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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.

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.

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.

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.