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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.

Within the scope of its health products control mission, the French Health Products Safety Agency (Afssaps) collaborating with two expert's sites, has assessed the 16 tests available on the French market in 2002 for rapid diagnosis of the Streptococcus A tonsillitis. The purpose of this study was to verify the reliability and rapidity of these tests and to give some information to the users about their analytical criteria and practicability characteristics. The analytical study has been performed on a same panel of four reference strains of Streptococcus pyogenes dilutions to determine the limit of detection of all the reagents in the same condition of methodology. The limit of detection has been calculated with the results expressed in colony forming unit by ml (CFU/ml). The practicability study has permitted to analyze the quality of the presentation, the easiness of the final reading and of performing tests. A score has been established for each rapid test. A classification of the analytical sensitivity (limit of detection) and practicability (score) of these 16 devices has been established. The limit of detection of the reagents giving the best results allows the detection of the lowest bacterial concentration of the panel which is 10(5) CFU/ml. Regarding practicability, the results suggest that, the immunochromatographic strip methods have the best score in a view with the use by a non medical laboratory.

A total of 206 recent throat isolates of Streptococcus pyogenes collected between 2002 and 2004 from children were tested for their susceptibility to penicillin, amoxycillin, erythromycin, clarythromycin and clindamycin. The erythromycin resistant isolates were further studied for their genetic mechanism of resistance by means of PCR. In all, 14.5% of the strains were erythromycin resistant and 13.5 and 1% expressed the constitutive MLS(B) and M resistance phenotypes and harbored the ermB and mef A genes respectively.

High incidence of nosocomial infections in children is largely explained by immunodeficiency, particularly in newborns. Central venous catheter is the main risk factor and coagulase negative staphylococcus the main pathogen in cause. Large variations of nosocomial infection incidences are observed by Nososcomial Infection Surveillance Networks and depend on the pediatric speciality. The highest rate is observed in neonatal intensive care, where incidence density of catheter-related sepsis varies from four to 23 infections for 1000 catheter-days. Local surveillance in each ward, risk factors and knowledge of bacterial epidemiology allow the development of rational preventive and therapeutic protocols. However, prophylactic use of vancomycin is dangerous and immunoglobulins are inefficient.

Nosocomial diarrhea are an important cause of childhood morbidity and mortality. Rotavirus has been recognized as the most important cause of nosocomial gastroenteritidis particularly in infants during winter months. Nosocomial diarrhea are also, caused by bacterial pathogen like Clostridium difficile, Salmonella, Shigella, Campylobacter. Clostridium difficile toxin assay should be considered for patients who are receiving antibiotics. Modifications of hygiene procedures and preventive measures are necessary in order to reduce nosocomial infection.

Between April and October 1997, 21 children of 4 days to 13 years old were admitted to the Pedatric Unit of Aulnay Sous Bois's Hospital for viral meningitidis. The number of white blood cells in the cerebrospinal fluid (CSF) was between 1 and 612 cells/mm3, with, on an average, 56% of segmented cells, 34% lymphocytes and 34% monocytes. Proteins and glucose of CSF were standard. One CSF was normal. Viral meningitidis was confirmed by viral culture of CSF onto MRC5. Enterovirus were identified by direct immunofluorescence (Monoclonal Mouse Anti-Enterovirus, Dako). Serotyping (Enterovirus antisera, Eurobio, Trousses 4) identified an echovirus 30 in all cases. A highly conserved 154 bp sequence at the 5'non-coding region was studied by reverse transcription-polymerase chain reaction (RT-PCR) followed by single-strand conformation polymorphism (SSCP) (GenPhor, Pharmacia) analysis. Two dominant SSCP patterns were observed: the first contained 4/21 strains and the other 10/21 strains. The SSCP patterns of the 7 other strains were different. These results show that 2 echovirus 30 dominant clones were responsible of viral meningitidis admitted to the Pediatric Unit of Aulnay Sous Bois's hospital, between april and october 1997. The PCR-SSCP of the 5'non-coding region of echovirus 30 is a convenient, simple, reproducible epidemiologic method and it's easily applicable in a general hospital.

Microorganisms that are resistant to multiple antimicrobial agents are of great concern to pediatric clinicians. Children infected with antibiotic-resistant bacteria are at risk to not respond to initial therapy. Nosocomial infection of pediatric patients with multidrug-resistant organisms are similar to those found in adults especially for Staphylococcus aureus, coagulase-negative staphylococci, vancomycin resistant Enterococcus and Enterobacteriaceae resistant to third-generation cephalosporin. However on the contrary to adult patients, multidrug-resistant bacteria are found in community infections with penicillin-resistant Streptococcus pneumoniae and P. aeruginosa in CF patients.

Nosocomial infection cause severe morbidity and mortality in pediatric patients. To find the cause of an infectious disease outbreak, epidemiologists need to determine early on whether a single strain of microorganism is responsible for the majority of cases. Phenotypic characteristics have been widely used in epidemiologic studies. However with most systems poor reproducibility, poor sensitivity have been reported and they do not reliably show enough strain-to-strain variation to be sufficiently discriminative. Molecular approaches like the analysis of the restriction fragment length polymorphism of the total DNA and of the rDNA regions and pulsed-field gel electrophoresis typing have now been applied with success to a large number of bacterial species associated with epidemics.

Moraxella catarrhalis is a Gram-negative diplococcus of the Neisseriaceae family now recognized as a cause of acute otitis media in pediatric patients and of lower respiratory tract infections in debilitated adults. The finding that 80% to 90% of strains produce beta-lactamases together with reports of cases of bacteremia and arthritis due to M. catarrhalis suggest an increasing pathogenic role for this organism. Antibiotic susceptibility testing suggests that the greatest bactericidal effect is provided by amoxicillin-clavulanic acid.

The minimum inhibitory concentrations (MICs) of vancomycin and teicoplanin were determined for 32 coagulase-negative staphylococci strains recovered from blood specimens from pediatric intensive care patients. All the strains were susceptible to vancomycin (MIC less than 4 mg/l). Sixteen strains were susceptible to teicoplanin (MIC less than 4 mg/l) and the sixteen remaining strains exhibited intermediate susceptibility, with MICs of 8 mg/l (10 strains) or 16 mg/l (6 strains). Inhibition zone diameters seen with vancomycin during agar diffusion susceptibility testing were consistently greater than 17 mm. Inhibition zones obtained with teicoplanin were 17 mm or more in diameter for 30 strains and under 17 mm in diameter for 2 strains. For 14 strains (44%), agar diffusion testing failed to detect the decreased susceptibility to teicoplanin revealed by MIC determinations. The agar diffusion method does not seem reliable for the determination of in vitro susceptibility to teicoplanin.

Vancomycin is a narrow-spectrum glycopeptide antibiotic which is primarily active against Gram-positive organisms. Bacterial resistance develops rarely due to its numerous modes of action. The mode of action of vancomycin involves the inhibition of peptidoglycan synthesis. Vancomycin forms a stoichiometric complex with the peptidoglycan precursor UDP-N-acetylmuramyl pentapeptide by forming hydrogen bonds. In patients with renal insufficiency vancomycin clearance is reduced and elimination half-life prolonged. Vancomycin is the drug of choice in the treatment of methicillin-resistant staphylococcal infections and in the treatment of Gram-positive endocarditis and has been used as alternative therapy in the treatment or prophylaxis of Gram-positive infections in penicillin-allergic patients.

Rapid eradication of bacteria in bloodstream is critical for the outcome in neonatal bacterial sepsis. Two groups of neonates with E. coli K1 sepsis without purulent meningitis were studied. Group I (n = 14) received cefotaxime IV (100 mg.kg-1 D-1) plus netilmicin (4 mg.kg-1 D-1); group II (n = 8) received amoxicillin/clavulanic acid IV (100/10 mg.kg-1 D-1) plus netilmicin (4 mg.kg-1 D-1). Both groups were identical. For all strains MICs of cefotaxime, amoxicillin/clavulanic acid, netilmicin were less than 0.2, 4 and 1 mg/l respectively. Serum bactericidal activity (SBA) was determined for each patient (peak sample). The SBA was defined as the greatest dilution in which 99,99% of the inoculum was killed. Time-kill curves were performed with 1:16 dilutions of peak serum samples to measure the kinetic of bacterial killing. The minimal bactericidal time of serum (MBTS) was defined as the minimal time required to observe a decrease of more than 4 log CFU/ml of the bacterial inoculum. Samples (10 microliters) were taken at 1 h intervals over a 6 h period and at 24 h for quantitative culture. All patients cured. Median SBA were respectively 1/128 and 1/64 for group I and II. However, mean MBTS for groups I and II were respectively 1.2 h +/- 0.8 and 3.9 h +/- 1.4. Killing was more rapid in group I (p less than 0.01). The MBTS may be a clinically useful adjunctive test when optimal therapy would be expected.

We have used an enzyme immunoassay method to measure serum levels of gentamicin in neonates according to gestational age, birth weight, dosage and intervals of intramuscular injections. Peak, predose serum levels and half-life decrease when gestational age increase. For the same gestational age, peak serum levels decrease in small weight neonates. Toxic postdose serum levels were obtained in preterm neonates with a daily regimen exceeding 5 mg/kg. Serum concentration monitoring of gentamicin is recommended to ensure adequate dosage and interval of injections.

The effect of an amoxycillin-clavulanic acid combination on the intestinal bacterial ecosystem was studied by differential quantitative analysis of the fecal flora in 11 hospitalized pediatric patients aged 8 months to 4 years. The antibiotic combination was given orally to 7 patients and intravenously to 4. The modifications in the intestinal flora are more important with oral route than intravenous route. After treatment, an increase in ampicillin-resistant E. coli with overgrowth of Klebsiella were found. Our previous studies have shown that this microbial overgrowth carries a risk of secondary septicemia. A strain of amoxycillin-resistant Serratia marcescens emerged but did not pullulate . The other aerobic or anaerobic bacteria were not significantly modified. Most strains which emerged after therapy remained susceptible to the combination. Yeasts emerged in two patients. Thus, the amoxycillin-clavulanic acid combination results in intestinal Klebsiella overgrowth, requiring monitoring of the intestinal bacterial ecosystem.

MIC 90 and MBC 90 of amoxicillin, cefaclor, cefadroxil and cefuroxime axetil have been determined by the microdilution method against 48 organisms responsible of acute respiratory tract infections in children: 17 E. coli, 15 K. pneumoniae, 16 H. influenzae. An inoculum effect and an inhibitory index in blood and bronchial secretions were determined. An inoculum effect was more important for amoxicillin and cefadroxil than for cefuroxime axetil and cefaclor. Against H. influenzae, cefuroxime axetil and cefaclor have a similar activity. Against Enterobacteria, cefuroxime axetil has the lowest MIC 90 and MBC 90 and the highest inhibitory index.

We evaluate a new and quick micromethod which detect strains of Salmonella, Shigella and Yersinia from suspected colonies isolated of stool specimens. In our study, from 2 384 non pathogen bacteria or as Salmonella. And we determined 297 strains of Salmonella, two hours after isolation on selective medium. This method is very easy and permits to have a rapid diagnostic of strains of Salmonella.

Morbidity and mortality among neonates with group B streptococcal infections remain high. As delays in bacterial killing may be responsible for these poor results, there is a need for studies into killing kinetics. We investigated antimicrobial sensitivity and killing effect time lags for penicillin, ampicillin and mezlocillin, alone and in combination with gentamicin or amikacin, against 20 strains of group B streptococci isolated in cultures of blood and cerebrospinal fluid from neonates. A culture of each strain (10(5) germs/ml) was exposed to the antibiotics individually or in combination. Antibiotics were used in the concentrations achieved clinically. Surviving bacteria were counted after 2 h 30, 4 h 30 and 24 h. incubation. Mean killing curves showed that the time interval until onset of a killing effect was 24 hours with either penicillin or ampicillin alone, against 4 h 30 with penicillin-amikacin or ampicillin-gentamicin. The most rapid killing effect (2 h 30) was observed with mezlocillin alone and ampicillin-amikacin. No antagonism was found between mezlocillin and aminoglycosides. Choice of the best antibiotic treatment for group B streptococcal infections should be based on both the rapidity of the in vitro killing effect and the antibiotic's diffusion into the site of the infection.

Morbidity and mortality among children with Pseudomonas aeruginosa infection in Pediatric Intensive Care Unit remains high. Delays in bacterial killing may be responsible for the poor outcome. Antimicrobial sensitivity and timed-killing assays were determined for ticarcillin, azlocillin, piperacillin, cefsulodin, ceftazidime, gentamicin, tobramycin and amikacin alone and in combination against 40 strains of Pseudomonas aeruginosa isolated from blood cultures and tracheal aspirate. Antibiotic concentrations used were at clinically achievable level. None bactericidal effect was observed with each beta-lactamin alone. However with the combinations azlocillin or piperacillin or cefsulodin or ceftazidime plus amikacin a bactericidal effect was observed at 4.5 hours.

Twelve children (mean age: 4) are treated in practice office with nitroxoline (200 mg/kg/24 h) during 10 days for urinary tract infection. A study on the effect of nitroxoline against the fecal flora is undertaken in a group of 21 children who receive nitroxoline during 4 days or a long period. Bacteriological and clinical efficacy is checked when urinary concentrations of nitroxoline are greater than 16 mg/l. The results show 8 clinical and bacteriological success one true failure and 3 failure due to a non-compliance to the treatment. No qualitative and quantitative modification of the fecal flora is observed after treatment with nitroxoline.

Because of the frequency of Haemophilus influenzae and Staphylococcus aureus in joint and bone sepsis in children, a prospective study of first line antibiotic therapy was performed. In a series of 23 cases, including 8 osteomyelitis and 15 arthritis, Gram stain on joint fluid or antigen detection was helpful in reaching a decision about initial therapy in only 3 cases (Haemophilus influenzae). In 20 of the 23 patients, the first line antibiotic therapy was cefotaxime (100 mg/kg/day) and fosfomycin (100 mg/kg/day) in combination. In 6 of them, the bacteriologic culture was positive (3 Staphylococcus aureus, 1 Haemophilus influenzae and 2 Streptococcus pneumoniae) and the initial antibiotic therapy was changed according to the antibiotic susceptibility testing. In the others 14 cases, from whom no agent was isolated, this combination was continued during about 15 days, then followed by pristinamycin and amoxicillin-clavulanic acid in combination during one month. The C. reactive protein dosage was performed in each patient. All children cured. In view of these first results, cefotaxime and fosfomycin in combination seems to us to be interesting in first line antibiotic treatment without initial orientation.

Loperamide has been recently indicated in the management of infants with severe protracted diarrhea. The purpose of this study was to determine the effect of loperamide on fecal flora in severe protracted diarrheas. 19 children aged 1 to 36 months, with severe protracted diarrhea were studied: 14 received loperamide (0.5 mg/kg/d) and 5 were without loperamide treatment. Criteria analysed were: clinical tolerance (vomiting and abdominal distention); efficacy (number of stools, transit time and Na+/K+ in stools) and effect on fecal flora, with differential qualitative and quantitative analysis method (complete identification and counts of aerobic and strict anaerobic bacteria in fresh fecal samples before and 4 to 8 days after the beginning of loperamide). Parenteral and/or oral alimentation remain constant during the study. Results show a rapid (24 h) efficacy in 9/14. Although important changes in specific fecal flora counts was noticed for streptococcus D and Proteus as compared to five controls, no bacterial overgrowth appeared or was increased during loperamide treatment.

A differential quantitative study of the fecal flora was used to appreciated the intestinal bacterial flora of 41 hospitalized children, with no infection and receiving no antibiotic treatment. Thus, it was possible to establish the bacterial normal profile of intestinal of hospitalized child. Because of the stability of the fecal flora in those controls, it was possible to study the effect of colistin on the intestinal ecosystem, in 21 children. Colistin per os, with intestinal nutrition and colistin IM in combination with gentamicin IM did not have any quantitative or qualitative effect on fecal flora. Colistin per os, with exclusive parenteral nutrition caused by disappearance of sensitive strains and their replacement by resistant organisms; this was followed by secondary septicaemia. This fact being a very high infectious risk, it leads us to abstain from administering colistin per os, when the intestine does not receive any food.

A differential quantitative study was used to evaluate the effects of parenteral cefoperazone upon children's fecal flora. Fecal specimens were obtained from 16 patients, before, during and after therapy. Cefoperazone therapy was associated with major changes in fecal flora. There was marked reduction or suppression to undetectable levels of Enterobacteria, Staphylococci and Streptococci in 13 patients. During therapy, yeasts were selected or acquired in 7 cases. 5 to 10 days after cefoperazone was discontinued, the fecal flora was virtually the same as before treatment. Thus, cefoperazone should prove very useful in the treatment of septicemia due to intestinal overgrowth.