Emerging non-13-valent pneumococcal conjugate vaccine (PCV13) serotypes causing adult invasive pneumococcal disease in the late-PCV13 period in Spain.

OBJECTIVE: An early reduction of adult invasive pneumococcal disease (IPD) was observed after the 13-valent pneumococcal conjugate vaccine (PCV13) introduction for children in Spain. We analysed the epidemiology of adult IPD in the late-PCV13 period. METHODS: This was a prospective multicentre study of adult IPD involving six hospitals. Strains were serotyped, genotyped and studied for antimicrobial susceptibility. The late-PCV13 period was compared with the pre- and early-PCV13 periods. RESULTS: A total of 2197 episodes were collected-949 in 2008-2009, 609 in 2012-2013 and 639 in 2015-2016. The initial decrease of IPD observed (from 12.3/100 000 to 8.1/100 000; 2008-2009 versus 2012-2013) plateaued in 2015-2016 (8.3/100 000). IPD due to PCV13 serotypes decreased (from 7.7 to 3.5 to 2.3/100 000; p < 0.05), whereas IPD caused by non-PCV13 serotypes increased (from 4.5 to 4.6 to 6.0/100 000; p < 0.05). The most frequent serotypes in the late-PCV13 period were: 8 (15.1%), 3 (10.5%), 12F (7.9%) and 9N (5.4%). These serotypes were related to major genotypes: CC53 (59.8%) and CC404 (30.4%) for serotype 8, CC180 (64.1%) and CC260 (28.1%) for serotype 3, CC989 (91.7%) for serotype 12F and CC67 (84.8%) for serotype 9N. Penicillin-non-susceptibility (21.2%) was associated with serotypes 11A (CC156), 14 (CC156) and 19A (CC320), and macrolide-resistance was related to serotypes 24F and 19A. Rates of pneumococcal meningitis remained stable throughout the periods (ranges 0.9, 0.8 and 1.0/100 000). CONCLUSIONS: The initial decrease of adult IPD observed after PCV13 introduction for children has been balanced by the rise of non-PCV13 serotypes. The spread of antibiotic-resistant lineages related to non-PCV13 serotypes (11A and 24F) could be a threat for the treatment of serious pneumococcal diseases.

Zoonotic approach to Shiga toxin-producing Escherichia coli: integrated analysis of virulence and antimicrobial resistance in ruminants and humans.

In 2014-2016, we conducted a cross-sectional survey in 115 sheep, 104 beef and 82 dairy cattle herds to estimate Shiga toxin-producing Escherichia coli (STEC) prevalence, and collected data on human clinical cases of infection. Isolates were characterised (stx1, stx2, eae, ehxA) and serogroups O157 and O111 identified by PCR, and their antimicrobial resistance (AMR) profiles were determined by broth microdilution. STEC were more frequently isolated from beef cattle herds (63.5%) and sheep flocks (56.5%) than from dairy cattle herds (30.5%) (P < 0.001). A similar but non-significant trend was observed for O157:H7 STEC. In humans, mean annual incidence rate was 1.7 cases/100 000 inhabitants for O157 STEC and 4.7 for non-O157 STEC, but cases concentrated among younger patients. Distribution of virulence genes in STEC strains from ruminants differed from those from human clinical cases. Thus, stx2 was significantly associated with animal STEC isolates (O157 and non-O157), ehxA to ruminant O157 STEC (P = 0.004) and eae to human non-O157 STEC isolates (P < 0.001). Resistance was detected in 21.9% of human and 5.2% of animal O157 STEC isolates, whereas all non-O157 isolates were fully susceptible. In conclusion, STEC were widespread in ruminants, but only some carried virulence genes associated with severe disease in humans; AMR in ruminants was low but profiles were similar to those found in human isolates.

Thirty years of human infections caused by Yersinia enterocolitica in northern Spain: 1985-2014.

Yersinia enterocolitica infection is a zoonosis with worldwide distribution, gastroenteritis being by far the most common clinical manifestation of human infection. In Gipuzkoa, northern Spain, human Y. enterocolitica infections increased from the mid-1980s to the beginning of the 21st century (from 7·9 to 23·2 annual episodes per 100 000 population) to decrease to 7·2 annual episodes per 100 000 population in the last years of the study. The hospital admission rate due to yersiniosis during the last 15 years of the study was 7·3%. More than 99% of isolates were serotype O:3. Infection affected mainly children under 5 years of age (average rate: 140 episodes per 100 000 population). The incidence in adults was low but hospitalisation increased with age, exceeding 50% in people over 64 years old.

Streptococcus pneumoniae ocular infections, prominent role of unencapsulated isolates in conjunctivitis.

The aim of this study was to determine the characteristics and shifts in serotype distribution of pneumococcal isolates causing ocular infections in a region of northern Spain in two periods: 1999-2010 for episodes of conjunctivitis (n = 612) and 1980-2010 for uncommon and more severe non-conjunctival ocular infections (n = 36). All isolates were serotyped and non-typeable isolates were confirmed as unencapsulated by multiplex-PCR of the lytA, ply and cpsA genes. Genotyping was done by pulsed-field gel electrophoresis and multi-locus sequence typing. Most conjunctivitis cases occurred in children under 5 years old (89.5%), and more severe non-conjunctival ocular infections occurred in patients older than 25 years (86.1%). Unencapsulated isolates were detected in 213 conjunctivitis episodes (34.8%) and one non-conjunctival infection (2.8%). Rates of unencapsulated isolates were similar throughout the study. Among 399 conjunctival encapsulated isolates, the most prevalent were serotypes 19A (n = 53), 15B (n = 30), 6A (n = 27), 19F (n = 25), 23F (n = 21) and 6B (n = 17). The most prevalent serotypes in non-conjunctival infections were serotype 3 (n = 4), 23F (n = 4), 6B (n = 3) and 19A (n = 3). Conjunctivitis caused by serotypes included in the hepta-valent pneumococcal conjugate vaccine steadily decreased, accounting for 34.9% (22/63) in 1999-2001, 19.7% (23/117) in 2002-04, 13.6% (33/242) in 2005-07 and 3.2% (6/190) in 2008-10. Among the 213 unencapsulated isolates, 31 different pulsed-field gel electrophoresis patterns were identified. The main clonal complexes (CC) were CC941 (ST941, ST942), CC448 (ST448) and CC344 (ST344, ST3097). CC941 was the predominant CC in 1999-2001, 2002-04 and 2005-07, being replaced by CC448 in 2008-10. The multidrug-resistant CC344 was present throughout the study.

Molecular characterization of Streptococcus pneumoniae invasive serotype 19A isolates from adults in two Spanish regions (1994-2009).

From 1994 to 2009, the incidence of invasive serotype 19A pneumococci isolated from adults in Barcelona and San Sebastian almost doubled every 4 years. Genotyping of the 167 invasive isolates studied showed serotype 19A to be highly heterogeneous, with 35 different sequence types (STs) and a different clonal structure in each region and time period. Multiresistance, defined as non-susceptibility to three or more antimicrobials, was found in 86 (51.5%) isolates. The most frequent ST was the multidrug-resistant ST276 (n = 28), which is a single-locus variant of the Denmark(14)-ST230 global clone. The ST276 clone, only present in San Sebastian before 2001, was successfully disseminated from 2002 in both cities and was the main contributor to the overall increase of serotype 19A infections.

Visually triggered K-complexes: a study in New Zealand rabbits.

K-complexes are the EEG elements recorded during the state of developing sleep and during slow wave sleep. They are the only EEG components which can be elicited by sensory stimulation during sleep. The peculiarity of New Zealand rabbits to sleep with their eyes open allows the use of visual stimuli to elicit K-complexes. Experiments were performed with three rabbits. For visual stimulation, an elongated screen illuminated by LED flashes was attached to an implant on the animal's skull. The screen covered 20-120° of the visual field of one eye, and moved with the head during animal motion. One-millisecond flashes (15-s interval) were used during daytime in an illuminated room. Flashes elicited evoked responses, which, during the first stages of sleep, were often accompanied by K-complexes. The induced K-complexes were recorded from electrodes located both above visual and somatosensory areas. Evoked responses to visual stimuli were also recorded from both pairs of electrodes, although they were generated exclusively in the visual cortex. Correlation analysis showed that visual evoked responses and K-complexes induced by this stimulation were generated in visual cortex, and passively spread to the electrodes above the somatosensory area. Investigation of the latencies of induced K-complexes revealed two time windows when these complexes could be seen. Within each window there was no correlation between latency and amplitude of K-complexes. There was also no correlation between amplitudes of the visual evoked responses and K-complexes elicited by these responses. We propose that visual stimulation in light sleep temporarily opens a gate for some independent external signals, which evoke activation of the visual cortex, reflected in K-complexes.

Prevalence and molecular characterization of Streptococcus pneumoniae serotype 6C causing invasive disease in Gipuzkoa, northern Spain, 1990-2009.

The recently discovered pneumococcus serotype 6C was responsible for ten of the 1,530 invasive isolates studied between 1990 and 2009. These ten isolates belonged to seven sequence types (STs) and were isolated only from adult patients: six with bacteremia, three with meningitis, and one with peritonitis. All isolates but one were fully penicillin-susceptible.

Antibody microarray typing, a novel technique for Streptococcus pneumoniae serotyping.

The use of antibody-based miniaturized devices for microbiological applications is a field poorly investigated in the era of more developed molecular amplification techniques. A novel antibody microarray for Streptococcus pneumoniae serotyping was developed, by printing nanolitre volumes of pneumococcal serotype-specific antibodies on multi-well slides. This microarray, which showed high specificity when tested against reference and clinical S. pneumoniae isolates, can be applicable as a faster, cost-effective and accurate serotyping technique for pneumococcal epidemiological studies.

Clonal structure and 21-year evolution of Streptococcus pneumoniae serotype 1 isolates in northern Spain.

This study of 135 serotype 1 pneumococcal isolates (88 invasive and 47 non-invasive), collected between 1987 and 2007, gave eight sequence types (217, 227, 228, 304, 305, 306, 3860 and 3861) that group, using eBurst, into three different lineages and one singleton. The annual incidence of serotype 1 invasive episodes per million inhabitants increased from 1.8 in 1987-1993 to 4.0 in 1994-2000, and to 25.6 in 2001-2007. ST228 was the predominant clone until 1998. ST306 first appeared in 1998 and became the most prevalent sequence type (>80%) after the introduction, in June 2001, of the heptavalent pneumococcal conjugate vaccine.

Invasive Streptococcus pneumoniae infections in children and older adults in the north of Spain before and after the introduction of the heptavalent pneumococcal conjugate vaccine.

In the last two decades, an increasing trend in the incidence of pneumococcal disease in Europe has been reported. We investigated the effect of the use of the heptavalent pneumococcal conjugate vaccine (PCV7) in an area of northern Spain, where all recorded cases of invasive pneumococcal diseases (IPD) were included (n = 450; 91 between 1996-2007 in children aged <5 years and 359 between 1998-2007 in adults aged >64 years). All isolates were serotyped. In children, the overall IPD incidence did not significantly decrease after the introduction, in late 2001, of PCV7. However, the incidence of PCV7 serotypes significantly decreased by 137.2% from 31.59 cases/100,000 population in 1996-2001 to 13.42 in 2002-2007 (95% confidence interval [CI] -27.2 to -342.4%), as did the overall rates of penicillin resistance (from 45.6 to 18.6%) and multiresistance (from 30.3 to 11%). In older adults, the overall IPD incidence showed a non-significant increase due to non-PCV7 serotypes, which seemed to continue a previous trend in our region.

Antimicrobial susceptibilities of amoxycillin-non-susceptible and susceptible isolates among penicillin-non-susceptible Streptococcus pneumoniae.

Antimicrobial susceptibilities of 244 amoxycillin-non-susceptible and 81 amoxycillin-susceptible pneumococcal isolates from 15 Spanish hospitals were determined and clonal relationships were investigated by pulsed-field gel electrophoresis after SmaI restriction. Amoxycillin-non-susceptible isolates exhibited higher rates of resistance to cefuroxime, cefixime, cefpodoxime and clarithromycin, but not to levofloxacin and cefotaxime. Cefditoren exhibited MIC(90) values one dilution lower than those of cefotaxime. Higher numbers of the Spain(14)-5 and Spain(6B)-2 clones, but not the Spain(9V)-3 and Spain(23F)-1 clones, were found among amoxycillin-non-susceptible isolates. Spain(14)-5 was the most problematic clone in terms of antibiotic resistance.

Neisseria meningitidis W-135 in the Basque Country, northern Spain.

Neisseria meningitidis W-135 accounted for nine (1.6%) of 562 cases of invasive meningococcal disease and 17 (3.9%) of 430 meningococcal isolates from healthy carriers. There was no mortality associated with the invasive nine isolates, which belonged to subtype P1.6 and geno-subtype P1.18-1. All invasive isolates and 15 of the 17 isolates from healthy carriers belonged to sequence type 22 by multilocus sequence typing, and showed a similarity of > 85% by pulsed-field gel electrophoresis following digestion with NheI. These results demonstrate that W-135 isolates in the Basque region of northern Spain have a high degree of similarity and are almost clonal.

Histological basis of the porcine femoral artery for vascular research.

The similarities between the porcine and human cardiovascular systems make the pig a useful animal model for the study of vascular biology. However, a standardized method is needed to describe the normal histological properties of porcine arteries in order to evaluate pathologic lesions in future studies. Descriptive and morphometric analyses were done on 16 porcine femoral arteries. For these purposes, three histological stains (haematoxylin eosin, Masson's trichrome, and orcein), four immunohistochemical methods (using antibodies anti-alpha-actin, anti-CD3, anti-L1 and anti-lysozyme), and a glycohistochemical method (using Dolichos biflorus lectin) were performed. The porcine femoral arteries evaluated had a mean total area of 6.25 +/- 1.99 mm(2) and a diameter of 2.79 +/- 0.41 mm. The majority of the total area was occupied by the medial layer (42.97 +/- 5.38%) and was mainly constituted by smooth muscle cells (94.58 +/- 2.65%). All the cell markers used reacted with porcine paraffin-embedded tissue. However, the anti-lysozyme antibody was excluded from this histological analysis because of cytoplasmatic reactivity in smooth muscle cells. In summary, this study proposes histological methods to describe the normal characteristics of the porcine femoral artery and raises the possibility of applying this methodology in future studies on porcine vascular research.

Dissemination of emm28 erythromycin-, clindamycin- and bacitracin-resistant Streptococcus pyogenes in Spain.

Reported here is an unusual cluster of non-invasive infections caused by an emm28 Streptococcus pyogenes strain resistant to bacitracin, erythromycin and clindamycin detected in Santander, Spain. Since one of the characteristics of group A streptococci is their almost uniform susceptibility to bacitracin, this finding was unusual, and a search for bacitracin-resistant Streptococcus pyogenes strains was conducted in two other distant cities of Spain (Madrid and San Sebastián) where their presence was confirmed. These strains were frequently associated with erythromycin- and clindamycin-resistance, and most of them belonged to a unique emm28 T28, ST52 clone.

Genetic relatedness of recently collected Spanish respiratory tract Streptococcus pneumoniae isolates with reduced susceptibility to amoxicillin.

Of Streptococcus pneumoniae isolates from 17 hospitals, 9.8% were amoxicillin nonsusceptible (MIC > or = 4 microg/ml). The genetic relatedness of 138 isolates was studied by pulsed-field gel electrophoresis. Although 44 different clones were detected, more than 62% of these isolates were related to four clones (Spain(23F)-1, Spain(6B)-2, Spain(9V)-3, and Spain(14)-5).

Salmonella enteric infections in Gipuzkoa, Spain, 1983-2000.

The incidence of Salmonella enteric infections in Gipuzkoa, Spain, was estimated by studying a stable population between 1983 and 2000. Only stool culture confirmed cases were included. The annual mean rate of infection in children under 2 years old was 1121 per 100,000 (CI 95%; 1060-1181). This age group had the highest relative risk (RR), 16.2-fold higher than the RR of those aged over 14 years. Salmonella Enteritidis was the most prevalent serovar (80.4% of all patients), followed by Salmonella Typhimurium (11.7%).

CMY-2-producing Salmonella enterica, Klebsiella pneumoniae, Klebsiella oxytoca, Proteus mirabilis and Escherichia coli strains isolated in Spain (October 1999-December 2000).

CMY-2 plasmid-mediated AmpC beta-lactamase (CMY-2) was detected in 21 isolates from two hospitals located in different geographical regions of Spain between October 1999 and December 2000. The isolates comprised two Salmonella enterica serovars (Mikawasima and Montevideo), 16 Escherichia coli, one Klebsiella pneumoniae, one Klebsiella oxytoca and one Proteus mirabilis. In addition to the expected resistance to beta-lactams, including extended-spectrum cephalosporins and cefoxitin, all isolates showed a broad spectrum of associated resistance. All were resistant to sulfamethoxazole, chloramphenicol, tetracycline and streptomycin, and all but two were also resistant to gentamicin. Five isolates were studied in detail and all transferred CMY-2 and other resistance determinants by conjugation. Genomic DNA restriction pattern analysis of the E. coli isolates excluded the dissemination of a single clone. To the best of our knowledge this is the first time that CMY-2 has been detected in P. mirabilis, K. oxytoca and S. enterica serovars Mikawasima and Montevideo. It is also the first time that CMY-2 has been described in Spain.

High proportion of pharyngeal carriers of commensal streptococci resistant to erythromycin in Spanish adults.

The presence of erythromycin-resistant (ErR) commensal streptococci in the throat of 110 healthy subjects and 87 patients with pharyngitis was investigated. The resistance determinants were studied by PCR using the primers for mef and erm genes, followed by hybridization and sequencing analysis. Overall, 94.4% of the subjects carried one or more ErR strains in their pharynx. A total of 253 ErR strains was studied: 127 (50.2%) showed constitutive or inducible resistance to clindamycin (MLS(B) phenotype) and 126 (49.8%) were susceptible to clindamycin (M phenotype). In 50 subjects (25.4%) both phenotypes were detected. The ermB gene was predominant among the MLS(B) phenotype strains (97.6%). The mefA (mefA/mefE) gene was detected in 100% of the strains with the M phenotype. One Streptococcus oralis strain bearing the MLS(B) phenotype carried both mefA and ermB genes. The mefA gene from clinical isolates of Streptococcus mitis and S. oralis was transferred by conjugation to an erythromycin-susceptible Streptococcus pneumoniae strain.