Isa Serrano

Changes in Streptococcus pneumoniae serotypes causing invasive disease with non-universal vaccination coverage of the seven-valent conjugate vaccine

The pneumococcal seven-valent conjugate vaccine (PCV7) has been administered in Portugal since late 2001 through the private sector. To evaluate the impact of PCV7 use, the serotypes and antimicrobial susceptibility of pneumococci causing invasive disease in Portugal during 2003-2005 were determined and compared with available data for the period 1999-2002. Changes in serotype distribution compatible with the introduction of PCV7 were shown for children <or=5 years of age from 2003 onwards and for adults from 2004 onwards. PCV7 use with coverage of 43% of children with four doses in the 2004 birth cohort, although substantially below universal coverage, seems to have contributed to greatly reducing the proportion of invasive infections due to vaccine serotypes 4, 6B, 14 and 23F. Similarly, significant indirect effects on the serotype distribution of pneumococci causing infections in adults were noted, with reductions in the proportion of invasive infections caused by serotypes 4, 5 and 14. These changes were accompanied by an increase in the proportion of two non-vaccine serotypes: 19A isolates in all age groups and 7F isolates in adults. Whereas serotypes 6B, 14 and 19A were associated with multidrug resistance, isolates expressing serotypes 4 and 7F were fully susceptible for the most part. There were no changes in the proportion of resistant isolates within each serotype and, in spite of the changes in serotype prevalence, there was not an overall reduction in the proportion of infections caused by resistant pneumococci.

The presence of the pilus locus is a clonal property among pneumococcal invasive isolates

BackgroundPili were recently recognized in Streptococcus pneumoniae and implicated in the virulence of this bacterium, which led to the proposal of using these antigens in a future pneumococcal vaccine. However, pili were found to be encoded by the rlrA islet that was not universally distributed in the species. We examined the distribution of the pilus islet, using the presence of the rlrA gene as a marker for the locus, among a collection of invasive isolates recovered in Portugal and analyzed its association with capsular serotypes, clusters defined by the pulsed-field gel electrophoretic profiles (PFGE) and multilocus sequence types.ResultsOnly a minority of the isolates were positive for the presence of the rlrA gene (27%). There was a high correspondence between the serotype and the presence or absence of rlrA (Wallace coefficient, W = 0.778). In particular, there was an association between the presence of rlrA and the vaccine serotypes 4, 6B, 9V and 14 whereas the gene was significantly absent from other serotypes, namely 1, 7F, 8, 12B and 23F, a group that included a vaccine serotype (23F) and serotype 1 associated with enhanced invasiveness. Even within serotypes, there was variation in the presence of the pilus islet between PFGE clones and a higher Wallace coefficient (W = 0.939) indicates that carriage of the islet is a clonal property of pneumococci. Analysis of rlrA negative isolates revealed heterogeneity in the genomic region downstream of the rfl gene, the region where the islet is found in other isolates, compatible with recent loss of the islet in some lineages.ConclusionThe pilus islet is present in a minority of pneumococcal isolates recovered from human invasive infections and is therefore not an essential virulence factor in these infections. Carriage of the pilus islet is a clonal property of pneumococci that may vary between isolates expressing the same serotype and loss and acquisition of the islet may be ongoing.

Characterization of the Genetic Lineages Responsible for Pneumococcal Invasive Disease in Portugal

ABSTRACT The availability of a conjugate vaccine has the potential to reduce the disease burden of pneumococci and to alter the serotype frequency in the disease-causing population through immunoselection. These changes will probably be reflected in the distributions of individual genetic lineages within the population. We present a characterization of a collection of recent (1999 to 2002) invasive isolates from Portugal (n = 465) by macrorestriction profiling with pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing. During this time, serotypes 14, 1, 3, 4, 8, 9V, 23F, 7F, 19A, and 12B were the 10 most prevalent overall by decreasing rank order. By combining the PFGE data with the sequence types (STs) of 104 isolates, we were able to identify the genetic lineages of the majority of the isolates. We found 66 STs, including 20 novel STs, corresponding to 47 different lineages by e-BURST analysis. We found in our collection a number of previously identified internationally disseminated lineages, especially among macrolide-resistant and penicillin-resistant isolates, and these accounted for most of the isolates. Most of the major lineages (17 of 25) were identified in all years of the study, suggesting that the pneumococcal population associated with invasive disease was stable. This study provides a characterization of the pneumococcal population associated with invasive disease that will be useful for detecting potential selective effects of the novel conjugate vaccine.

Denmark14-230 Clone as an Increasing Cause of Pneumococcal Infection in Portugal within a Background of Diverse Serotype 19A Lineages

ABSTRACT Pneumococci of serotype 19A are increasingly found to be the cause of infection in various geographic regions. We have characterized the serotype 19A isolates (n = 288) found among pneumococci responsible for infections (n = 1,925) and pneumococci recovered from asymptomatic carriers (n = 1,973) in Portugal between 2001 and 2006. We show that despite the existence of serotype 19A clones that have a greater potential to cause invasive disease or an enhanced colonization capacity, the lineage that is increasing as a cause of infection in Portugal is a multiresistant clone that is competent at both. The expanding Denmark14-230 clone found in Portugal is disseminated in other Mediterranean countries, where it is also increasingly responsible for invasive infections in both children and adults. The lineages driving the rise of serotype 19A infections in Asia and the United States (sequence type 320 [ST320] and ST199) are either absent or account for only a small proportion of isolates in Portugal. These data highlight the importance of locally circulating clones with the ability to compete in the nasopharyngeal niche in the emergence of the serotype 19A lineages which are an increasing cause of infection in various geographic regions.

Drug–lipid interaction evaluation : why a 19th century solution?

The affinity of a drug candidate for a biological membrane (its lipophilicity) is closely related to the pharmacologically crucial events of absorption, biodistribution, metabolization and excretion. The evolution of knowledge of biological membranes during the past two decades contrasts with the rudimentary parameter most commonly used to assess lipophilicity: P(o/w), the octanol-water partition coefficient. P(o/w) is especially unrealistic when testing molecules that are polar or partially charged. By contrast, lipid vesicle-based methods determine the extent of the actual partition of a drug to a membrane much more accurately, and have the additional advantage of enabling the choice of the lipid composition considered most suitable to answer a specific biological or pharmaceutical question. In addition, some of these methods are appropriate for high throughput screening, thus shifting determinations of membrane partition to a more preliminary stage of drug development. This streamlines research and development, by saving the time and money that would be spent on unpromising leads.

Chemical Conjugation of the Neuropeptide Kyotorphin and Ibuprofen Enhances Brain Targeting and Analgesia

The pharmaceutical potential of natural analgesic peptides is mainly hampered by their inability to cross the blood-brain barrier, BBB. Increasing peptide-cell membrane affinity through drug design is a promising strategy to overcome this limitation. To address this challenge, we grafted ibuprofen (IBP), a nonsteroidal anti-inflammatory drug, to kyotorphin (l-Tyr-l-Arg, KTP), an analgesic neuropeptide unable to cross BBB. Two new KTP derivatives, IBP-KTP (IbKTP-OH) and IBP-KTP-amide (IbKTP-NH(2)), were synthesized and characterized for membrane interaction, analgesic activity and mechanism of action. Ibuprofen enhanced peptide-membrane interaction, endowing a specificity for anionic fluid bilayers. A direct correlation between anionic lipid affinity and analgesic effect was established, IbKTP-NH(2) being the most potent analgesic (from 25 μmol · kg(-1)). In vitro, IbKTP-NH(2) caused the biggest shift in the membrane surface charge of BBB endothelial cells, as quantified using zeta-potential dynamic light scattering. Our results suggest that IbKTP-NH(2) crosses the BBB and acts by activating both opioid dependent and independent pathways.

Heterogeneity of pneumococcal phase variants in invasive human infections

BackgroundStreptococcus pneumoniae can be carried asymptomatically in the nasopharynx of its human host but can also cause a wide range of infections. A role for pneumococcal phase variants in the different lifestyles of this bacterium has been suggested but no systematic survey of the colony phenotypes of isolates associated with human infections has been undertaken.ResultsWe report the colony opacity phenotypes of a genetically diverse set of 304 invasive isolates representing 10 serotypes. Over half of the isolates (52%) presented the opaque phenotype whereas transparent variants accounted for only 26% of the total. However, the frequency of recovery of each phase variant was not uniform, while serotypes 1, 4, 12B and 23F presented the opaque phenotype more frequently than expected by chance, serotypes 3 and 14 where less frequently associated with this phenotype.ConclusionThe opaque phenotype was the most frequent phenotype found among invasive isolates. An unexpected and equally important finding is the variability of the dominant opacity phenotype found among serotypes. This observation highlights the heterogeneity of opacity phenotypes in invasive isolates and lends further support to the proposal that other factors, in addition to the site of isolation, determine the opacity phenotype of a given isolate. The association between serotype and colonial opacity could help explain epidemiological differences observed among pneumococcal serotypes such as a higher invasive disease potential.

Endothelium-Mediated Action of Analogues of the Endogenous Neuropeptide Kyotorphin (Tyrosil-Arginine): Mechanistic Insights from Permeation and Effects on Microcirculation

Kyotorphin (KTP) is an endogenous peptide with analgesic properties when administered into the central nervous system (CNS). Its amidated form (l-Tyr-l-Arg-NH2; KTP-NH2) has improved analgesic efficacy after systemic administration, suggesting blood-brain barrier (BBB) crossing. KTP-NH2 also has anti-inflammatory action impacting on microcirculation. In this work, selected derivatives of KTP-NH2 were synthesized to improve lipophilicity and resistance to enzymatic degradation while introducing only minor changes in the chemical structure: N-terminal methylation and/or use of d amino acid residues. Intravital microscopy data show that KTP-NH2 having a d-Tyr residue, KTP-NH2-DL, efficiently decreases the number of leukocyte rolling in a murine model of inflammation induced by bacterial lipopolysaccharide (LPS): down to 46% after 30 min with 96 μM KTP-NH2-DL. The same molecule has lower ability to permeate membranes (relative permeability of 0.38) and no significant activity in a behavioral test which evaluates thermal nociception (hot-plate test). On the contrary, methylated isomers at 96 μM increase leukocyte rolling up to nearly 5-fold after 30 min, suggesting a proinflammatory activity. They have maximal ability to permeate membranes (relative permeability of 0.8) and induce long-lasting antinociception.

Improvement of the pharmacological properties of amidated kyotorphin by means of iodination

Amidated kyotorphin (L-Tyr-L-Arg-NH2; KTP-NH2) shows analgesic properties following systemic administration. Although KTP-NH2 does not have toxic effects in the liver, its biodistribution is unknown. KTP-NH2 was radioiodinated to evaluate its biological fate in vivo. Mono-radioiodinated KTP-NH2 ([125I]MIK) was radiochemically stable in vitro, namely in saline at 4 °C up to 1 week, and moderately stable when incubated with human serum at 37 °C. Although the radioiodinated peptide could translocate a cellular model of the blood–brain barrier (BBB), the levels of radioactivity in the brain were minimal, 5 and 10 min after intraperitoneal injection (i.p.). Significant accumulation of 125I was found in the thyroid probably reflecting the hydrolysis of the iodine–tyrosine bond by liver deiodinases. HPLC analysis of plasma samples collected 5 min post-injection showed that intact [125I]MIK accounted for 72.5% of the total radioactivity, whereas the remaining radioactivity was associated with free radioiodide (I−). These findings were subsequently confirmed by the low radioactivity found in the liver and kidney homogenates from rats sacrificed 10 min after i.p. administration. The analgesic activity of mono- and di-iodinated KTP-NH2 was also evaluated by a hot plate assay showing a delayed peak of maximal efficacy compared to KTP-NH2 (30 vs. 15 minutes). Overall, the peripheral effects of the peptides cannot be excluded.