PCR-ribotying, a typing method based on polymorphism in the 16S-23S intergenic spacer region, has been recently used to investigate outbreaks due to Clostridium difficile. However, this method generates bands of high and close molecular masses which are difficult to separate on agarose gel electrophoresis. To improve reading of banding patterns of PCR-ribotyping applied to C. difficile, a partial sequencing of the rRNA genes (16S and 23S) and intergenic spacer region has been performed, then a new set of primers located closer to the intergenic spacer region has been defined. The new PCR gave reproducible patterns of bands easy to separate on agarose gel electrophoresis. Each of the 10 serogroups and 11 subgroups of serogroup A produced a different pattern. This typing method has evidenced major qualities such as easiness, rapidity and reproducibility. However, its discriminatory power has to be evaluated to validate its importance as a typing tool for C. difficile.
Clinical features and molecular characterization of 109 group B streptococci causing neonatal invasive infections were determined over an 18-month period in France. Sixtyfour percent of the strains were from late-onset infections, and 75% were capsular type III. The hypervirulent clone ST-17 was recovered in 80% of meningitis cases. G roup B Streptococcus (GBS)is the leading cause of infectious illness among newborns. Invasive infections in neonates can result in pneumonia, sepsis, or meningitis. Early-onset disease (EOD) occurs within the fi rst week. Lateonset disease (LOD) occurs after the fi rst week and accounts for most meningitis cases and deaths (1). Because recommendations for intrapartum antibioprophylaxis (IAP) for mothers in labor at risk for GBS infection have been widely implemented in many countries, the incidence of EOD has declined to <1/1,000 births, but the incidence of LOD has remained unchanged (2). To date, 10 capsular serotypes have been described (Ia, Ib, and II-IX). Among these, serotype III is of particular importance because it is responsible for a substantial proportion of EOD and most cases of . Different studies have suggested that most neonatal invasive diseases and almost all cases of meningitis are caused by a limited number of strains belonging to a homogeneous serotype III clone. This clone is defi ned by multilocus sequence typing (MLST) analyses as ST-17, the so-called highly virulent clone (4-8). However, data available in Europe are limited regarding the distribution of GBS genotypes among invasive isolates recovered from neonates.We describe clinical characteristics, capsular type, and MLST allelic and antimicrobial drug-susceptibility profi les of 109 nonredundant GBS isolates that caused neonatal invasive infections. These isolates were collected during an active surveillance performed in France from May 2006 through December 2007. The StudyClinical data on 109 infants up to 4 months of age were analyzed. Sepsis was defi ned as GBS bacteremia in the presence of consistent clinical signs and symptoms. Meningitis was diagnosed if GBS was recovered from cerebrospinal fl uid. GBS isolates were identifi ed by using a commercial Lancefi eld group-specifi c latex agglutination test. Capsular typing was performed by a multiplex PCR as described (9), and the hypervirulent ST-17 clone was detected by real-time PCR, as reported (6). Susceptibility testing, antibiograms, and MICs were performed according to Clinical and Laboratory Standards Institute recommendations (www.clsi.org). Antimicrobial drug-resistance genes were detected by using the multiplex PCR as described (10). Statistical analysis was performed according to the Fisher exact and χ 2 tests. A p value of <0.05 was used as the threshold for statistical signifi cance.We studied 109 GBS strains responsible for neonatal invasive infections; 36% (n = 39) and 64% (n = 70) were responsible for EOD and LOD, respectively (Table). Eighty percent of EOD cases occurred during the fi rst 24 hours after birth, with a male:female rati...
Clostridium difficile is now recognized as the major agent responsible for nosocomial diarrhea in adults. Among the genotyping methods available, arbitrarily primed PCR (AP-PCR), PCR-ribotyping, and pulsed-field gel electrophoresis (PFGE) have been widely used for investigating outbreaks of C. difficileinfections. However, the comparative typing ability, reproducibility, discriminatory power, and efficiency of these methods have not been fully investigated. We compared the results of three methods—AP-PCR with three different primers (AP3, AP4, and AP5), PCR-ribotyping, and PFGE (with SmaI endonuclease)—to differentiate 99 strains of C. difficile that had been previously serogrouped. Typing abilities were 100% for PCR-ribotyping and AP-PCR with AP3 and 90% for PFGE, due to early DNA degradation in strains from serogroup G. Reproducibilities were 100% for PCR-ribotyping and PFGE but only 88% for AP-PCR with AP3, 67% for AP-PCR with AP4, and 33% for AP-PCR with AP5. Discriminatory power for unrelated strains was >0.95 for all the methods but was lower for PCR-ribotyping among serogroups D and C. PCR-based methods were easier and quicker to perform, but their fingerprints were more difficult to interpret than those of PFGE. We conclude that PCR-ribotyping offers the best combination of advantages as an initial typing tool for C. difficile.
We studied a collection of 110 serotype III group B streptococcus (GBS) isolates causing neonatal meningitis, by means of both pulsed-field gel electrophoresis (PFGE) with SmaI and Southern hybridization with probes for genes potentially associated with virulence (neuA, cpsA, scpB, and hylB and, for mobile genetic elements [MGEs], GBSi1 and IS1548), in comparison with 44 serotype III GBS isolates colonizing healthy neonates. Using polymerase chain reaction, we assessed both the insertion of MGEs downstream of the scpB gene and the insertion of IS1548 within the hylB gene. PFGE clustered the isolates into 3 main groups. One PFGE group accounted for 80% of typeable cerebrospinal fluid (CSF) isolates, versus 24% of colonization isolates (P=1.8 x 10-9). GBSi1 was found in 67% of CSF isolates and in only 23% of colonization isolates (P=5.3 x 10-7). A 15-kbp SmaI restriction-DNA fragment bearing the neuA gene was significantly associated with CSF isolates (P=1.1 x 10-11).
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