J Liñares

University of Barcelona, Barcino, Catalonia, Spain

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Publications (169)1082.01 Total impact

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    ABSTRACT: Nontypeable Haemophilus influenzae (NTHi) is a common cause of respiratory infections in adults, who are frequently treated with fluoroquinolones. The aims of this study were to characterize the genotypes of fluoroquinolone-resistant NTHi isolates and their mechanisms of resistance. Among 7267 H. influenzae isolates collected from adult patients (2000-2013), 28 (0.39%) were ciprofloxacin-resistant according to Clinical and Laboratory Standards Institute (CLSI) criteria. In addition, a nalidixic acid screening during 2010-2013 detected five (0.23%) isolates, which were ciprofloxacin-susceptible but nalidixic acid-resistant. Sequencing of their quinolone resistance-determinant regions and genotyping by pulse-field gel electrophoresis and multi locus sequence typing was performed in the 25 ciprofloxacin-resistant isolates available and in all five nalidixic acid-resistant isolates. In the NTHi studied, two mutations producing changes in two GyrA residues (Ser84, Asp88), and/or two ParC residues (Ser84, Glu88), were associated with increased fluoroquinolone MICs. Strains with one or two mutations (n=15) presented ciprofloxacin and levofloxacin MICs of 0.12-2 μg/ml while those with three or more mutations (n=15) presented MICs of 4-16 μg/ml. Long persistence of fluoroquinolone-resistant strains was observed in three COPD patients. High genetic diversity was observed among fluoroquinolone-resistant NTHi. Although fluoroquinolones are commonly used to treat respiratory infections, the proportion of resistant NTHi remains low. The nalidixic acid disk is a useful test for detecting the first changes in GyrA or in GyrA plus ParC among fluoroquinolone-susceptible strains which are a potential risk for the development of resistance under selective pressure by fluoroquinolone treatments.
    Antimicrobial Agents and Chemotherapy 11/2014; · 4.57 Impact Factor
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    ABSTRACT: Since 2004, a total of 131 isolates of Streptococcus pneumoniae multidrug-resistant invasive serotype 8 have been detected in Spain. These isolates showed resistance to erythromycin, clindamycin, tetracycline, and ciprofloxacin. All isolates were obtained from adult patients and shared a common genotype (sequence type [ST]63; penicillin-binding protein 1a [pbp1a], pbp2b, and pbp2x gene profiles; ermB and tetM genes; and a ParC-S79F change). Sixty-eight isolates that required a ciprofloxacin MIC ≥16 μg/mL had additional gyrA gene changes. Serotype 8-ST63 pbp2x sequences were identical with those of antimicrobial drug-susceptible serotype 8-ST53 isolates. Serotype 8-ST63 pbp2b sequences were identical with those of the multidrug-resistant Sweden 15A-ST63 clone. Recombination between the capsular locus and flanking regions of an ST53 isolate (donor) and an ST63 pneumococcus (recipient) generated the novel 15A-ST63 clone. One recombination point was upstream of pbp2x and another was within pbp1a. A serotype 8-ST63 clone was identified as a cause of invasive disease in Spain.
    Emerging infectious diseases. 11/2014; 20(11):1848-56.
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    ABSTRACT: Biofilm formation by non-typeable Haemophilus influenzae (NTHi) remains a controversial topic. Nevertheless, biofilm-like structures have been observed in the middle-ear mucosa of experimental chinchilla models of otitis media (OM). To date, there have been no studies of biofilm formation in large collections of clinical isolates. This study aimed to investigate the initial adhesion to a solid surface and biofilm formation by NTHi by comparing isolates from healthy carriers, those with non-invasive respiratory disease, and those with invasive respiratory disease. We used 352 isolates from patients with non-bacteremic community acquired pneumonia (NB-CAP), chronic obstructive pulmonary disease (COPD), OM, invasive disease, and a group of healthy colonized children. We then determined the speed of initial adhesion to a solid surface by the biofilm ring test methodology, and quantified biofilm formation by crystal violet staining. Isolates from different clinical sources displayed high levels of biofilm formation on a static solid support after growth for 24 hours. We observed clear differences in initial attachment and biofilm formation depending on the pathology associated with NTHi isolation, with significantly increased biofilm formation for NTHi isolates collected from patients with invasive disease and OM compared with NTHi isolates from patients with NB-CAP, COPD or healthy colonization. In all cases, biofilm structures were detached by proteinase K treatment, suggesting an important role for proteins in the initial adhesion and static biofilm formation measured by crystal violet staining.
    Applied and Environmental Microbiology 09/2014; · 3.95 Impact Factor
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    ABSTRACT: Haemophilus influenzae colonizes the upper respiratory tract and can spread causing otitis and sinusitis. This work aimed to study the oropharyngeal carriage rate in healthy <5-year-old children attending day care centers in Oviedo, Spain in two consecutive years (January to March 2004-2005). The carriage rate was 42% (400/960) and highly variable among centers (range, 12% to 83%). Isolates were mainly identified as nontypeable H. influenzae (NTHi, 99%). Epidemiologically, 127 different genotypes were identified by PFGE with a minimum of two genotypes per center. One hundred fourteen children (12%) were included in both studies and none of them harbored the same strain over a period of time. The isolates only showed resistance to cotrimoxazol and ampicillin, presenting a shift in the level of ampicillin reduced susceptibility, showing a predominance of PBP3 mutations in 2004 and a predominance of β-lactamase production in 2005. This study proved the great genetic variability of NTHi isolates that present similar genotypic patterns in both years with no long-term carriage of the same strain.
    Microbial drug resistance (Larchmont, N.Y.) 04/2014; · 1.99 Impact Factor
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    ABSTRACT: In Spain, rates of ciprofloxacin resistance in pneumococci were low during the last decade (2.6% in 2002; 2.3% in 2006). In 2012 the rate remained at 2.3%, equivalent to 83 of 3,621 isolates. Of the 83 resistant isolates, 15 showed a low level (MIC of 4-8 μg/ml) and 68 a high level (MIC of 16-128 μg/ml) of ciprofloxacin resistance. Thirteen low-level resistant isolates had single changes in ParC, one had a single ParE change, and one did not present any mutations. High-level resistant isolates had GyrA changes, plus additional ParC and/or ParE changes: 51, 15, and 2 isolates had 2, 3, or 4 mutations, respectively. Although 24 different serotypes were observed, six serotypes accounted for 51.8% of ciprofloxacin-resistant isolates: 8 (14.5%), 19A (10.8%), 11A (7.2%), 23A (7.2%), 15A (6.0%), and 6B (6.0%). A decrease in PCV7 serotypes was observed from 2006 (35.7%) to 2012 (16.9%), especially of serotype 14 (from 16.3% to 2.1%; P<0.001). In comparison with 2006, multidrug resistance was greater in 2012 (P=0.296), mainly due to the increased presence and/or emergence of clonal complexes associated with non-PCV7 serotypes: CC63 expressing serotypes 8, 15A, 19A and 19F; CC320 (with serotype 19A); CC42 (with serotype 23A). Although rates of ciprofloxacin resistance remained low and stable throughout the last decade, changes in serotype and genotype distributions were observed in 2012, notably the expansion of a pre-existing multidrug-resistant clone, CC63, and the emergence of the CC156 clone expressing serotype 11A.
    Antimicrobial Agents and Chemotherapy 02/2014; · 4.57 Impact Factor
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    ABSTRACT: Non-typeable Haemophilus influenzae (NTHi) is an opportunistic pathogen causing otitis media in children and community acquired pneumonia or exacerbations of chronic obstructive pulmonary disease in adults. A large variety of studies suggest that biofilm formation by NTHi may be an important step in the pathogenesis of this bacterium.The objective of this report was to determine the relationship between presence of phosphorylcholine in the lipooligosaccharide of NTHi and the level of biofilm formation. The study was performed on 111 NTHi clinical isolates collected from oropharyngeal samples of healthy children, middle ear fluid of children with otitis media and sputum samples of patients with chronic obstructive pulmonary disease or community acquired pneumonia.NTHi clinical isolates presented a large variation in level of biofilm formation and phosphorylcholine content. Isolates collected from the oropharynx and middle ear fluid of children tended to have more phosphorylcholine and made denser biofilms compared to isolates collected from sputum of patients with chronic obstructive pulmonary disease or community acquired pneumonia. Contrary to previous literature, no correlation was observed between biofilm formation and presence of phosphorylcholine in the lipooligosaccharide, either in planktonic or biofilm growth. This lack of correlation was confirmed by abrogating phosphorylcholine incorporation into lipooligosaccharide through licA gene deletion, which had strain-specific effects on biofilm formation.Altogether, we present strong evidence to conclude that there is no correlation between biofilm formation and presence of phosphorylcholine in lipooligosaccharide in a large collection of clinical NTHi isolates collected from different groups of patients.
    Infection and immunity 01/2014; · 4.21 Impact Factor
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    ABSTRACT: Nontypable Haemophilus influenzae (NTHi) has emerged as an important opportunistic pathogen causing infection in adults suffering obstructive lung diseases. Existing evidence associates chronic infection by NTHi to the progression of the chronic respiratory disease, but specific features of NTHi associated with persistence have not been comprehensively addressed. To provide clues about adaptive strategies adopted by NTHi during persistent infection, we compared sequential persistent isolates with newly acquired isolates in sputa from six patients with chronic obstructive lung disease. Pulse field gel electrophoresis (PFGE) identified three patients with consecutive persistent strains and three with new strains. Phenotypic characterisation included infection of respiratory epithelial cells, bacterial self-aggregation, biofilm formation and resistance to antimicrobial peptides (AMP). Persistent isolates differed from new strains in showing low epithelial adhesion and inability to form biofilms when grown under continuous-flow culture conditions in microfermenters. Self-aggregation clustered the strains by patient, not by persistence. Increasing resistance to AMPs was observed for each series of persistent isolates; this was not associated with lipooligosaccharide decoration with phosphorylcholine or with lipid A acylation. Variation was further analyzed for the series of three persistent isolates recovered from patient 1. These isolates displayed comparable growth rate, natural transformation frequency and murine pulmonary infection. Genome sequencing of these three isolates revealed sequential acquisition of single-nucleotide variants in the AMP permease sapC, the heme acquisition systems hgpB, hgpC, hup and hxuC, the 3-deoxy-D-manno-octulosonic acid kinase kdkA, the long-chain fatty acid transporter ompP1, and the phosphoribosylamine glycine ligase purD. Collectively, we frame a range of pathogenic traits and a repertoire of genetic variants in the context of persistent infection by NTHi.
    PLoS ONE 01/2014; 9(5):e97020. · 3.53 Impact Factor
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    ABSTRACT: The epidemiology of invasive Haemophilus influenzae (Hi) has changed since the introduction of the Hi type b (Hib) vaccine. The aim of this study was to analyze the clinical and molecular epidemiology of Hi invasive disease in adults.
    PLoS ONE 01/2014; 9(11):e112711. · 3.53 Impact Factor
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    ABSTRACT: Antibiotic resistance in Streptococcus pneumoniae has increased worldwide by the spread of a few clones. Fluoroquinolone resistance occurs mainly by alteration of their intracellular targets, the type II DNA topoisomerases, which is acquired either by point mutation or by recombination. Increase in fluoroquinolone-resistance may depend on the balance between antibiotic consumption and the cost that resistance imposes to bacterial fitness. In addition, pneumococcal prophages could play an important role. Prophage induction by fluoroquinolones was confirmed in 4 clinical isolates by using Southern blot hybridization. Clinical isolates (105 fluoroquinolone-resistant and 160 fluoroquinolone-susceptible) were tested for lysogeny by using a PCR assay and functional prophage carriage was studied by mitomycin C induction. Fluoroquinolone-resistant strains harbored fewer inducible prophages (17/43) than fluoroquinolone-susceptible strains (49/70) (P = 0.0018). In addition, isolates of clones associated with fluoroquinolone resistance [CC156 (3/25); CC63 (2/20), and CC81 (1/19)], had lower frequency of functional prophages than isolates of clones with low incidence of fluoroquinolone resistance [CC30 (4/21), CC230 (5/20), CC62 (9/21), and CC180 (21/30)]. Likewise, persistent strains from patients with chronic respiratory diseases subjected to fluoroquinolone treatment had a low frequency of inducible prophages (1/11). Development of ciprofloxacin resistance was tested with two isogenic strains, one lysogenic and the other non-lysogenic: emergence of resistance was only observed in the non-lysogenic strain. These results are compatible with the lysis of lysogenic isolates receiving fluoroquinolones before the development of resistance and explain the inverse relation between presence of inducible prophages and fluoroquinolone-resistance.
    PLoS ONE 01/2014; 9(4):e94358. · 3.53 Impact Factor
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    ABSTRACT: Objectives To determine the prevalence of smoking and alcohol abuse among patients with invasive pneumococcal disease (IPD) in order to promote prevention strategies. Methods We prospectively studied all culture-proven IPD cases in patients aged ≥18 years during the period 1997–2011. The habits of smoking and alcohol abuse were evaluated. Pneumococcal serotyping was performed. Results There were 1378 IPD cases, with a mean age of 61 ± 17 years; 65% were males. Compared to the general population aged 18–64 years, patients with IPD of the same age group were more often current smokers (57% vs. 35%, p < 0.001) and alcohol abusers (21% vs. 6%, p < 0.001). Among patients with IPD, young adults (aged 18–49 vs. 50–64 vs. ≥65 years) were more commonly current smokers (71% vs. 40% vs.14%, p < 0.001) and alcohol abusers (23% vs. 18% vs. 6%, p < 0.001). Males were more frequently smokers and alcohol abusers than females. Smokers and alcohol abusers more often had underlying diseases such as HIV infection and chronic liver disease. Pneumonia was more common in smokers and peritonitis in alcohol abusers. Alcohol abuse conferred higher mortality. Certain pneumococcal serotypes, such as serotypes 1, 8, and 23F, more frequently caused IPD in smokers, and serotypes 4, 11A, and 19F in alcohol abusers. Conclusions Smoking and alcohol abuse are the most preventable risk factors for IPD. Implementing smoking and alcohol abuse cessation programs and a pneumococcal vaccination schedule are essential to diminish the burden of pneumonia and other pneumococcal infections.
    International Journal of Infectious Diseases. 01/2014;
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    ABSTRACT: Pneumococci are an important cause of acute exacerbations in patients with chronic obstructive pulmonary disease (COPD). In the last decade, the pneumococcal population has changed, mainly due to the introduction of the 7-valent conjugate vaccine (PCV7). We analysed the antimicrobial susceptibility (microdilution), serotype (PCR) and genotype (PFGE/multilocus sequence typing) of pneumococci causing acute exacerbations during the period 2009-12. Results were compared with two previously published historic periods (2001-04 and 2005-08). A total of 206 pneumococci were collected from 162 COPD patients with acute exacerbations. Compared with previous periods, no significant changes in the rate of multidrug resistance were observed (36.2% in the 2001-04 period to 33.5% in the 2009-12 period, P = 0.644). The most frequent serotypes in the 2009-12 period were 15A (9.6%), 3 (8.1%), 19F (6.6%), 11A (6.1%) and 6C (5.6%), which accounted for 36.0%. A drastic decrease in PCV7 serotypes was observed throughout the study period (from 39.7% in 2001-04 to 10.9% in 2009-12, P < 0.001); non-PCV13 serotypes increased from 44.9% to 71.2%, especially 15A (from 2.2% to 9.6%) and 6C (from 0.0% to 5.6%) (P < 0.05). The most frequent genotypes (clonal complexes, CCs) in the 2009-12 period were CC63(15A,19F,15F) (9.1%), CC180(3) (4.5%), CC62(11A) (4.0%), CC97(10A) (4.0%), CC386(6C) (3.5%), CC260(3) (3.5%) and CC30(16F) (3.5%). Serotypes 19F, 19A, 6A and 6C were genetically diverse. PCV7 serotypes have decreased dramatically. In parallel, two non-PCV7 serotypes (15A and 6C) and their related genotypes (CC63 and CC386) showed a significant increase. Although resistance rates to β-lactams decreased over time, multidrug resistance remained stable.
    Journal of Antimicrobial Chemotherapy 12/2013; · 5.34 Impact Factor
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    ABSTRACT: We aimed to analyse the clinical epidemiology and genetic diversity of invasive pneumococcal disease (IPD) episodes attributed to the Spain(23F)-ST81 (PMEN1) clone. Fifty-eight (2.7%) of 2117 invasive pneumococci isolated from adult patients during the 1990-2012 period shared a PFGE pattern related to the PMEN1 clone. The genotype was confirmed by multilocus sequence typing. The pbp2x, pbp1a, pbp2b and pspA genes were PCR-amplified and sequenced. Polymorphisms in the pspC gene were identified by PCR restriction fragment length polymorphism. The presence of transposons with erythromycin and tetracycline resistance determinants was detected by PCR. The prevalence of the PMEN1 clone increased from 0.8% in 1991 to 6.2% in 2001, and decreased to 0% in 2010-12, concomitant with the introduction of the seven-valent pneumococcal conjugate vaccine for children. A total of 93.1% of patients had pneumonia, meningitis or peritonitis; 87.9% of patients had associated underlying diseases, mainly cancer, chronic obstructive pulmonary disease and diabetes. Two closely related sequence types (STs) (ST81, n = 52; ST85, n = 6) were detected, with different serotypes: 23F (n = 42), 19A (n = 9) and 19F (n = 6). All the isolates were resistant to penicillin, co-trimoxazole and chloramphenicol. All the isolates also shared the same pbp1a allele, whereas multiple alleles of pbp2b, pbp2x, pspA and pspC were detected. Of the isolates, 89.7% were tetracycline resistant and 60.3% (n = 35) were macrolide resistant, and resistance was associated with different Tn916-like transposons. Adult IPD caused by this clone was mainly detected in patients with underlying conditions, and genetic variability was observed among PMEN1 isolates collected in our area over the past 20 years.
    Journal of Antimicrobial Chemotherapy 12/2013; · 5.34 Impact Factor
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    ABSTRACT: Multi-drug resistant Klebsiella pneumoniae isolates are associated with nosocomial infections, in which colonized patients act as a reservoir and source of cross-infection for other patients. In this study, the antimicrobial susceptibility of K. pneumoniae was tested by microdilution using the commercial method MicroScan (Siemens). The genetic relatedness of K. pneumoniae strains was determined by pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). PCR experiments were carried out to obtain primer sets and positive PCR products were purified and sequenced. From May 2007 until December 2009, 98 clonally related K. pneumoniae isolates were detected from clinical samples of 38 patients admitted to the University Hospital of Bellvitge, Barcelona, Spain, including 27 admitted to the intensive care unit (ICU). The most important sources of the isolates were: lower respiratory tract (n = 12), urine (n = 12), and blood (n = 11). The strains were resistant to amoxicillin/clavulanic acid, piperacillin/tazobactam, tobramycin, amikacin, and ciprofloxacin, and had diminished susceptibility to cefepime. All the isolates shared a common PFGE pattern related to sequence type 14 after MLST analysis. In K. pneumoniae isolates and their transconjugants, the bla(OXA-1) gene was located in the variable region of a class I integron that also contains the aac(6')Ib-cr gene. Sequencing of the quinolone resistance determinant regions of gyrA and parC revealed a S83F change in GyrA and no changes in ParC.
    Int Microbiol. 12/2013; 16(4):227-33.
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    ABSTRACT: Since the new GOLD guidelines were implemented no data have been published about the etiology of acute exacerbations (AECOPD) in severe COPD patients with a different frequency of annual episodes. One hundred and eleven COPD patients (FEV1<50%) were prospectively followed up for a year. Good-quality sputum samples recovered during AECOPD were processed, including quantitative culture and PCR detection of atypical bacteria. A total of 188 sputum samples were obtained from AECOPD episodes. Forty patients had a single episode, and 71 patients had ≥2. In 128 episodes a single pathogen was isolated, while 42 episodes were polymicrobial (≥2 pathogens). Overall, the most frequent pathogen isolated was Pseudomonas aeruginosa (n=54), followed by Haemophilus influenzae (n=37), Streptococcus pneumoniae (n=31), Moraxella catarrhalis (n=29) and Staphylococcus aureus (n=12. P. aeruginosa was the most frequent in both groups of patients (35% and 27% in those with 1 and ≥2 AECOPD, respectively). H. influenzae was associated with patients with a single annual AECOPD (33% vs. 16%; P=0.006), while Enterobacteriaceae were associated with frequent exacerbators (0% vs. 12%; P<0.044). Overall, P. aeruginosa was the most frequent pathogen isolated from exacerbations. However, different bacterial etiology was observed depending on the number of annual episodes.
    The Journal of infection 09/2013; · 4.13 Impact Factor
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    ABSTRACT: In this study, we analyzed the clinical and molecular epidemiology of invasive serotype 5 (Ser5) pneumococcal isolates, in four teaching Hospitals in the Barcelona area (1997-2011 period). Among 5,093 invasive pneumococci collected, 134 (2.6%) Ser5 were detected. Although the overall incidence of Ser5 IPD was low (0.25 cases/100,000 inhabitants), three incidence peaks were detected: 0.63/100,000 in 1999, 1.15/100,000 in 2005 and 0.37/100,000 in 2009. Rates of Ser5 IPD were higher among young adults (18-64 years) and older adults (over 64 years) in the first two peaks, whereas they were higher among children in 2009. The majority (88.8%) of patients had pneumonia. Co-morbid conditions were present in young (47.6%) and older adults (78.7%), the most common being chronic obstructive pulmonary disease (20.6% vs 38.3%, respectively) and cardiovascular diseases (11.1% vs 38.3%, respectively). Mortality rates were higher among older adults (8.5%). All Ser5 pneumococci were fully susceptible to penicillin, cefotaxime, erythromycin, and ciprofloxacin. The resistance rates were: cotrimoxazole (48.5 %), chloramphenicol (6.7%) and tetracycline (6%). Two major related sequence types (STs) were detected: ST1223 (n=65), and ST289 (n=61). The Colombia(5)-ST289 clone was responsible for all the cases of the Ser5 outbreak in 1999, whereas ST1223 clone accounted for 73.8% and 61.5% of isolates in 2005 and 2009, respectively. Ser5 pneumococci are a frequent cause of IPD outbreaks in the community involving children and adults with or without co-morbidities. The implementation of the new pneumococcal conjugated-vaccines (PCV10 and PCV13) could prevent such outbreaks.
    Journal of clinical microbiology 08/2013; · 4.16 Impact Factor
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    ABSTRACT: NotI, the most prevalent restriction enzyme used for typing Moraxella catarrhalis, failed to digest genomic DNA from respiratory samples. An improved PFGE methodology determined SpeI as the best choice for typing this bacterial species, with a good restriction of clinical samples and a good clustering correlation with NotI.
    Journal of clinical microbiology 05/2013; · 4.16 Impact Factor
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    ABSTRACT: To analyze the role of the capsular type in pneumococci causing relapse and reinfection episodes of acute exacerbation in COPD patients. A total of 79 patients with 116 recurrent episodes of acute exacerbations caused by S. pneumoniae were included into this study (1995-2010). A relapse episode was considered when two consecutive episodes were caused by the same strain (identical serotype and genotype); otherwise it was considered reinfection. Antimicrobial susceptibility testing (microdilution), serotyping (PCR, Quellung) and molecular typing (PFGE/MLST) were performed. Among 116 recurrent episodes, 81 (69.8%) were reinfections, caused by the acquisition of a new pneumococcus, and 35 (30.2%) were relapses, caused by a pre-existing strain. Four serotypes (9V, 19F, 15A and 11A) caused the majority (60.0%) of relapses. When serotypes causing relapses and reinfection were compared, only two serotypes were associated with relapses: 9V (OR 8.0; 95% CI, 1.34-85.59) and 19F (OR 16.1; 95% CI, 1.84-767.20). Pneumococci isolated from relapses were more resistant to antimicrobials than those isolated from the reinfection episodes: penicillin (74.3% vs. 34.6%, p<0.001), ciprofloxacin (25.7% vs. 9.9%, p<0.027), levofloxacin (22.9% vs. 7.4%, p = 0.029), and co-trimoxazole (54.3% vs. 25.9%, p<0.001). Although the acquisition of a new S. pneumoniae strain was the most frequent cause of recurrences, a third of the recurrent episodes were caused by a pre-existing strain. These relapse episodes were mainly caused by serotypes 9V and 19F, suggesting an important role for capsular type.
    PLoS ONE 01/2013; 8(3):e59027. · 3.53 Impact Factor
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    ABSTRACT: We aimed to obtain insights on the nature of a collection of isolates presumptively identified as atypical Streptococcus pneumoniae recovered from invasive and non-invasive infections in Spain. One-hundred and thirty-two isolates were characterized by: optochin susceptibility in ambient and CO(2)-enriched atmosphere; bile solubility; PCR-based assays targeting pneumococcal genes lytA, ply, pspA, cpsA, Spn9802, aliB-like ORF2, and a specific 16S rRNA region; multilocus sequence analysis; and antimicrobial susceptibility. By multilocus sequence analysis, 61 isolates were S. pseudopneumoniae, 34 were pneumococci, 13 were S. mitis, and 24 remained unclassified as non-pneumococci. Among S. pseudopneumoniae isolates, 51 (83.6%) were collected from respiratory tract samples; eight isolates were obtained from sterile sources. High frequency of non-susceptibility to penicillin (60.7%) and erythromycin (42.6%) was found. Only 50.8% of the S. pseudopneumoniae isolates displayed the typical optochin phenotype originally described for this species. None harbored the cpsA gene or the pneumococcal typical lytA restriction fragment length polymorphism. The Spn9802 and the specific 16S rRNA regions were detected among the majority of the S. pseudopneumoniae isolates (n = 59 and n = 49, respectively). The ply and pspA genes were rarely found. A high genetic diversity was found and 59 profiles were identified. Among the S. pneumoniae, 23 were capsulated and 11 were non-typeable. Three non-typeable isolates, associated to international non-capsulated lineages, were recovered from invasive disease sources. In conclusion, half of the atypical pneumococcal clinical isolates were, in fact, S. pseudopneumoniae and one-fourth were other streptococci. We identified S. pseudopneumoniae and non-typeable pneumococci as cause of disease in Spain including invasive disease.
    PLoS ONE 01/2013; 8(2):e57047. · 3.53 Impact Factor
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    ABSTRACT: Nontypeable Haemophilus influenzae (NTHi) is an opportunistic pathogen which causes a variety of respiratory infections. The objectives of the study were to determine its antimicrobial susceptibility, to characterize the β-lactam resistance, and to establish a genetic characterization of NTHi isolates. Ninety-five NTHi isolates were analyzed by pulsed field gel electrophoresis (PFGE) and multi locus sequence typing (MLST). Antimicrobial susceptibility was determined by microdilution, and the ftsI gene (encoding penicillin-binding protein 3, PBP3) was PCR amplified and sequenced. Thirty (31.6%) isolates were non-susceptible to ampicillin (MIC≥2 mg/L), with 10 of them producing β-lactamase type TEM-1 as a resistance mechanism. After ftsI sequencing, 39 (41.1%) isolates showed amino acid substitutions in PBP3, with Asn526→ Lys being the most common (69.2%). Eighty-four patients were successfully treated with amoxicillin/clavulanic acid, ceftriaxone and levofloxacin. Eight patients died due either to aspiration or complication of their comorbidities. In conclusion, NTHi causing CAP in adults shows high genetic diversity and is associated with a high rate of reduced susceptibility to ampicillin due to alterations in PBP3. The analysis of treatment and outcomes demonstrated that NTHi strains with mutations in the ftsI gene could be successfully treated with ceftriaxone or fluoroquinolones.
    PLoS ONE 01/2013; 8(12):e82515. · 3.53 Impact Factor
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    ABSTRACT: Background. Changes in serotype prevalence among pneumococcal populations result from both serotype replacement and serotype (capsular) switching. Temporal changes in serotype distributions are well documented, but the contribution of capsular switching to such changes is unknown. Furthermore, it is unclear to what extent vaccine-induced selective pressures drive capsular switching.Methods. Serotype and multi-locus sequence typing data for 426 pneumococci dated 1937 to 2007 were analysed. Whole-genome sequence data for a subset of isolates were used to investigate capsular switching events.Results. We identified 36 independent capsular switch events, 18 of which were explored in detail with whole-genome sequence data. Recombination fragment lengths were estimated for 11 events and ranged from ~19.0 to ≥58.2 kb. Two events took place no later than 1960 and the imported DNA included the capsular locus and the nearby penicillin-binding protein genes pbp2x and pbp1a.Conclusions. Capsular switching has been a regular occurrence throughout the last 7 decades of pneumococcal history. Recombination of large DNA fragments (>30 kb), sometimes including the capsular locus and penicillin binding-protein genes, pre-dated both vaccine introduction and widespread antibiotic use. This type of recombination has likely been an intrinsic feature throughout the history of pneumococcal evolution.
    The Journal of Infectious Diseases 11/2012; · 5.85 Impact Factor

Publication Stats

4k Citations
1,082.01 Total Impact Points

Institutions

  • 1983–2014
    • University of Barcelona
      • • Department of Microbiology
      • • Departament de Medicina
      Barcino, Catalonia, Spain
  • 2003–2012
    • Instituto de Salud Carlos III
      Madrid, Madrid, Spain
  • 1989–2012
    • Hospital Universitari de Bellvitge
      • Department of Infectious Diseases
      l'Hospitalet de Llobregat, Catalonia, Spain
  • 2007
    • Hospital General Universitario Gregorio Marañón
      Madrid, Madrid, Spain
  • 2005
    • University Hospital Donostia
      San Sebastián, Basque Country, Spain
    • Institut Marqués, Spain, Barcelona
      Barcino, Catalonia, Spain