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The bacterial speciesMoraxella catarrhalishas been hypothesized as being composed of two distinct lineages (referred to as the seroresistant [SR] and serosensitive [SS]) with separate evolutionary histories based on several molecular typing methods, whereas 16S ribotyping has suggested an additional split within the SS lineage. Previously, we characterized whole-genome sequences of 12 SR-lineage isolates, which revealed a relatively small supragenome when compared with other opportunistic nasopharyngeal pathogens, suggestive of a relatively short evolutionary history. Here, we performed whole-genome sequencing on 18 strains from both ribotypes of the SS lineage, an additional SR strain, as well as four previously identified highly divergent strains based on multilocus sequence typing analyses. All 35 strains were subjected to a battery of comparative genomic analyses which clearly show that there are three lineages-the SR, SS, and the divergent. The SR and SS lineages are closely related, but distinct from each other based on three different methods of comparison: Allelic differences observed among core genes; possession of lineage-specific sets of core and distributed genes; and by an alignment of concatenated core sequences irrespective of gene annotation. All these methods show that the SS lineage has much longer interstrain branches than the SR lineage indicating that this lineage has likely been evolving either longer or faster than the SR lineage. There is evidence of extensive horizontal gene transfer (HGT) within both of these lineages, and to a lesser degree between them. In particular, we identified very high rates of HGT between these two lineages for ß-lactamase genes. The four divergent strains aresui generis, being much more distantly related to both the SR and SS groups than these other two groups are to each other. Based on average nucleotide identities, gene content, GC content, and genome size, this group could be considered as a separate taxonomic group. The SR and SS lineages, although distinct, clearly form a single species based on multiple criteria including a large common core genome, average nucleotide identity values, GC content, and genome size. Although neither of these lineages arose from within the other based on phylogenetic analyses, the question of how and when these lineages split and then subsequently reunited in the human nasopharynx is explored., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Objectives: In blood culture-proven pneumococcal infections, streamlining empirical therapy to monotherapy with a penicillin is preferred in order to reduce the use of broad-spectrum antibiotics. However, adherence to this international recommendation is poor, and curiously it is unclear whether antibiotic streamlining may be harmful to individual patients. We investigated whether streamlining in bacteraemic pneumococcal infections is associated with mortality., Methods: Adults admitted to two Dutch hospitals between 2001 and 2011 with bacteraemic pneumococcal infections were retrospectively included. Detailed clinical data on patient characteristics, comorbidities and severity and outcome of disease were obtained in addition to data on antibiotic treatment. Those eligible for streamlining were selected for further analyses., Results: In the 45.8% of cases (126 of 275) where antibiotic treatment was streamlined, a lower mortality rate was observed (6.3% versus 15.4%, P = 0.021). The decision to streamline was only marginally explained by the 38 determinants accounted for. After correction for potential confounders, the OR for death while streamlining was 0.45 (95% CI: 0.18-1.11, P = 0.082) in all cases and 0.35 (95% CI: 0.12-0.99, P = 0.048) specifically in pneumonia cases., Conclusions: Our results suggest that streamlining in eligible pneumococcal bacteraemia cases is safe, irrespective of patient characteristics, severity of disease or empirical treatment regimen., (© The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Nontypeable Haemophilus influenzae (NTHi) is a Gram-negative microbe that frequently colonizes the human host without obvious signs of inflammation, but is also a frequent cause of otitis media in children and exacerbations in chronic obstructive pulmonary disease patients. Accumulating data suggest that NTHi can reside in biofilms during both colonization and infection. Recent literature proposes roles for phosphorylcholine, sialic acid, bacterial DNA, but also eukaryotic DNA in the development of NTHi biofilms. However, many questions remain. Until now, there are insufficient data to explain how NTHi forms biofilms. Here, we review the recent advances in NTHi biofilm formation with particular focus on the role that neutrophils may play in this process. We propose that recruitment of neutrophils facilitates NTHi biofilm formation on mucosal sites by the initiation of neutrophil extracellular traps., (© 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

Invasive aspergillosis is a major threat to patients with chronic granulomatous disease (CGD). Fungal pathogenesis is the result of a diminished antifungal capacity and dysregulated inflammation. A deficient NADPH-oxidase complex results in defective phagolysosomal alkalization. To investigate the contribution of defective pH regulation in phagocytes among patients with CGD during fungal pathogenesis, we evaluated the effect of the acidotropic, antimalarial drug chloroquine (CQ) on the antifungal capacity of polymorphonuclear cells (PMNs) and on the inflammatory response of peripheral blood mononuclear cells (PBMCs). Chloroquine exerted a direct pH-dependent antifungal effect on Aspergillus fumigatus and Aspergillus nidulans; it increased the antifungal activity of PMNs from patients with CGD at a significantly lower concentration, compared with the concentration for PMNs from healthy individuals; and decreased the hyperinflammatory state of PBMCs from patients with CGD, as observed by decreased tumor necrosis factor α and interleukin 1β release. Chloroquine targets both limbs of fungal pathogenesis and might be of great value in the clearance of invasive aspergillosis in patients with CGD.

Myasthenia gravis is a paralytic disorder with autoantibodies against acetylcholine receptors at the neuromuscular junction. A proportion of patients instead has antibodies against muscle-specific kinase, a protein essential for acetylcholine receptor clustering. These are generally of the immunoglobulin-G4 subclass and correlate with disease severity, suggesting specific myasthenogenic activity. However, immunoglobulin-G4 subclass antibodies are generally considered to be 'benign' and direct proof for their pathogenicity in muscle-specific kinase myasthenia gravis (or other immunoglobulin-G4-associated disorders) is lacking. Furthermore, the exact electrophysiological synaptic defects caused at neuromuscular junctions by human anti-muscle-specific kinase autoantibodies are hitherto unknown. We show that purified immunoglobulin-G4, but not immunoglobulin-G1-3, from patients with muscle-specific kinase myasthenia gravis binds to mouse neuromuscular junctions in vitro, and that injection into immunodeficient mice causes paralysis. Injected immunoglobulin-G4 caused reduced density and fragmented area of neuromuscular junction acetylcholine receptors. Detailed electrophysiological synaptic analyses revealed severe reduction of postsynaptic acetylcholine sensitivity, and exaggerated depression of presynaptic acetylcholine release during high-rate activity, together causing the (fatigable) muscle weakness. Intriguingly, compensatory transmitter release upregulation, which is the normal homeostatic response in acetylcholine receptor myasthenia gravis, was absent. This conveys extra vulnerability to neurotransmission at muscle-specific kinase myasthenia gravis neuromuscular junctions. Thus, we demonstrate that patient anti-muscle-specific kinase immunoglobulin-G4 is myasthenogenic, independent of additional immune system components, and have elucidated the underlying electrophysiological neuromuscular junction abnormalities.

Background: Tuberculosis (TB) transmission is associated with patient-related risk factors. However, DNA fingerprint analysis has provided anecdotal evidence suggesting a role for bacteriological factors., Methods: To examine the importance of the bacteriological component in TB transmission, we investigated the number of tuberculin skin test-positive (TST induration, ≥ 10 mm) contacts and secondary cases observed in contact investigations around TB cases in relation to the size of the genotype cluster the patient belonged to at the time of diagnosis. We also compared the number of TST-positive contacts and secondary cases of patients with drug-resistant and drug-susceptible TB., Results: Larger clusters were independently associated with an increased number of positive contacts. The mean number of positive contacts ranged from 3.8 for clusters of 2 cases, to 4.7 for clusters of 3-10 cases, to 6.0 for cases in clusters of >10 cases (mean increase in number of positive contacts for every extra case in the cluster, 0.21; 95% confidence interval, 0.09-0.26). The mean number of positive contacts was significantly lower among index cases with isoniazid-monoresistant TB (1.6) than among index cases with pan-susceptible TB (4.6; relative number, 0.45; 95% confidence interval, 0.22-0.92)., Conclusion: These results suggest that spread of tuberculosis also depends on bacteriological factors.

Pertussis, or whooping cough, is a highly contagious, acute respiratory disease of humans that is caused by the Gram-negative bacterial pathogen Bordetella pertussis. In the face of extensive global vaccination, this extremely monomorphic pathogen has persisted and re-emerged, causing approximately 300,000 deaths each year. In this review, we discuss the interaction of B. pertussis with the host mucosal epithelium and immune system. Using a large number of virulence factors, B. pertussis is able to create a niche for colonization in the human respiratory tract. The successful persistence of this pathogen is mainly due to its ability to interfere with almost every aspect of the immune system, from the inhibition of complement- and phagocyte-mediated killing to the suppression of T- and B-cell responses. Based on these insights, we delineate ideas for the rational design of improved vaccines that can target the 'weak spots' in the pathogenesis of this highly successful pathogen., (© 2011 RIVM. FEMS Microbiology Reviews © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd.)

Background: The prevalence of Streptococcus gallolyticus subsp gallolyticus ( Streptococcus bovis biotype I) endocarditis is in general low but very often linked to colorectal cancer. Therefore, this study aimed to reveal the virulence characteristics that distinguish this opportunistic pathogen from a panel of (closely related) intestinal bacteria., Methods: The route of infection was reconstructed in vitro with adhesion, invasion, and translocation assays on differentiated Caco-2 cells. Furthermore, cellular immune responses upon infection and bacterial biofilm formation were analyzed in a comparative manner., Results: S. gallolyticus subsp gallolyticus strains were demonstrated to have a relative low adhesiveness and could not internalize epithelial cells. However, these bacteria were uniquely able to paracellularly cross a differentiated epithelium without inducing epithelial interleukin 8 or 1β responses. Importantly, they had an outstanding ability to form biofilms on collagen-rich surfaces, which in vivo are found at damaged heart valves and (pre)cancerous sites with a displaced epithelium., Conclusions: Together, these data show that S. gallolyticus subsp gallolyticus has a unique repertoire of virulence factors that facilitate infection through (pre)malignant colonic lesions and subsequently can provide this bacterium with a competitive advantage in (1) evading the innate immune system and (2) forming resistant vegetations at collagen-rich sites in susceptible patients with colorectal cancer., (© The Author 2011. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved.)

Background: Staphylococcus aureus is capable of persistently colonizing the vestibulum nasi. We hypothesized that polymorphisms in host inflammatory response genes and genetic variation in S. aureus contribute to susceptibility to S. aureus carriage and infection., Methods: The prevalence of persistent nasal carriage of S. aureus in 3851 participants aged 61-101 years was 18% (678 of 3851 participants), whereas 73% of volunteers (2804 of 3851) were not colonized. A total of 1270 individuals had boils. Polymorphisms in TNFA (C -863T), IL4 (C -542T), CFH (Tyr402His), and CRP (C1184T, C2042T, and C2911G) were determined. Genetic similarity among 428 S. aureus isolates was determined by use of amplified fragment length polymorphism analysis (AFLP)-mediated genotyping., Results: The IL4 -524 C/C host genotype was associated with an increased risk of persistent S. aureus carriage, irrespective of S. aureus AFLP genotype. The CRP haplotype 1184C; 2042C; 2911C was overrepresented in individuals who were not colonized . In individuals with boils, carriers of the CFH Tyr402 variant, and the CRP 2911 C/C genotype were overrepresented., Conclusion: Persistent carriage of S. aureus is influenced by genetic variation in host inflammatory response genes. As would be expected in multifactorial host-microbe interactions, these effects are limited. Interestingly, host genotype was associated with the carriage of certain S. aureus genotypes. Apparently, a close interaction between host and bacterial determinants are prerequisites for long-term colonization.

Little attention has been paid to pneumococcal carriage and disease in Amerindians from Latin America. The Warao people, an indigenous population from Venezuela, live in the delta of the Orinoco River in geographically isolated communities with difficult access to medical care. To obtain insight into pneumococcal carriage and the theoretical coverage of pneumococcal vaccines in this population, we investigated pneumococcal colonization, serotype, and genotype distribution among Warao children in 9 distinct, geographically isolated communities in the Delta Amacuro area in the northeast of Venezuela. From April 2004 through January 2005, a total of 161 Streptococcus pneumoniae isolates were recovered from single nasopharyngeal swab samples obtained from 356 children aged 0-72 months. The overall pneumococcal carriage rate was 49%, ranging from 13% to 76%, depending on the community investigated and the age of the children (50% among children aged <2 years and 25% among children aged >2 years). The most frequent serotypes were 23F (19.5% of isolates), 6A (19.5%), 15B (10.4%), 6B (9.1%), and 19F (7.2%). The theoretical coverage of the 7-valent pneumococcal conjugate vaccine, including the cross-reactive nonvaccine serotype 6A, was 65%. A total of 26% of the isolates were resistant to first-line antibiotics, with 70% of these strains being covered by the 7-valent pneumococcal conjugate vaccine. Restriction fragment end labelling analysis revealed 65 different genotypes, with 125 (80%) of the isolates belonging to 27 different genetic clusters, suggesting a high degree of horizontal spread of pneumococcal strains in and between the villages. The high colonization rates and high (registered) acute respiratory tract infection morbidity and mortality in this part of Venezuela suggest that Warao children are at increased risk for pneumococcal disease and, therefore, benefit from vaccination.

Background: Identification of new therapeutic targets remains an imperative goal to improve the morbidity and mortality associated with severe sepsis and septic shock. Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine and counterregulator of glucocorticoids, has recently emerged as a critical mediator of innate immunity and experimental sepsis, and it is an attractive new target for the treatment of sepsis., Methods: Circulating concentrations of MIF were measured in 2 clinical trial cohorts of 145 pediatric and adult patients who had severe sepsis or septic shock caused predominantly by infection with Neisseria meningitidis or other gram-negative bacteria, to study the kinetics of MIF during sepsis, to analyze the interplay between MIF and other mediators of sepsis or stress hormones (adrenocorticotropic hormone and cortisol), and to determine whether MIF is associated with patient outcome., Results: Circulating concentrations of MIF were markedly elevated in 96% of children and adults who had severe sepsis or septic shock, and they remained elevated for several days. MIF levels were correlated with sepsis severity scores, presence of shock, disseminated intravascular coagulation, urine output, blood pH, and lactate and cytokine levels. High levels of MIF were associated with a rapidly fatal outcome. Moreover, in meningococcal sepsis, concentrations of MIF were positively correlated with adrenocorticotropic hormone levels and negatively correlated with cortisol levels and the cortisol:adrenocorticotropic hormone ratio, suggesting an inappropriate adrenal response to sepsis., Conclusions: MIF is markedly and persistently up-regulated in children and adults with gram-negative sepsis and is associated with parameters of disease severity, with dysregulated pituitary-adrenal function in meningococcal sepsis, and with early death.

Plasminogen activator inhibitor type 1 (PAI-1) is a 50-kilodalton glycoprotein of the serine protease inhibitor family. The primary role of PAI-1 in vivo is the inhibition of both tissue- and urokinase-type plasminogen activators. In addition to this function, PAI-1 acts as an acute-phase protein during acute inflammation. PAI-1 is a pivotal player in the pathogenesis of sepsis, a complex clinical syndrome that results from a systemic inflammatory response. In patients with sepsis, the levels of PAI-1 are positively related to poor outcome, increased severity of disease, and increased levels of various cytokines, acute-phase proteins, and coagulation parameters. The 4G/5G insertion/deletion promoter polymorphism, which leads to differences in PAI-1 production, has been demonstrated to affect the risk of developing severe complications and dying from sepsis during meningococcal infection and multiple trauma.

We investigated the prevalence and determinants of nasopharyngeal carriage of Neisseria meningitidis in 3200 healthy children aged 1-19 years. The incidence of meningococcal carriage was, on average, 1.5%. Peak incidences were seen at age 1 year and after age 15 years. The independent determinants of meningococcal carriage included age, regular visits to youth clubs (odds ratio [OR], 2.2) and discotheques (OR, 4.3), and pneumococcal carriage (OR, 4.1).

Background: We recently showed that vaccination with a 7-valent pneumococcal conjugate vaccine (PCV7) followed by a 23-valent pneumococcal polysaccharide vaccine (PPSV23) failed to prevent new episodes of acute otitis media (AOM) in previously unvaccinated toddlers and children with a history of recurrent AOM. We describe in detail the impact of pneumococcal vaccinations on nasopharyngeal carriage of S. pneumoniae in this study population., Methods: The impact of vaccination with PCV7 followed by PPSV23 on pneumococcal nasopharyngeal carriage was studied in a prospective, randomized trial involving 383 children (age range, 1-7 years) with previous AOM. Nasopharyngeal swab specimens were collected at the time of first vaccination and at 6-7-month intervals during the 26-month follow-up period., Results: Overall, pneumococcal carriage rates did not diminish, remaining at approximately 50% in both PCV7/PPSV23 and control vaccinees. A significant shift from conjugate vaccine- to nonconjugate vaccine-type pneumococci was observed in children aged 1-2 years, who received the conjugate vaccine twice before the polysaccharide vaccine was administered. Conjugate vaccine serotype carriage was not influenced in older children, who received the conjugate vaccine once before receiving the polysaccharide booster., Conclusions: The administration of conjugate vaccines at least twice also after 2 years of age may be mandatory for reducing the carriage of conjugate vaccine serotypes in children with recurrent AOM. Polysaccharide booster vaccination did not affect nasopharyngeal colonization with serotypes not included in the conjugate vaccine.

A multidrug-resistant strain of Streptococcus pneumoniae was isolated in The Netherlands during a nosocomial outbreak among 36 patients who mainly had chronic obstructive pulmonary disease. After the commencement of barrier nursing and short-term ceftriaxone-rifampin eradication therapy, the epidemic ceased. However, eradication therapy failed in 3 patients, and follow-up investigation of these patients showed the emergence of rifampin-resistant isolates.

The molecular epidemiologic characteristics of penicillin-resistant pneumococci in the Netherlands were investigated in 1995. Dutch electronic surveillance data showed that 0.7% of all pneumococci were intermediately resistant and 0.4% were highly resistant to penicillin. From March 1995 to March 1996, 89 penicillin-resistant isolates were collected by 39 medical microbiology laboratories. Thirty different genotypes were observed by restriction fragment end labeling. Twenty-one DNA types were unique, whereas 9 distinct genotypes were shared by > or = 2 isolates. Different serogroups were found within 6 of the 9 genetically identical clusters of penicillin-resistant isolates, suggesting that horizontal transfer of capsular genes is common. Finally, nosocomial transmission of penicillin-resistant pneumococci was observed among 21 elderly adults with chronic obstructive pulmonary disease. This study demonstrates that multiple clones of penicillin-resistant pneumococci have been introduced in the Netherlands, a country with a low prevalence of pneumococcal infection. Some clones spread among the population in and outside hospitals.

The genetic heterogeneity among Mycobacterium tuberculosis isolates from 501 patients in Ethiopia, Tunisia, and the Netherlands was compared by analysis of DNA polymorphism driven by insertion element IS6110. The percentage of isolates displaying two or more identical patterns differed greatly in the three countries: It was highest among Tunisian isolates and lowest in Dutch isolates. In contrast to isolates from Dutch subjects infected with M. tuberculosis, the majority of strains from Ethiopia and Tunisia were from a few families of genetically highly related strains. Furthermore, little overlap was observed among isolates from the three countries, indicating strict isolation of the bacterial reservoirs in the countries. A few strains from the Netherlands matched strains from Ethiopia and Tunisia. Those strains were invariably isolated from refugees, immigrants, or persons who visited Ethiopia or Tunisia.