Bacteremia during tonsillectomy.

To determine the risk of bacteremia during tonsillectomy, we cultured blood specimens that were taken from 32 children during surgery and tonsillar swabs that were obtained just before excision, and compared the results with quantitative cultures of the excised tonsillar tissue. Twenty-five children had Haemophilus influenzae within the tonsillar tissue (density range, 10(3) to 10(8) colony-forming units per gram), and seven had Streptococcus pyogenes (density, 10(3) colony-forming units per gram in one case, 10(5) colony-forming units per gram in one case, and 10(6) colony-forming units per gram in five cases). Twelve perioperative blood cultures were positive; H influenzae was found nine times, and Micrococcus species was found one time, and alpha-hemolytic streptococci were found two times. Haemophilus influenzae was always present in the corresponding tonsillar specimens, although there was no apparent relationship between the density of colonization of the tonsillar tissue and a positive blood culture.

Use of ribotyping in epidemiological surveillance of nosocomial outbreaks.

Over the past few years, genotypic methods based on the study of bacterial DNA polymorphism have shown high discriminatory power for strain differentiation and superiority over most phenotypic methods commonly available in the clinical microbiology laboratory. Some of the methods used, however, required either a high level of technology and sophisticated equipment (e.g., pulsed-field gel electrophoresis) or species-specific reagents of restricted availability (randomly cloned DNA probes or gene-specific probes). Because ribotyping uses a universal probe (rRNA) and is a rather simple technology, particularly since the advent of nonradioactive labelling systems, it has been widely used for strain differentiation of most bacterial species involved in nosocomial outbreaks. In vitro and in vivo stability of the markers studied has been demonstrated. Although there may be limitation to this approach, ribotyping was found to be highly discriminative, particularly for typing members of the family Enterobacteriaceae, Pseudomonas cepacia, and Xanthomonas maltophilia. In many cases, it has improved the understanding of the mechanism of nosocomial acquisition of organisms by allowing a distinction between endogenous and exogenous infections. Among exogenous infections, it has distinguished between individual and epidemic strains, thus differentiating cross-infection from independent acquisition.

Evidence for the genetic unrelatedness of nosocomial vancomycin-resistant Enterococcus faecium strains in a pediatric hospital.

Vancomycin-resistant enterococci are now found with increasing frequency. Up to now, epidemiological studies of enterococci have been limited by the lack of convenient and accessible methods for comparing strains. In this study, we report an epidemiological investigation on 16 nosocomial vancomycin-resistant Enterococcus faecium strains isolated from 15 patients in four different wards of a children's hospital over a period of 17 months. Analysis of restriction fragment length polymorphism (RFLP) of total DNA and of ribosomal DNA regions (ribotyping) was used as a typing approach. Each strain produced a different total DNA RFLP pattern after HindIII and PvuII digestion, except for two strains that were isolated from a single patient and that gave indistinguishable patterns. In our system, ribotyping was less discriminative than RFLP of total DNA. This approach, therefore, shows the genetic unrelatedness of the nosocomial strains studied and excludes patient-to-patient strain transmission either in the same ward or between wards.

Bactericidal activity against intermediately cephalosporin-resistant Streptococcus pneumoniae in cerebrospinal fluid of children with bacterial meningitis treated with high doses of cefotaxime and vancomycin.

Cerebrospinal fluid (CSF) was taken from 19 children with bacterial meningitis treated with cefotaxime (300 mg/kg of body weight/day) and vancomycin (60 mg/kg/day). Median levels of drugs in CSF were smaller than expected, as follows: 4.4 microg/ml for cefotaxime, 3.2 microg/ml for desacetylcefotaxime, and 1.7 microg/ml for vancomycin. The median CSF bactericidal titer against an intermediately cefotaxime-resistant pneumococcus was 1:4. Our data suggest at least an additive interaction between the drugs used in this study.

Molecular epidemiology of plasmid spread among extended broad-spectrum beta-lactamase-producing Klebsiella pneumoniae isolates in a pediatric hospital.

Over a 12-month period, 43 children in eight different wards of our hospital (Hôpital Robert Debré) were infected or colonized with Klebsiella pneumoniae strains producing extended broad-spectrum beta-lactamases. The epidemiology of the outbreak was studied by a molecular approach including the determination of the beta-lactamase physicochemical parameters and plasmid profiles, as well as analysis of the restriction fragment length polymorphisms of the rDNA regions (ribotyping). The last approach produced 12 and 5 different patterns with EcoRI and HindIII, respectively, thus identifying 15 different ribotypes among the 43 clinical K. pneumoniae strains. However, 60% of the strains in six wards belonged to only two ribotypes, whereas nine ribotypes were observed only once. Twelve isolates from different wards that were representative of the eight most common ribotypes showed four different beta-lactamase isoelectric focusing patterns and seven different plasmid profiles by direct analysis or after EcoRI digestion. Thus, at least two genetically unrelated strains in the same ward were found to have the same plasmid content. Our results show the complexity of the outbreak, which was associated with patient-to-patient cross-contamination with several epidemic strains with different plasmid contents, interspersed sporadic cases with nonepidemic strains, and the possible spread of a plasmid. The combination of plasmid profile analysis and ribotyping therefore seems to be powerful at deciphering the details of such outbreaks.

In vitro killing activities of antibiotics at clinically achievable concentrations in cerebrospinal fluid against penicillin-resistant Streptococcus pneumoniae isolated from children with meningitis.

We evaluated the in vitro killing activities of ceftriaxone, imipenem, vancomycin, gentamicin, fosfomycin, and rifampin, alone and in combination, against 26 Streptococcus pneumoniae strains (penicillin G MICs, > 0.125 to 2 micrograms/ml) isolated from the cerebrospinal fluid of children with meningitis. The antibiotics were tested at clinically achievable concentrations in cerebrospinal fluid. After 5 h of incubation, imipenem was the most effective drug. None of the combinations had synergistic activity. Killing by beta-lactam antibiotics or vancomycin was enhanced by the addition of gentamicin, reduced by the addition of rifampin, and unaffected by the addition of fosfomycin.

Ribotyping provides efficient differentiation of nosocomial Serratia marcescens isolates in a pediatric hospital.

Ribotyping with a nonradioactive probing system was used for the epidemiological evaluation of 15 Serratia marcescens nosocomial strains isolated from the stools of 12 children with no apparent illness in five different hospital wards over a 20-day period. Our results indicate that the occurrence of S. marcescens colonization was the result of the spread of a single epidemiological strain in the hematology ward, the oncology ward, and the gastroenterology ward and in two neonates in the neonatology ward, suggesting cross-contamination between the patients in these four wards. This isolate was genotypically unrelated to the bacterial strain found in the three other patients in the neonatology ward. Interestingly, one patient in the neonatology ward harbored these two genotypically different strains. Finally, the patient in the intensive care unit was colonized with a different strain. We find ribotyping to be a more reliable technique than biochemical typing. The results of ribotyping are more easily interpreted than are those of total DNA analysis, with an equivalent degree of discrimination.

DNA restriction fragment length polymorphism differentiates crossed from independent infections in nosocomial Xanthomonas maltophilia bacteremia.

Restriction fragment length polymorphisms of total DNA and rDNA were used to study the relationship between 11 isolates of Xanthomonas maltophilia, obtained from seven patients with nosocomial bacteremia in four distinct wards of a single hospital, and the type strain of the species, ATCC 13637. Our results indicated that there were episodes of cross-infection among the patients of two wards, but there were also independent infectious episodes in the two other wards.

Molecular epidemiology unravels the complexity of neonatal Escherichia coli acquisition in twins.

Combined analysis of restriction fragment length polymorphism of regions of genes coding for rRNA (ribotyping) and esterase electrophoretic typing was used to document neonatal acquisition of Escherichia coli in twins. Our study shows vertical mother-to-infant transmission of one strain of E. coli to one twin and the development of neonatal septicemia with a distinct nonvirulent carboxylesterase type B1 E. coli strain for the other twin.

Arbitrarily primed polymerase chain reaction as a rapid method to differentiate crossed from independent Pseudomonas cepacia infections in cystic fibrosis patients.

We used DNA fingerprinting by the arbitrarily primed polymerase chain reaction (AP-PCR) technique for an epidemiological investigation of 23 Pseudomonas cepacia isolates obtained from 11 cystic fibrosis (CF) patients attending our CF center. This approach was compared with ribotyping, pulsed-field gel electrophoresis (PFGE), and conventional phenotypic typing. AP-PCR and ribotyping were identical in resolving power, since the two methods generated four different profiles and identified the same group of strains. Six patients on the one hand and four on the other harbored strains of the same genotype, thus raising the possibility of either patient-to-patient transmission or acquisition from a common hospital environmental source. PFGE results were in good agreement with those of the other two methods, but PFGE seems more discriminative since it generated a fifth profile for a single strain in a group of four. Our results show in vivo stability for the three methods during a period extending from 3 to 41 months. These genotypic techniques are particularly promising for clinical laboratories to help to clarify the epidemiology of P. cepacia in CF patients. The AP-PCR method constitutes an easier alternative to the well-established ribotyping method. AP-PCR provides the quickest results with minimal technical complexity. However, our results suggest that it is less discriminative than the labor-intensive PFGE method.