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Competence development in the human pathogen Streptococcus pneumoniae controls several features such as genetic transformation, biofilm formation, and virulence. Competent bacteria produce so-called "fratricins" such as CbpD that kill noncompetent siblings by cleaving peptidoglycan (PGN). CbpD is a choline-binding protein (CBP) that binds to phosphorylcholine residues found on wall and lipoteichoic acids (WTA and LTA) that together with PGN are major constituents of the pneumococcal cell wall. Competent pneumococci are protected against fratricide by producing the immunity protein ComM. How competence and fratricide contribute to virulence is unknown. Here, using a genome-wide CRISPRi-seq screen, we show that genes involved in teichoic acid (TA) biosynthesis are essential during competence. We demonstrate that LytR is the major enzyme mediating the final step in WTA formation, and that, together with ComM, is essential for immunity against CbpD. Importantly, we show that key virulence factors PspA and PspC become more surface-exposed at midcell during competence, in a CbpD-dependent manner. Together, our work supports a model in which activation of competence is crucial for host adherence by increased surface exposure of its various CBPs. Competence development in the human pathogen Streptococcus pneumoniae controls several features such as genetic transformation, biofilm formation and virulence. This study shows that competence activation is crucial for optimal host adherence, via a mechanism dependent on the fratricin CbpD, which exposes virulence factors to the cell surface. [ABSTRACT FROM AUTHOR]

Streptococcus pneumoniae (pneumococcus) is an opportunistic pathogen that causes otitis media, sinusitis, pneumonia, meningitis and sepsis. The progression to this pathogenic lifestyle is preceded by asymptomatic colonization of the nasopharynx. This colonization is associated with biofilm formation; the competence pathway influences the structure and stability of biofilms. However, the molecules that link the competence pathway to biofilm formation are unknown. Here, we describe a new competence-induced gene, called briC, and demonstrate that its product promotes biofilm development and stimulates colonization in a murine model. We show that expression of briC is induced by the master regulator of competence, ComE. Whereas briC does not substantially influence early biofilm development on abiotic surfaces, it significantly impacts later stages of biofilm development. Specifically, briC expression leads to increases in biofilm biomass and thickness at 72h. Consistent with the role of biofilms in colonization, briC promotes nasopharyngeal colonization in the murine model. The function of BriC appears to be conserved across pneumococci, as comparative genomics reveal that briC is widespread across isolates. Surprisingly, many isolates, including strains from clinically important PMEN1 and PMEN14 lineages, which are widely associated with colonization, encode a long briC promoter. This long form captures an instance of genomic plasticity and functions as a competence-independent expression enhancer that may serve as a precocious point of entry into this otherwise competence-regulated pathway. Moreover, overexpression of briC by the long promoter fully rescues the comE-deletion induced biofilm defect in vitro, and partially in vivo. These findings indicate that BriC may bypass the influence of competence in biofilm development and that such a pathway may be active in a subset of pneumococcal lineages. In conclusion, BriC is a part of the complex molecular network that connects signaling of the competence pathway to biofilm development and colonization. [ABSTRACT FROM AUTHOR]

Streptococcus pneumoniae becomes competent for genetic transformation when exposed to an autoinducer peptide known as competence-stimulating peptide (CSP). This peptide was originally described as a quorum-sensing signal, enabling individual cells to regulate competence in response to population density. However, recent studies suggest that CSP may instead serve as a probe for sensing environmental cues, such as antibiotic stress or environmental diffusion. Here, we show that competence induction can be simultaneously influenced by cell density, external pH, antibiotic-induced stress, and cell history. Our experimental data is explained by a mathematical model where the environment and cell history modify the rate at which cells produce or sense CSP. Taken together, model and experiments indicate that autoinducer concentration can function as an indicator of cell density across environmental conditions, while also incorporating information on environmental factors or cell history, allowing cells to integrate cues such as antibiotic stress into their quorum-sensing response. This unifying perspective may apply to other debated quorum-sensing systems. [ABSTRACT FROM AUTHOR]

The diverse pneumococcal diseases are associated with different pneumococcal lineages, or clonal complexes. Nevertheless, intra-clonal genomic variability, which influences pathogenicity, has been reported for surface virulence factors. These factors constitute the communication interface between the pathogen and its host and their corresponding genes are subjected to strong selective pressures affecting functionality and immunogenicity. First, the presence and allelic dispersion of 97 outer protein families were screened in 19 complete pneumococcal genomes. Seventeen families were deemed variable and were then examined in 216 draft genomes. This procedure allowed the generation of binary vectors with 17 positions and the classification of strains into surfotypes. They represent the outer protein subsets with the highest inter-strain discriminative power. A total of 116 non-redundant surfotypes were identified. Those sharing a critical number of common protein features were hierarchically clustered into 18 surfogroups. Most clonal complexes with comparable epidemiological characteristics belonged to the same or similar surfogroups. However, the very large CC156 clonal complex was dispersed over several surfogroups. In order to establish a relationship between surfogroup and pathogenicity, the surfotypes of 95 clinical isolates with different serogroup/serotype combinations were analyzed. We found a significant correlation between surfogroup and type of pathogenic behavior (primary invasive, opportunistic invasive, and non-invasive). We conclude that the virulent behavior of S. pneumoniae is related to the activity of collections of, rather than individual, surface virulence factors. Since surfotypes evolve faster than MLSTs and directly reflect virulence potential, this novel typing protocol is appropriate for the identification of emerging clones. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Capsular switching allows pre-existing clones of Streptococcus pneumoniae expressing vaccine serotypes to escape the vaccine-induced immunity by acquisition of capsular genes from pneumococci of a non-vaccine serotype. Here, we have analysed the clonal composition of 492 clinical isolates of serotype 11A causing invasive disease in Spain (2000–2012), and their ability to evade the host immune response. Antibiograms, serotyping and molecular typing were performed. The restriction profiles of pbp2x, pbp1a and pbp2b genes were also analysed. Interaction with the complement components C1q, C3b, C4BP, and factor H was explored whereas opsonophagocytosis assays were performed using a human cell line differentiated to neutrophils. Biofilm formation and the polymorphisms of the major autolysin LytA were evaluated. The main genotypes of the 11A pneumococci were: ST62 (447 isolates, 90.6%), followed by ST6521 (35 isolates, 7.3%) and ST838 (10 isolates, 2.1%). Beta lactam resistant serotype 11A variants of genotypes ST838 and ST6521 closely related to the Spain9V-ST156 clone were first detected in 2005. A different pattern of evasion of complement immunity and phagocytosis was observed between genotypes. The emergence of one vaccine escape variant of Spain9V-ST156 (ST652111A), showing a high potential to avoid the host immune response, was observed. In addition, isolates of ST652111A showed higher ability to produce biofilms than ST83811A or ST6211A, which may have contributed to the emergence of this PEN-resistant ST652111A genotype in the last few years. The emergence of penicillin-resistant 11A invasive variants of the highly successful ST156 clonal complex merits close monitoring. [ABSTRACT FROM AUTHOR]

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 24 h. 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Objectives: To analyze the role of the capsular type in pneumococci causing relapse and reinfection episodes of acute exacerbation in COPD patients. Methods: 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. Results: 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). Conclusions: 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. [ABSTRACT FROM AUTHOR]

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 CO2-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. [ABSTRACT FROM AUTHOR]

Background: Adult invasive pneumococcal disease (IPD) occurs mainly in the elderly and patients with co-morbidities. Little is known about the clinical characteristics, serotypes and genotypes causing IPD in healthy adults. Methods: We studied 745 culture-proven cases of IPD in adult patients aged 18--64 years (1996--2010). Patients were included in two groups: 1.) adults with co- morbidities, and 2.) healthy adults, who had no prior or coincident diagnosis of a chronic or immunosuppressive underlying disease. Microbiological studies included pneumococcal serotyping and genotyping. Results: Of 745 IPD episodes, 525 (70%) occurred in patients with co-morbidities and 220 (30%) in healthy adults. The healthy adults with IPD were often smokers (56%) or alcohol abusers (18%). As compared to patients with co-morbidities, the healthy adults had (P,0.05): younger age (43.5+/213.1 vs. 48.7+/211.3 years); higher proportions of women (45% vs. 24%), pneumonia with empyema (15% vs. 7%) and infection with non- PCV7 serotypes including serotypes 1 (25% vs. 5%), 7F (13% vs. 4%), and 5 (7% vs. 2%); and lower mortality (5% vs. 20%). Empyema was more frequently caused by serotype 1. No death occurred among 79 patients with serotype 1 IPD. There was an emergence of virulent clonal-types Sweden¹-ST306 and Netherlands7F-ST191. The vaccine serotype coverage with the PCV13 was higher in healthy adults than in patients with co-morbidities: 82% and 56%, respectively, P,0.001. Conclusion: In this clinical study, one-third of adults with IPD had no underlying chronic or immunosuppressive diseases (healthy adults). They were often smokers and alcohol abusers, and frequently presents with pneumonia and empyema caused by virulent clones of non-PCV7 serotypes such as the Sweden¹-ST306. Thus, implementing tobacco and alcohol abuse- cessation measures and a proper pneumococcal vaccination, such as PCV13 policy, in active smokers and alcohol abusers may diminish the burden of IPD in adults. [ABSTRACT FROM AUTHOR]