia.

Ralstonia paucula (Formerly CDC group IV c-2): unsuccessful strain differentiation with PCR-based methods, study of the 16S-23S spacer of the rRNA operon, and comparison with other Ralstonia species (R. eutropha, R. pickettii, R. gilardii, and R. solanacearum).

Ralstonia paucula (formerly CDC group IV c-2) can cause serious human infections. Confronted in 1995 with five cases of nosocomial bacteremia, we found that pulsed-field gel electrophoresis could not distinguish between the isolates and that randomly amplified polymorphic DNA analysis was poorly discriminatory. In this study, we used PCR-ribotyping and PCR-restriction fragment length polymorphism analysis of the spacer 16S-23S ribosomal DNA (rDNA); both methods were unable to differentiate R. paucula isolates. Eighteen strains belonging to other Ralstonia species (one R. eutropha strain, six R. pickettii strains, three R. solanacearum strains, and eight R. gilardii strains) were also tested by PCR-ribotyping, which failed to distinguish between the four species. The 16S-23S rDNA intergenic spacer of R. paucula contains the tRNA(Ile) and tRNA(Ala) genes, which are identical to genes described for R. pickettii and R. solanacearum.

Pseudomonas (Burkholderia) cepacia in children with cystic fibrosis: epidemiological investigation by analysis or restriction fragment length polymorphism.

Since 1987, Pseudomonas cepacia has been isolated with an increasing frequency in the expectorants of children with cystic fibrosis followed at the Hôpital d'enfants Armand Trousseau (Paris, France). Colonization by P. cepacia may be responsible for serious secondary infections and rapid deterioration in respiratory function in these patients. Among the 130 children attending our centre, 14 (8 girls and 6 boys) aged 3 to 18, exhibited chronic colonization. 132 isolates, originating from sputum obtained between 1992 and 1994 were studied. Nine biochemical patterns and 6 antibiotic susceptibility patterns at least were defined, therefore exhibiting great polymorphism. Chromosome restriction patterns with Xba I after pulsed field gel electrophoresis enabled 4 pulsotypes to be identified: A, B, C and D. Thirteen patients harboured pulsotypes A, C and D, and 1 patient pulsotype B, the last being quite distinct from the first three. Pulsotypes A, C and D were almost similar, suggesting that closely related strains, probably the same strain, was harboured by 13 of the 14 patients. The origin could be contamination from a single source, or stem from patient-to-patient crossed transmission.

Comparison of randomly amplified polymorphic DNA analysis and pulsed-field gel electrophoresis for typing of Moraxella catarrhalis strains.

Randomly amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) for the analysis of 13 Moraxella catarrhalis isolates, 11 successive strains isolated from sputa of five children and 2 isolates obtained the same day from twins, were compared. RAPD and PFGE both yielded nine types from the 13 isolates, showing a chronic colonization with one strain in three patients and a successive colonization with different strains in two patients. The promising results obtained with RAPD should be confirmed with a larger number of strains, but RAPD seems as suitable as PFGE for the typing of M. catarrhalis.

CDC group IV c-2: a new Ralstonia species close to Ralstonia eutropha.

CDC group IV c-2, an environmental gram-negative bacillus recently proposed for inclusion in the genus Ralstonia, has been isolated in several human infections. Biochemical characterization and 16S ribosomal DNA (rDNA) sequencing with phylogenetic analysis were used to characterize eight clinical isolates and four type strains. Other typing tools, such as pulsed-field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA (RAPD) analysis, were also used. PFGE typing of clinical isolates was unsuccessful because the DNA was degraded, and RAPD analysis was poorly discriminatory. In contrast, the type strains were clearly distinguished with both PFGE and RAPD analysis. All of the 16S rDNA sequences were identical. Comparison of the 16S rDNA sequences to the GenBank sequences showed that they were consistent with CDC group IV c-2 belonging to the genus Ralstonia. The closest matches were obtained with Ralstonia eutropha. However, four differences in 32 biochemical tests separated R. eutropha from CDC group IV c-2, which suggests that CDC group IV c-2 is a new species of the genus Ralstonia.

Molecular epidemiology of Streptococcus pneumoniae with decreased susceptibility to penicillin in a Paris children's hospital.

Among pneumococci with decreased susceptibility or pneumococci resistant to penicillin (PRP) isolated at Armand-Trousseau children's hospital, those expressing capsular serotypes 23F, 9V, and 14 were the most frequently isolated. We compared 53 clinical isolates (14 type 9V, 26 type 23F, and 13 type 14) by analysis of chromosomal macrorestriction patterns and DNA restriction patterns of the penicillin-binding protein (PBP) genes pbp 2b, pbp 2x, and pbp 1a. All 9V isolates originated from the same clone. Five 23F clones were distinguished, the largest of which comprised 20 isolates. The main type 14 clone comprised nine isolates; three other type 14 strains were closely related to the 9V clone, probably by horizontal transfer of capsular biosynthesis genes. Most 23F and type 14 isolates shared the same PBP gene restriction patterns as the 9V clone, suggesting horizontal transfer of altered PBP genes.

[Molecular epidemiology of pneumococci with decreased susceptibility to penicillin isolated in a Parisian pediatric hospital]. / Epidémiologie moléculaire des pneumocoques de sensibilité diminuée a la pénicilline isolés dans un hôpital pédiatrique parisien.

Pneumococci with decreased susceptibility or resistant to penicillin (PRP) have been isolated with an increasing frequency in France. Among PRP, isolates of serotypes 23F and 9V were the most frequently recovered in our children's hospital. Penicillin-resistance is due to the appearance of altered penicillin binding proteins (PBPs) with reduced affinity for beta-lactam antibiotics. 3 PBPs have been well studied, 2b, 2x and 1a, and the sequences of their genes have been determined. Our molecular epidemiological study of 14 PRP 9V and 26 PRP 23F isolated mainly from otitis in 1993-94, consisted of determining chromosomic restriction patterns (Apa I) by pulsed-field gel electrophoresis, and restriction patterns (Hinf I) of PBP genes pbp 2b, pbp 2x and pbp 1a after PCR. All the PRP 9V exhibited the same pulsotype and identical patterns for each of the genes pbp 2b, pbp 2x and pbp 1a, suggesting a clonal origin. The origins of PRP 23F were more heterogenous: 5 clones could be defined, with one predominant clone composed of 20 isolates. Most of the PRP 23F shared identical profiles for the genes pbp 2b, pbp 2x and pbp 1a with the PRP 9V, suggesting a horizontal transfer of DNA. Molecular markers, which provide more informations than serotyping, were useful to clarify the complex epidemiology of PRP.

Nosocomial CDC group IV c-2 bacteremia: epidemiological investigation by randomly amplified polymorphic DNA analysis.

The CDC group IV c-2 bacterium is a gram-negative bacillus rarely isolated from clinical specimens. This organism caused catheter-related bacteremia in five immunocompromised children hospitalized in two distinct wards of our institution between November 1993 and October 1994. Three patients recovered on empiric antibacterial chemotherapy combining ceftazidime and amikacin, and a fourth patient required imipenem instead of ceftazidime. The fifth patient recovered without treatment. Catheter removal was never necessary. The randomly amplified polymorphic DNA technique with three different primers was applied to nine isolates recovered by culturing blood from the five children and showed that all of the patients harbored isolates of the same genotype. The source of the outbreak could not be determined.

Investigation of an outbreak of wound infections due to Alcaligenes xylosoxidans transmitted by chlorhexidine in a burns unit.

Alcaligenes xylosoxidans, an environmental gram-negative bacillus, was isolated within a 1-month period from six patients in a pediatric burns unit. Twelve isolates were studied, one from each of the six patients (five from wound cultures and one from a blood culture) and one from each of six contaminated atomizers containing chlorhexidine diluted to 600 mg/l. The biochemical and susceptibility patterns of all the isolates were similar, and their DNA enzyme restriction patterns were identical. The epidemic strain of Alcaligenes xylosoxidans was probably introduced into the atomizers during handling of the diluted solution, which failed to eliminate it.