Epidemiological surveillance of colonising group B Streptococcus epidemiology in the Lisbon and Tagus Valley regions, Portugal (2005 to 2012): emergence of a new epidemic type IV/clonal complex 17 clone.

This study presents the serotype distribution and the antibiotic resistance profile of 953 colonising group B Streptococcus (GBS) recovered from women of child bearing age (15 to 49 years) between 2005 and 2012 in the Lisbon and Tagus Valley region, Portugal. Overall, serotypes Ia, II, III, and V were the most common, accounting 752 of the 953 isolates (about 80%). However, there were changes in GBS distribution, in particular in the two last years of the study. Of note, the proportion of serotype IV isolates increased from 1% (2/148) in 2006 to 20% (19/97) in 2012. Also, considerable proportions of serotype IV isolates from 2010 to 2012 were respectively resistant to erythromycin (9/43; 21%) or clindamycin (6/43; 14%). The identification of nine serotype IV isolates presenting a novel association with the clonal complex (CC) 17 lineage, involving a putative capsular switch, may accentuate their virulence potential and ecological success. Molecular analysis of this subgroup of isolates revealed the presence of rib, IS (insertion sequence) 861 and GBSi1 group II intron within the C5a peptidase gene (scpB) ­ laminin-binding protein gene (lmb) region, reflecting high clonality and a putative common origin. A close surveillance of the emergent type IV/CC17 isolates is crucial considering the potential impact over GBS treatment guidelines and capsular vaccine development.

Cystic fibrosis F508del patients have apically localized CFTR in a reduced number of airway cells.

Present state of knowledge, mostly based on heterologous expression studies, indicates that the cystic fibrosis transmembrane conductance regulator (CFTR) protein bearing the F508del mutation is misprocessed and mislocalized in the cytoplasm, unable to reach the cell surface. Recently, however, it was described that protein levels and localization are similar between F508del and wild-type CFTR in airway and intestinal tissues, but not in the sweat glands. In this study, we used immunocytochemistry with three different anti-CFTR antibodies to investigate endogenous CFTR expression and localization in nasal epithelial cells from F508del homozygous patients, F508del carriers, and non-CF individuals. On average, 300 cells were observed per individual. No significant differences were observed for cell type distributions among CF, carrier, and non-CF samples; epithelial cells made up approximately 80% to 95% of all cells present. CFTR was detected mostly in the apical region (AR) of the tall columnar epithelial (TCE) cells, ciliated or nonciliated. By confocal microscopy analysis, we show that the CFTR apical region-staining does not overlap with either anti-calnexin (endoplasmic reticulum), anti-p58 (Golgi), or anti-tubulin (cilia) stainings. The median from results with three antibodies indicate that the apical localization of CFTR happens in 22% of TCE cells from F508del homozygous patients with CF (n = 12), in 42% of cells from F508del carriers (n = 20), and in 56% of cells from healthy individuals (n = 12). Statistical analysis indicates that differences are significant among all groups studied and for the three antibodies (p < 0.05). These results confirm the presence of CFTR in the apical region of airway cells from F508del homozygous patients; however, they also reveal that the number of cells in which this occurs is significantly lower than in F508del carriers and much lower than in healthy individuals. These findings may have an impact on the design of novel pharmacological strategies aimed at circumventing the CF defect caused by the F508del mutation.

Cystic fibrosis patients with the 3272-26A-->G mutation have mild disease, leaky alternative mRNA splicing, and CFTR protein at the cell membrane.

We characterized the 3272-26A-->G mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, creating an alternative acceptor splice site in intron 17a, that competes with the normal one, although we predict from consensus values, with lower efficiency. We analyzed five Cystic Fibrosis (CF) Portuguese patients with the 3272-26A-->G/F508del genotype. Besides clinical and haplotype characterization of those patients, we report here results from CFTR transcript analysis in nasal brushings from all five patients. RT-PCR analysis supports alternative splicing in all patients and carriers, but not in controls. By sequencing, we determined that the alternative transcript includes 25 nucleotides from intron 17a, which predictively cause frameshift and a premature stop codon. The use of this alternative splice site causes a reduction in the levels of normal transcripts from the allele with this mutation and, most probably, of normal protein as well. By immunocytochemistry of both epithelial primary cell cultures and slices from CF polyps, CFTR protein is detected at the cell membrane, with three different antibodies. Ussing chamber analysis of one nasal polyp shows a high sodium absorption, characteristic of CF. Altogether, the results suggest that the main defect caused by the 3272-26A-->G mutation is a reduction in normal CFTR transcripts and protein and therefore this mutation should be included in class V, according to Zielenski and Tsui.