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This study provides an update on invasive Haemophilus influenzae disease in Bellvitge University Hospital (2014–2019), reporting its evolution from a previous period (2008–2013) and analysing the non-typeable H. influenzae (NTHi) population structure using a clade-related classification. Clinical data, antimicrobial susceptibility and serotyping were studied and compared with those of the previous period. Population structure was assessed by multilocus sequence typing (MLST), SNP-based phylogenetic analysis and clade-related classification. The incidence of invasive H. influenzae disease remained constant between the two periods (average 2.07 cases per 100 000 population), while the 30 day mortality rate decreased (20.7–14.7 %, respectively). Immunosuppressive therapy (40 %) and malignancy (36 %) were the most frequent comorbidities. Ampicillin and fluoroquinolone resistance rates had increased between the two periods (10–17.6 % and 0–4.4 %, respectively). NTHi was the main cause of invasive disease in both periods (84.3 and 85.3 %), followed by serotype f (12.9 and 8.8 %). NTHi displayed high genetic diversity. However, two clusters of 13 (n=20) and 5 sequence types (STs) (n=10) associated with clade V included NTHi strains of the most prevalent STs (ST3 and ST103), many of which showed increased frequency over time. Moreover, ST103 and ST160 from clade V were associated with β-lactam resistance. Invasive H. influenzae disease is uncommon, but can be severe, especially in the elderly with comorbidities. NTHi remains the main cause of invasive disease, with ST103 and ST160 (clade V) responsible for increasing β-lactam resistance over time.

Aim: The bronchial microbiome of severe chronic obstructive pulmonary disease patients colonized by Pseudomonas aeruginosa was analyzed using 16S rRNA gene sequencing to identify differences related to biofilm-forming capacity. Patients & methods: Patient sputum samples from 21 patients were studied. Results: Statistically significant differences related to biofilm-forming capacity were only found for genera with relative abundances 50% which increased from baseline were observed in 10/14 exacerbations, but corresponded to Pseudomonas only in three episodes, while other pathogenic genera were identified in seven. Conclusion: The bronchial microbiome shows differences according with P. aeruginosa biofilm-forming capacity. Pathogenic microorganisms other than P. aeruginosa cause a significant part of the exacerbations in colonized chronic obstructive pulmonary disease patients.

Objectives The objectives of this study were to establish the frequency of Haemophilus haemolyticus in clinical samples, to determine the antimicrobial resistance rate and to identify the mechanisms of resistance to β-lactams and quinolones. Methods An updated database was used to differentiate between MALDI-TOF MS results for Haemophilus influenzae and H. haemolyticus. Antimicrobial susceptibility was studied by microdilution, following EUCAST criteria. The β-lactamase types were identified by PCR analysis of isolates that tested positive for nitrocefin hydrolysis. Mutations in the ftsI gene were identified in isolates with ampicillin MICs ≥0.25 mg/L. Mutations in the quinolone resistance-determining region (QRDR) were identified in isolates with ciprofloxacin MICs ≥0.5 mg/L. Results Overall, we identified 69 H. haemolyticus isolates from 1706 clinical isolates of Haemophilus spp. from respiratory, genital, invasive, and other infection sources. The frequency of H. haemolyticus was low in respiratory samples compared with that of H. influenzae, but in genital-related samples, the frequency was similar to that of H. influenzae. We found low antimicrobial resistance rates among H. haemolyticus isolates, with 8.7% for ampicillin, 8.7% for co-trimoxazole, 7.2% for tetracycline and 4.3% for ciprofloxacin. Mutations in the ftsI gene classified the isolates into four groups, including the newly described Group Hhae IV, which presents mutations in the ftsI gene not identified in H. influenzae and H. haemolyticus type strains. Three ciprofloxacin-resistant H. haemolyticus isolates with mutations affecting GyrA and ParC were identified. Conclusions The frequency of H. haemolyticus was low, especially in respiratory samples, where H. influenzae is the main pathogen of this genus. Although antimicrobial resistance rates were low, three ciprofloxacin-resistant H. haemolyticus clinical isolates have been identified for the first time.

The clinical significance of high levels of homocysteine in autism spectrum disorder (ASD) is unknown. An experimental study was conducted in order to evaluate the concentration of homocysteine in children with ASD and typically developing children and to analyse any relationships with the severity of core symptoms of ASD and other clinical features (drugs, co-morbidities, gender, age, diet). Core symptoms of autism were evaluated by DSM-IV criteria. Homocysteine, glutathione, methionine, 3-nitrotyrosine were measured in urine. The increase in homocysteine concentration was significantly and directly correlated with the severity of the deficit in communication skills, but was unrelated to deficit in socialisation or repetitive/restricted behaviour. Urinary homocysteine concentration may be a possible biomarker for communication deficits in ASD and a potential diagnostic tool useful to evaluate new treatment options since no treatment for core symptoms of ASD are available.

Among epigenetic factors leading to increased prevalence of juvenile neuropsychiatric disorders, including autism spectrum disorder, exposure to metals, such as lead (Pb) have led to conflicting results. The aim of the present study was to determine the levels of Pb in the urine of children with autism spectrum disorder (ASD) compared with typically developing children (TD) age- and sex-matched, and to analyze any association between core symptoms of ASD, special diets, supplements intake or prescription drugs and the concentration of Pb. The study was performed in a group of children with ASD (n = 35, average age 7.4 ± 0.5 years) and TD (n = 34, average age 7.7 ± 0.9 years). Measurement of lead in urine was performed by atomic absorption spectrometry, symptoms of ASD were analyzed by diagnostic and statistical manual of mental disorders (DMS-IV) using the questionnary ADI-R. Careful clinical evaluation was also undertaken and statistical analysis was done taking into account any possible confounding factor.

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 7,267 H. influenzae isolates collected from adult patients from 2000 to 2013, 28 (0.39%) were ciprofloxacin resistant according to Clinical and Laboratory Standards Institute (CLSI) criteria. In addition, a nalidixic acid screening during 2010 to 2013 detected five (0.23%) isolates that were ciprofloxacin susceptible but nalidixic acid resistant. Sequencing of their quinolone resistance-determining regions and genotyping by pulse-field gel electrophoresis and multilocus sequence typing of the 25 ciprofloxacin-resistant isolates available and all 5 nalidixic acid-resistant isolates were performed. In the NTHi isolates 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) had ciprofloxacin and levofloxacin MICs of 0.12 to 2 μg/ml, while those with three or more mutations (n=15) had MICs of 4 to 16 μg/ml. Long persistence of fluoroquinolone-resistant strains was observed in three chronic obstructive pulmonary disease patients. High genetic diversity was observed among fluoroquinolone-resistant NTHi isolates. Although fluoroquinolones are commonly used to treat respiratory infections, the proportion of resistant NTHi isolates remains low. The nalidixic acid disk test is useful for detecting the first changes in GyrA or in GyrA plus ParC among fluoroquinolone-susceptible strains that are at a potential risk for the development of resistance under selective pressure by fluoroquinolone treatment., This work was supported by grants from the Fondo de Investigaciones Sanitarias de la Seguridad Social (PI 0901904) and the Plan Nacional de IDI of Ministerio de Ciencia e Innovación (BIO2011-25343) and by CIBER de Enfermedades Respiratorias, CIBERES; (CB06/06/0037), run by the Instituto de Salud Carlos III, Madrid, Spain. C.P. was supported by FPU grant AP2010-3202 (Formación de Profesorado Universitario, Ministerio de Educación, Spain). S.M. was supported by Sara Borrell Postdoctoral contract CD10/00298 from the Instituto de Salud Carlos III, Madrid, Spain.

Nontypeable Haemophilus influenzae (NTHi) is an opportunistic pathogen that causes 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 the 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 the level of biofilm formation in a static assay and phosphorylcholine content. Isolates collected from the oropharynx and middle ear fluid of children tended to have more phosphorylcholine and made denser biofilms than isolates collected from sputum samples of patients with chronic obstructive pulmonary disease or community-acquired pneumonia. No correlation was observed between biofilm formation and the presence of phosphorylcholine in the lipooligosaccharide for either 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 in a static assay and the presence of phosphorylcholine in lipooligosaccharide in a large collection of clinical NTHi isolates collected from different groups of patients.

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.

Autism spectrum disorder (ASD) is a group of neurodevelopmental disorders characterized by a combination of reciprocal social deficits, communication impairment, and rigid ritualistic interest and stereotypies. The etiology is generally multifactorial, including genetic, immunological and/or environmental factors. A group of ASD has been linked to mitochondrial dysfunction with subsequent deficiency in energy production. Patients with ASD and mitochondrial disease often show signs and symptoms uncommon to idiopathic ASD such as cardiac, pancreatic or liver dysfunction, cardiac, growth retardation, fatigability, but in some cases semiology is different. We show two clinical cases of ASD associated to a deficiency of the mitochondrial respiratory chain (complex I+III and IV) with different clinical presentations. In one case, signs and symptoms of mitochondrial disorder were mild and the second diagnosis was attained many years after that of ASD. These findings support the recent growing body of evidence that ASD can be associated with mitochondrial disorder. Children with ASD and abnormal neurologic or systemic findings should be evaluated for mitochondrial disorder.

Most, if not all, bacteria form a biofilm, a multicellular structure that protects them from antimicrobial actions of the host immune system and affords resistance to antibiotics. The latter is especially disturbing with the increase in multiresistant bacterial clones worldwide. Bacterial biofilm formation is a multistep process that starts with surface adhesion, after which attached bacteria divide and give rise to biomass. The actual steps required for Haemophilus influenzae biofilm formation are largely not known. We show that interference with peptidoglycan biosynthesis increases biofilm formation because of the release of bacterial genomic DNA. Subinhibitory concentrations of β-lactam antibiotics, which are often prescribed to treat H. influenzae infections, increase biofilm formation through a similar mechanism. Therefore, when β-lactam antibiotics do not reach their MIC in vivo, they might not only drive selection for β-lactam-resistant clones but also increase biofilm formation and resistance to other antimicrobial compounds., Nontypeable Haemophilus influenzae (NTHi) is an opportunistic pathogen that mainly causes 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. However, the underlying mechanisms involved in this process are poorly elucidated. In this study, we used a transposon mutant library to identify bacterial genes involved in biofilm formation. The growth and biofilm formation of 4,172 transposon mutants were determined, and the involvement of the identified genes in biofilm formation was validated in in vitro experiments. Here, we present experimental data showing that increased bacterial lysis, through interference with peptidoglycan synthesis, results in elevated levels of extracellular DNA, which increased biofilm formation. Interestingly, similar results were obtained with subinhibitory concentrations of β-lactam antibiotics, known to interfere with peptidoglycan synthesis, but such an effect does not appear with other classes of antibiotics. These results indicate that treatment with β-lactam antibiotics, especially for β-lactam-resistant NTHi isolates, might increase resistance to antibiotics by increasing biofilm formation. IMPORTANCE Most, if not all, bacteria form a biofilm, a multicellular structure that protects them from antimicrobial actions of the host immune system and affords resistance to antibiotics. The latter is especially disturbing with the increase in multiresistant bacterial clones worldwide. Bacterial biofilm formation is a multistep process that starts with surface adhesion, after which attached bacteria divide and give rise to biomass. The actual steps required for Haemophilus influenzae biofilm formation are largely not known. We show that interference with peptidoglycan biosynthesis increases biofilm formation because of the release of bacterial genomic DNA. Subinhibitory concentrations of β-lactam antibiotics, which are often prescribed to treat H. influenzae infections, increase biofilm formation through a similar mechanism. Therefore, when β-lactam antibiotics do not reach their MIC in vivo, they might not only drive selection for β-lactam-resistant clones but also increase biofilm formation and resistance to other antimicrobial compounds.

Dicarboxylic acids are an important source of information about metabolism and potential physiopathological alterations in children with autism spectrum disorders (ASDs). We measured the concentration between dicarboxylic adipic and suberic acids in children with an ASD and typically-developing (TD) children and analyzed any relationships between the severity of the core symptoms of ASDs and other clinical features (drugs, supplements, drugs, or diet). The core symptoms of autism were evaluated using the DSM-IV criteria, and adipic acid and suberic acid were measured in urine samples. Overall, no increase in the concentration of adipic acid in children with ASDs compared to TD children, however when considering vitamin B supplementation in ASD there were significantly increased level of urinary adipic acid in children with an ASD not taking vitamin B supplementation compared to supplemented children or to TD children. No significant difference were observed in suberic acid. Interestingly, the increase in adipic acid concentration was significantly and indirectly correlated with the severity of the deficit in socialization and communication skills in children with an ASD. Therefore, therapeutic treatments aimed at decreasing adipic acid concentration might not be beneficial for treating the core symptoms of ASDs.

The recovery of Pseudomonas aeruginosa from respiratory samples of COPD patients has been associated with impaired lung function and recurrent exacerbations, partly due to strain-dependent factors such as biofilm formation. Aims: To analyse the bronchial microbiome of COPD patients with positive cultures for P.aeruginosa associated with either colonization or infection, and differentiated according to biofilm production, both in stability and exacerbation. Methods: Sputum samples with positive cultures for P.aeruginosa were obtained from 21 COPD patients during stability (n=18) and exacerbation (n=23), and analysed by 16S rRNA gene amplification and pyrosequencing. Biofilm-forming capacity of P. aeruginosa was examined using crystal violet in a static microtiter plate assay. Sputum samples were classified according to the biofilm-forming capacity of P.aeruginosa (low/high), assessing the differences between both groups, both in stability and exacerbation.RESULTS:No significant differences in biodiversity parameters (Shannon and Chao1) were found between low and high biofilm-forming groups. However, the bronchial microbiome of the high biofilm-forming group showed higher relative abundance of bacterial Operational Taxonomic Units (12 and 9 OTUs during stability and exacerbation, respectively), and represented up to 10% of the relative abundance in the sample. Conclusions: In COPD patients colonized or infected by P. aeruginosa , biofilm production was associated with an overgrowth of some bacterial OTUs, which attained a relative abundance of up to 10%. Accordingly, a biofilm environment undermined a change in the bronchial microbiome identifiable through 16srRNA analyses.

Biofilm formation by nontypeable (NT) Haemophilus influenzae 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 NT H. influenzae by comparing isolates from healthy carriers, those with noninvasive respiratory disease, and those with invasive respiratory disease. We used 352 isolates from patients with nonbacteremic community-acquired pneumonia (NB-CAP), chronic obstructive pulmonary disease (COPD), OM, and 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 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 24h. We observed clear differences in initial attachment and biofilm formation depending on the pathology associated with NT H. influenzae isolation, with significantly increased biofilm formation for NT H. influenzae isolates collected from patients with invasive disease and OM compared with NT H. influenzae isolates from patients with NB-CAP or COPD and healthy colonized subjects. 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., This study was supported by grants from the Fondo de Investigaciones Sanitarias de la Seguridad Social (PI 0901904) and MINECO (SAF2012- 31166) and by CIBER de Enfermedades Respiratorias, CIBERES (CB06/ 06/0037), run by the Instituto de Salud Carlos III (ISCIII), Madrid, Spain. The work with the BioFilm ring test was performed in the framework of a collaboration with Biofilm Control. C.P. was supported by FPU grant (Formación de Profesorado Universitario, Ministerio de Educación, Spain). S.M. was supported by Sara Borrell postdoctoral contract CD10/ 00298 from the Instituto de Salud Carlos III (ISCIII), Madrid, Spain.

Objectives 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. Methods Clinical data of the 82 patients with Hi invasive infections were analyzed. Antimicrobial susceptibility, serotyping, and genotyping were studied (2008–2013). Results Men accounted for 63.4% of patients (whose mean age was 64.3 years). The most frequent comorbidities were immunosuppressive therapy (34.1%), malignancy (31.7%), diabetes, and COPD (both 22%). The 30-day mortality rate was 20.7%. The majority of the strains (84.3%) were nontypeable (NTHi) and serotype f was the most prevalent serotype in the capsulated strains. The highest antimicrobial resistance was for cotrimoxazole (27.1%) and ampicillin (14.3%). Twenty-three isolates (32.9%) had amino acid changes in the PBP3 involved in resistance. Capsulated strains were clonal and belonged to clonal complexes 6 (serotype b), 124 (serotype f), and 18 (serotype e), whereas NTHi were genetically diverse. Conclusions Invasive Hi disease occurred mainly in elderly and those with underlying conditions, and it was associated with a high mortality rate. NTHi were the most common cause of invasive disease and showed high genetic diversity.

The human respiratory tract contains a highly adapted microbiota including commensal and opportunistic pathogens. Noncapsulated or nontypable Haemophilus influenzae (NTHi) is a human-restricted member of the normal airway microbiota in healthy carriers and an opportunistic pathogen in immunocompromised individuals. The duality of NTHi as a colonizer and as a symptomatic infectious agent is closely related to its adaptation to the host, which in turn greatly relies on the genetic plasticity of the bacterium and is facilitated by its condition as a natural competent. The variable genotype of NTHi accounts for its heterogeneous gene expression and variable phenotype, leading to differential host-pathogen interplay among isolates. Here we review our current knowledge of NTHi diversity in terms of genotype, gene expression, antigenic variation, and the phenotypes associated with colonization and pathogenesis. The potential benefits of NTHi diversity studies discussed herein include the unraveling of pathogenicity clues, the generation of tools to predict virulence from genomic data, and the exploitation of a unique natural system for the continuous monitoring of long-term bacterial evolution in human airways exposed to noxious agents. Finally, we highlight the challenge of monitoring both the pathogen and the host in longitudinal studies, and of applying comparative genomics to clarify the meaning of the vast NTHi genetic diversity and its translation to virulence phenotypes.

NotI, the most prevalent restriction enzyme used for typing Moraxella catarrhalis , failed to digest genomic DNA from respiratory samples. An improved pulsed-field gel electrophoresis (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.

The birth rate in Spain has increased due to the continuous rise in the number of immigrants. Ethnic origin and socioeconomic position can be determining factors in differences in maternal and child health. The aim of the present study was to determine the possible existence of differences in neonatal diagnoses according to parental ethnic origin.We performed a retrospective, cross-sectional study of all live newborns delivered in Hospital del Mar (Barcelona) between 2003 and 2005. The variables studied were risk of admission, diagnostic classes, and parental ethnic group.Of the 2118 newborns included in this study, 46.7% were of immigrant origin (mainly from Central and South America) and 6.4% were gypsies. More than 60% of the 1445 admitted newborns were included in the diagnostic class of risk or suspicion of infection. The risk of pregnancy with little or no prenatal care was higher in non-native and gypsy newborns (OR = 2.58; 95%CI: 1.76-3.77, and OR = 5.84; 95%CI: 3.45-9.90, respectively). The risk of low birth weight and maternal drug use were lower in non-native newborns (OR = 0.17; 95%CI: 0.03-0.90, and OR = 0.12; 95%CI: 0.03-0.44, respectively).Differences in non-native and gypsy newborns compared with native newborns are not due to imported or genetic diseases but are probably due to differences in the social and cultural environment during pregnancy. Preventive measures should be promoted and reinforced and access to and the quality of primary care should be improved in these mothers and their infants.