Ricardo Dias

Risk factors for the nasopharyngeal carriage of respiratory pathogens by Portuguese children: phenotype and antimicrobial susceptibility of Haemophilus influenzae and Streptococcus pneumoniae.

Between 1997 and 2000 nasopharyngeal specimens were obtained from 466 children < or = 12 years old attending the Pediatric Emergency Department at S. Francisco Xavier Hospital, Lisbon, to evaluate risk factors for nasopharyngeal carriage of Haemophilus influenzae and Streptococcus pneumoniae and to characterize their phenotype and antimicrobial susceptibility. The attending pediatrician completed written questionnaires about the childrens demographic and clinical histories. Over half the children (52.8%) carried H. influenzae and/or S. pneumoniae. Forty-one percent of these children had H. influenzae, 22.8% had S. pneumoniae and 36.2% had both. Risk factors identified for carriage of respiratory pathogens were: age below 3 years (p < 0.05), black race (p < 0.01), attending a daycare center (p < 0.05), and having a lower respiratory infection (p < 0.05). Asthmatic children were less likely to be carriers (p = 0.004). About two-thirds of H. influenzae isolates were susceptible to all antibiotics tested, 7.9% were beta-lactamase producers, 16.4% were nonsusceptible to trimethoprim, and 6.9% were intermediately resistant to clarithromycin. Over half (57.1%) of S. pneumoniae isolates were susceptible to all antibiotics tested, 21.1% were multiresistant, 23.3% were nonsusceptible to penicillin, and about 20% were resistant to macrolides. Low-level resistance to third-generation cephalosporins was detected in 2.3%. The data reflect the controversy surrounding risk factors of nasopharyngeal colonization. These may have significant implications on clinical practice and on antimicrobial strategies to prevent the appearance of further resistant strains. Our findings highlight the importance to investigate the relationship between asthma and carriage.

Antimicrobial susceptibility of invasive Streptococcus pneumoniae isolates in Portugal over an 11-year period.

ABSTRACT This national surveillance study presents the in vitro activities of the main antimicrobial agents against 1,331 S. pneumoniae isolates as tested by an agar dilution method according to the guidelines of the Clinical and Laboratory Standards Institute (formerly NCCLS). The strains were isolated in several regions of Portugal from cases of invasive disease over an 11-year period (1994 to 2004). This study shows that the percentage of penicillin-nonsusceptible strains increased from 12% in 1994 to 28.5% in 2000. Then the rate declined to 17.7% in 2003 but increased again to 23.2% in 2004. Nevertheless, the rate of highly resistant isolates declined consistently, to 0.9% in 2001 to 2004. Ceftriaxone- and cefotaxime-nonsusceptible isolates became less frequent, from 4% and 8%, respectively, in 1994 to ≤1% in 2004. The macrolide-lincosamide-streptogramin B phenotype was the predominant macrolide phenotype found. The increase in the percentage of isolates that were only nonsusceptible to erythromycin (3.7% in 1994 to 1998 to 9.1% in 2002 to 2004) was similar to that for isolates with coresistance to penicillin and erythromycin (3.3% in 1994 to 1998 to 9.1% in 2002 to 2004). The nonsusceptibility to ciprofloxacin increased during recent years, from 0.5% in 2002 to 3.5% in 2004. Multidrug resistance also increased in recent years: from 7.9% in 2002 to 15.6% in 2004. The increasing use of macrolides could be causing the increase in penicillin and multidrug resistance, due to the coresistance to macrolides. The use of penicillin to treat empirical invasive pneumococci infections may need to be reconsidered.

The highly conserved serine threonine kinase StkP of Streptococcus pneumoniae contributes to penicillin susceptibility independently from genes encoding penicillin-binding proteins

BackgroundThe serine/threonine kinase StkP of Streptococcus pneumoniae is a major virulence factor in the mouse model of infection. StkP is a modular protein with a N-terminal kinase domain a C-terminal PASTA domain carrying the signature of penicillin-binding protein (PBP) and prokaryotic serine threonine kinase. In laboratory cultures, one target of StkP is the phosphoglucosamine mutase GlmM involved in the first steps of peptidoglycan biosynthesis. In order to further elucidate the importance of StkP in S. pneumoniae, its role in resistance to β-lactams has been assessed by mutational analysis in laboratory cultures and its genetic conservation has been investigated in isolates from infected sites (virulent), asymptomatic carriers, susceptible and non-susceptible to β-lactams.ResultsDeletion replacement mutation in stkP conferred hypersensitivity to penicillin G and was epistatic on mutations in PBP2X, PBP2B and PBP1A from the resistant 9V clinical isolate URA1258. Genetic analysis of 55 clinical isolates identified 11 StkP alleles differing from the reference R6 allele. None relevant mutation in the kinase or the PASTA domains were found to account for susceptibility of the isolates. Rather the minimal inhibitory concentration (MIC) values of the strains appeared to be determined by their PBP alleles.ConclusionThe results of genetic dissection analysis in lab strain Cp1015 reveal that StkP is involved in the bacterial response to penicillin and is epistatic on mutations PBP 2B, 2X and 1A. However analysis of the clinical isolates did not allow us to find the StkP alleles putatively involved in determining the virulence or the resistance level of a given strain, suggesting a strong conservation of StkP in clinical isolates.

Trends in resistance to penicillin and erythromycin of invasive pneumococci in Portugal

Antimicrobial resistance of pneumococci is influenced by serotypes, antimicrobial consumption and vaccine use. Serotyping of 697 out of 1331 pneumococcal isolates, recovered in Portugal from 1994 to 2004, showed that the theoretical rate of heptavalent conjugate vaccine coverage was 91.7% and 63.6% for penicillin and erythromycin non-susceptible strains, respectively, in children up to 1 year old. The use of amoxicillin and erythromycin decreased in the vaccine period 2001-2004 (P=0.04 and P<0.01, respectively) but azithromycin usage increased in the same period (P<0.01). By using linear regression models, we evaluated the role of antimicrobial and vaccine use in the trends of resistance to penicillin and erythromycin among the isolates. The models suggest that the use of macrolides was the main factor associated with an increase of penicillin and erythromycin non-susceptible isolates from adults (P<0.01) and erythromycin non-susceptible isolates among children (P=0.006). These models also suggest that heptavalent vaccine is failing to reduce antimicrobial resistance as expected, possibly due to the increased consumption of azithromycin (P=0.04). The efficient use of new antibiotics may reverse the present trends of antimicrobial resistance.

Neisseria meningitidis C:2b:P1.2,5 with Intermediate Resistance to Penicillin, Portugal

For 1 year, serogroup, serotype, serosubtype, and penicillin susceptibility of meningococci circulating in various regions in Portugal were evaluated. Most frequent phenotypes were B:4:P1.15 (13.4%) and C:2b:P1.2,5 (75.9%), which are also common in Spain. Overall, 27.5% of C:2b:P1.2,5 strains showed intermediate resistance to penicillin. Laboratory-based surveillance of meningococcal infection in Portugal provides important information to assess the adequacy of public health measures.

Antimicrobial susceptibility, serotype and genotype distribution of meningococci in Portugal, 2001-2002.

One hundred and eighteen Neisseria meningitidis isolates were recovered from patients with invasive meningococcal disease in Portugal, over one year. Our study was undertaken to evaluate antimicrobial susceptibility, serogroup, serotype and genotype of isolates. One quarter (24.6%) of the isolates showed moderate resistance to penicillin and 47.4% were resistant to sulphadiazine. The two most common serosubtypes were C:2b:P1.5,2 (31.3%) and B:4:P1.15 (3.4%). Half (53.6%) of the isolates with moderate resistance to penicillin were phenotype C:2b:P1.5,2 (n=14), C:2b:P1.2 (n=1) or C:2b:NST (n=1); Pulsed-field gel electrophoresis (PFGE) showed that all these isolates were genetically related. Multilocus sequence typing (MLST) analysis of representative clones from each PFGE pattern showed the predominance of the ST-8 complex/cluster A4 among N. meningitidis with moderate resistance to penicillin. This clonal complex has been principally found in Southern Europe. The apparent emergence and dissemination of the hypervirulent ST-8 complex/cluster A4 among serogroup C strains increases the need for a continued surveillance of antimicrobial susceptibility of meningococci and of genotypic markers in Portugal.