Meningococcal Two-Partner Secretion Systems and Their Association with Outcome in Patients with Meningitis.

Two-partner secretion (TPS) systems export large TpsA proteins to the surface and extracellular milieu. In meningococci, three different TPS systems exist, and of these, TPS system 2 (TPS2) and TPS3 can be detected by the host's immune system. We evaluated the distribution of TPS systems among clinical isolates from two prospective cohort studies comprising 373 patients with meningococcal meningitis. TPS system 1 was present in 91% of isolates, and system 2 and/or 3 was present in 67%. The TPS system distribution was related to clonal complexes. Infection with strains with TPS2 and/or TPS3 resulted in less severe disease and better outcomes than infection with strains without these systems. Using whole-blood stimulation experiments, we found no differences in the host cytokine response between patients infected with TPS system 2 and 3 knockout strains and patients infected with a wild-type strain. In conclusion, meningococcal TPS system 2 and/or 3 is associated with disease severity and outcome in patients with meningitis.

Streptococcus pneumoniae arginine synthesis genes promote growth and virulence in pneumococcal meningitis.

Streptococcus pneumoniae (pneumococcus) is a major human pathogen causing pneumonia, sepsis and bacterial meningitis. Using a clinical phenotype based approach with bacterial whole-genome sequencing we identified pneumococcal arginine biosynthesis genes to be associated with outcome in patients with pneumococcal meningitis. Pneumococci harboring these genes show increased growth in human blood and cerebrospinal fluid (CSF). Mouse models of meningitis and pneumonia showed that pneumococcal strains without arginine biosynthesis genes were attenuated in growth or cleared, from lung, blood and CSF. Thus, S. pneumoniae arginine synthesis genes promote growth and virulence in invasive pneumococcal disease.

Effect of growth rate and selection pressure on rates of transfer of an antibiotic resistance plasmid between E. coli strains.

Antibiotic resistance increases costs for health care and causes therapy failure. An important mechanism for spreading resistance is transfer of plasmids containing resistance genes and subsequent selection. Yet the factors that influence the rate of transfer are poorly known. Rates of plasmid transfer were measured in co-cultures in chemostats of a donor and a acceptor strain under various selective pressures. To document whether specific mutations in either plasmid or acceptor genome are associated with the plasmid transfer, whole genome sequencing was performed. The DM0133 TetR tetracycline resistance plasmid was transferred between Escherichia coli K-12 strains during co-culture at frequencies that seemed higher at increased growth rate. Modeling of the take-over of the culture by the transformed strain suggests that in reality more transfer events occurred at low growth rates. At moderate selection pressure due to an antibiotic concentration that still allowed growth, a maximum transfer frequency was determined of once per 10(11) cell divisions. In the absence of tetracycline or in the presence of high concentrations the frequency of transfer was sometimes zero, but otherwise reduced by at least a factor of 5. Whole genome sequencing showed that the plasmid was transferred without mutations, but two functional mutations in the genome of the recipient strain accompanied this transfer. Exposure to concentrations of antibiotics that fall within the mutant selection window stimulated transfer of the resistance plasmid most.

Full genome sequences of all nine Chlamydia psittaci genotype reference strains.

Chlamydia psittaci primarily infects birds, but zoonotic transmission occurs in people in close contact with infected birds. The clinical outcome ranges from inapparent disease to pneumonia. Here we report the genome sequences of all 9 Chlamydia psittaci genotype reference strains.

Clinical features, outcome, and meningococcal genotype in 258 adults with meningococcal meningitis: a prospective cohort study.

Meningococcal meningitis remains a life-threatening disease. Neisseria meningitidis is the leading cause of meningitis and septicemia in young adults and is a major cause of endemic bacterial meningitis worldwide. The Meningitis Cohort Study was a Dutch nationwide prospective observational cohort study of adults with community-acquired bacterial meningitis, confirmed by culture of cerebrospinal fluid, from October 1998 to April 2002. Patients underwent a neurologic examination at discharge, and outcome was graded with the Glasgow Outcome Scale. Serogrouping, multi-locus sequence typing, and susceptibility testing of meningococcal isolates were performed. The study identified 258 episodes of meningococcal meningitis in 258 patients. The prevalence of the classical triad of fever, neck stiffness, and change in mental status was low (70/258, 27%). When rash was added to the classical triad, 229 of 258 (89%) patients had at least 2 of 4 signs. Systolic hypotension was associated with rash (22/23 vs. 137/222, p = 0.002) and absence of neck stiffness (6/23 vs. 21/220, p = 0.05). Neuroimaging before lumbar puncture was an important cause of delay of therapy: antibiotics were not initiated before computed tomography (CT) scan in 85% of patients who underwent CT scan before lumbar puncture. Unfavorable outcome occurred in 30 of 258 (12%) patients, including a mortality rate of 7%. Neurologic sequelae occurred in 28 of 238 (12%) patients, particularly hearing loss (8%). Factors associated with sepsis and infection with meningococci of clonal complex 11 (cc11) are related with unfavorable outcome.

Meningitis caused by a lipopolysaccharide deficient Neisseria meningitidis.

OBJECTIVE: Lipopolysaccharide (LPS) is a major component of the Neisseria meningitidis outer membrane. Here we report a patient with meningococcal meningitis of which the causative isolate lacked LPS. Thus far, no naturally occurring LPS-deficient meningococcal isolate has been known to cause clinical disease. METHODS: We used SDS-PAGE, silver staining and LPS-specific antibodies in whole cell ELISA to determine LPS presence in the causative isolate. Meningococcal whole genome sequencing was performed using Roche 454-sequencing. The N. meningitidis strain MC58 was used to compare all LPS biosynthesis associated genes. We compared growth characteristics of Escherichia coli transformed with a plasmid containing 2 lpxH types. RESULTS: The patient presented with isolated thunderclap headache. Analysis of the causative N. meningitidis showed no LPS. Whole genome sequencing revealed a mutation located in lpxH explaining LPS-deficiency. Expression of this lpxH variant in E. coli resulted in growth impairment compared to E. coli expressing the meningococcal wild type lpxH variant. In addition, inactivating lpxH in N. meningitidis H44/76 by insertional inactivation with a kanamycin cassette resulted in a LPS-deficient phenotype. CONCLUSIONS: We describe invasive meningococcal disease caused by a naturally occurring LPS-deficient meningococcal isolate.

Meningococcal factor H binding protein fHbpd184 polymorphism influences clinical course of meningococcal meningitis.

Factor H Binding protein (fHbp) is an important meningococcal virulence factor, enabling the meningococcus to evade the complement system, and a main target for vaccination. Recently, the structure of fHBP complexed with factor H (fH) was published. Two fHbp glutamic acids, E(283) and E(304), form salt bridges with fH, influencing interaction between fHbp and fH. Fifteen amino acids were identified forming hydrogen bonds with fH. We sequenced fHbp of 254 meningococcal isolates from adults with meningococcal meningitis included in a prospective clinical cohort to study the effect of fHbp variants on meningococcal disease severity and outcome. All fHbp of subfamily A had E304 substituted with T304. Of the 15 amino acids in fHbp making hydrogen bonds to fH, 3 were conserved, 11 show a similar distribution between the two fHbp subfamilies as the polymorphism at position 304. The proportion of patients infected with meningococci with fHbp of subfamily A with unfavorable outcome was 2.5-fold lower than that of patients infected with meningococci with fHbp of subfamily B (2 of 40 (5%) vs. 27 of 213 (13%) (P = 0.28). The charge of 2 of 15 amino acids (at position 184 and 306) forming hydrogen bonds was either basic or acidic. The affinity of fHbp(K184) and of fHbp(D184) for recombinant purified human fH was assessed by Surface Plasmon Resonance and showed average K(D) of 2.60×10(-8) and 1.74×10(-8), respectively (ns). Patients infected with meningococci with fHbp(D184) were more likely to develop septic shock during admission (11 of 42 [26%] vs. 19 of 211 [9%]; P = 0.002) resulting in more frequent unfavorable outcome (9 of 42 [21%] vs. 20 of 211 [10%]; P = 0.026). In conclusion, we dentified fHBP(D184) to be associated with septic shock in patients with meningococcal meningitis.