Dominique A. Caugant

Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis—One Species on the Basis of Genetic Evidence

ABSTRACT Bacillus anthracis, Bacillus cereus, andBacillus thuringiensis are members of the Bacillus cereus group of bacteria, demonstrating widely different phenotypes and pathological effects. B. anthracis causes the acute fatal disease anthrax and is a potential biological weapon due to its high toxicity. B. thuringiensis produces intracellular protein crystals toxic to a wide number of insect larvae and is the most commonly used biological pesticide worldwide. B. cereus is a probably ubiquitous soil bacterium and an opportunistic pathogen that is a common cause of food poisoning. In contrast to the differences in phenotypes, we show by multilocus enzyme electrophoresis and by sequence analysis of nine chromosomal genes thatB. anthracis should be considered a lineage of B. cereus. This determination is not only a formal matter of taxonomy but may also have consequences with respect to virulence and the potential of horizontal gene transfer within the B. cereus group.

NadA, a Novel Vaccine Candidate of Neisseria meningitidis

Neisseria meningitidis is a human pathogen, which, in spite of antibiotic therapy, is still a major cause of mortality due to sepsis and meningitis. Here we describe NadA, a novel surface antigen of N. meningitidis that is present in 52 out of 53 strains of hypervirulent lineages electrophoretic types (ET) ET37, ET5, and cluster A4. The gene is absent in the hypervirulent lineage III, in N. gonorrhoeae and in the commensal species N. lactamica and N. cinerea. The guanine/cytosine content, lower than the chromosome, suggests acquisition by horizontal gene transfer and subsequent limited evolution to generate three well-conserved alleles. NadA has a predicted molecular structure strikingly similar to a novel class of adhesins (YadA and UspA2), forms high molecular weight oligomers, and binds to epithelial cells in vitro supporting the hypothesis that NadA is important for host cell interaction. NadA induces strong bactericidal antibodies and is protective in the infant rat model suggesting that this protein may represent a novel antigen for a vaccine able to control meningococcal disease caused by three hypervirulent lineages.

Population genetics and molecular epidemiology of Neisseria meningitidis

Under non‐epidemic conditions, Neisseria meningitidis causes disease primarily in children under the age of 5 and the cases are sporadic without any evident relationship between them. Occasionally, localized outbreaks of meningococcal disease occur, and sometimes epidemic waves of disease may spread to several countries or even continents and constitute a pandemic. In the past 10 years or so, population genetic analyses have provided insights into the biology of the bacterium and the epidemiology of meningococcal disease, improving our understanding of the cause of epidemics. Through the application of molecular methods, and especially multilocus enzyme electrophoresis, to N. meningitidis strains of worldwide origin, it has been possible to identify virulent clones and provide a surveillance system to warn of meningococcal epidemics. The characteristics of the predominant clones which are nowadays causing meningococcal disease in the world are summarized here and the importance of population genetics in interpreting the epidemiological data is illustrated.

Multilocus sequence typing scheme for bacteria of the Bacillus cereus group

ABSTRACT In this study we developed a multilocus sequence typing (MLST) scheme for bacteria of the Bacillus cereus group. This group, which includes the species B. cereus, B. thuringiensis, B. weihenstephanensis, and B. anthracis, is known to be genetically very diverse. It is also very important because it comprises pathogenic organisms as well as bacteria with industrial applications. The MLST system was established by using 77 strains having various origins, including humans, animals, food, and soil. A total of 67 of these strains had been analyzed previously by multilocus enzyme electrophoresis, and they were selected to represent the genetic diversity of this group of bacteria. Primers were designed for conserved regions of housekeeping genes, and 330- to 504-bp internal fragments of seven such genes, adk, ccpA, ftsA, glpT, pyrE, recF, and sucC, were sequenced for all strains. The number of alleles at individual loci ranged from 25 to 40, and a total of 53 allelic profiles or sequence types (STs) were distinguished. Analysis of the sequence data showed that the population structure of the B. cereus group is weakly clonal. In particular, all five B. anthracis isolates analyzed had the same ST. The MLST scheme which we developed has a high level of resolution and should be an excellent tool for studying the population structure and epidemiology of the B. cereus group.

Meningococcal carriage and disease—Population biology and evolution

Meningococcal disease occurs worldwide with incidence rates varying from 1 to 1000 cases per 100,000. The causative organism, Neisseria meningitidis, is an obligate commensal of humans, which normally colonizes the mucosa of the upper respiratory tract without causing invasive disease, a phenomenon known as carriage. Studies using molecular methods have demonstrated the extensive genetic diversity of meningocococci isolated from carriers, in contrast to a limited number of genetic types, known as the hyperinvasive lineages, associated with invasive disease. Population and evolutionary models that invoke positive selection can be used to resolve the apparent paradox of virulent lineages persisting during the global spread of a non-clonal and normally commensal bacterium. The application of insights gained from studies of meningococcal population biology and evolution is important in understanding the spread of disease, as well as in vaccine development and implementation, especially with regard to the challenge of producing comprehensive vaccines based on sub-capsular antigens and measuring their effectiveness.

Distribution of Serogroups and Genotypes among Disease-Associated and Carried Isolates of Neisseria meningitidis from the Czech Republic, Greece, and Norway

ABSTRACT The distribution of serogroups and multilocus sequence types (STs) in collections of disease-associated and carried meningococci from the period 1991 to 2000 in three European countries (the Czech Republic, Greece, and Norway) was investigated. A total of 314 patient isolates and 353 isolates from asymptomatic carriers were characterized. The frequency distributions of serogroups and clone complexes differed among countries and between disease and carrier isolate collections. Highly significant differentiation was seen at each housekeeping locus. A marked positive association of serogroup C with disease was evidenced. The ST-11 complex was strongly positively associated with disease; associations for other clone complexes were weaker. The genetic diversity of the clone complexes differed. A single ST dominated the ST-11 clone complex, while the ST-41/44 complex exhibited greater levels of diversity. These data robustly demonstrated differences in the distribution of meningococcal genotypes in disease and carrier isolates and among countries. Further, they indicated that differences in genotype diversity and pathogenicity exist between meningococcal clone complexes.

Predicted strain coverage of a meningococcal multicomponent vaccine (4CMenB) in Europe: a qualitative and quantitative assessment.

BACKGROUND A novel multicomponent vaccine against meningococcal capsular group B (MenB) disease contains four major components: factor-H-binding protein, neisserial heparin binding antigen, neisserial adhesin A, and outer-membrane vesicles derived from the strain NZ98/254. Because the public health effect of the vaccine, 4CMenB (Novartis Vaccines and Diagnostics, Siena, Italy), is unclear, we assessed the predicted strain coverage in Europe. METHODS We assessed invasive MenB strains isolated mainly in the most recent full epidemiological year in England and Wales, France, Germany, Italy, and Norway. Meningococcal antigen typing system (MATS) results were linked to multilocus sequence typing and antigen sequence data. To investigate whether generalisation of coverage applied to the rest of Europe, we also assessed isolates from the Czech Republic and Spain. FINDINGS 1052 strains collected from July, 2007, to June, 2008, were assessed from England and Wales, France, Germany, Italy, and Norway. All MenB strains contained at least one gene encoding a major antigen in the vaccine. MATS predicted that 78% of all MenB strains would be killed by postvaccination sera (95% CI 63-90, range of point estimates 73-87% in individual country panels). Half of all strains and 64% of covered strains could be targeted by bactericidal antibodies against more than one vaccine antigen. Results for the 108 isolates from the Czech Republic and 300 from Spain were consistent with those for the other countries. INTERPRETATION MATS analysis showed that a multicomponent vaccine could protect against a substantial proportion of invasive MenB strains isolated in Europe. Monitoring of antigen expression, however, will be needed in the future. FUNDING Novartis Vaccines and Diagnostics.

Sequence Diversity of the Factor H Binding Protein Vaccine Candidate in Epidemiologically Relevant Strains of Serogroup B Neisseria meningitidis

BACKGROUND Recombinant forms of Neisseria meningitidis human factor H binding protein (fHBP) are undergoing clinical trials in candidate vaccines against invasive meningococcal serogroup B disease. We report an extensive survey and phylogenetic analysis of the diversity of fhbp genes and predicted protein sequences in invasive clinical isolates obtained in the period 2000-2006. METHODS Nucleotide sequences of fhbp genes were obtained from 1837 invasive N. meningitidis serogroup B (MnB) strains from the United States, Europe, New Zealand, and South Africa. Multilocus sequence typing (MLST) analysis was performed on a subset of the strains. RESULTS Every strain contained the fhbp gene. All sequences fell into 1 of 2 subfamilies (A or B), with 60%-75% amino acid identity between subfamilies and at least 83% identity within each subfamily. One fHBP sequence may have arisen via inter-subfamily recombination. Subfamily B sequences were found in 70% of the isolates, and subfamily A sequences were found in 30%. Multiple fHBP variants were detected in each of the common MLST clonal complexes. All major MLST complexes include strains in both subfamily A and subfamily B. CONCLUSIONS The diversity of strains observed underscores the importance of studying the distribution of the vaccine antigen itself rather than relying on common epidemiological surrogates such as MLST.

Spread of Drug-Resistant Mycobacterium tuberculosis Strains of the Beijing Genotype in the Archangel Oblast, Russia

ABSTRACT A collection of 119 strains of Mycobacterium tuberculosis isolated from patients with pulmonary tuberculosis in the Archangel Oblast, Russia, in 1998 and 1999 were studied by using restriction fragment length polymorphism (RFLP) analysis with the IS6110 probe and spoligotyping. Resistance of the strains to antituberculosis drugs was analyzed by the BACTEC method, and mutations associated with rifampin resistance were detected by using the Inno-LiPA Rif. TB test. RFLP analysis and spoligotyping demonstrated that 53 (44.5%) of the strains belonged to the Beijing genotype. These strains showed a significantly higher rate of resistance than M. tuberculosis strains of other genotypes circulating in the region. In particular, 43.4% of the strains of the Beijing genotype were multidrug resistant; in contrast, only 10.6% of the other strains were. Of the strains of the Beijing genotype, 92.5% were part of a cluster, while only 33.3% of the remaining strains were clustered. Analysis of the medical records of the patients demonstrated that individuals infected with a strain of the Beijing genotype were significantly more likely to be alcohol abusers and to have chronic obstructive pulmonary disease prior to the tuberculosis diagnosis. Multivariate analysis showed that both variables were independently associated with infection by strains belonging to the Beijing genotype. Our study demonstrated that strains of the Beijing genotype are an important cause of tuberculosis in the Archangel Oblast and that dissemination of these strains is associated with the high incidence of drug resistance.

Effectiveness of a 2+1 dose schedule pneumococcal conjugate vaccination programme on invasive pneumococcal disease among children in Norway

The 7-valent pneumococcal conjugate vaccine (PCV-7) was licensed in Norway in 2001. In July 2006, PCV-7 was introduced in the Norwegian Childhood Vaccination Programme in a 2+1 dose schedule, with immunizations administered at 3, 5 and 12 months of age. PCV-7 was offered through the vaccination programme to all children born from January 2006, i.e. a catch-up for children aged 3-6 months. Prior to 2006 the use of PCV-7 was negligible. The effectiveness of the PCV-7 vaccination programme was assessed using data on invasive pneumococcal disease (IPD) incidence obtained from the Norwegian Surveillance System for Communicable Diseases, serotype distribution from the National Reference Laboratory for Pneumococci, and vaccine coverage and vaccination status from the Norwegian National Vaccination Register. Vaccine coverage quickly reached high levels; 95% of children >3 months born from January 2006 had received at least one immunization with PCV-7. The incidence rate of IPD among children <2 years rapidly declined; the rate of vaccine serotype IPD in this age group fell from an average of 47.1 cases/100,000 population in the 2 years prior to PCV-7 introduction to 13.7 cases/100,000 population in 2007. The incidence rate of nonvaccine serotype IPD remained stable. The vaccine programme effectiveness was estimated to be 74% (95% CI 57-85%). No vaccine failure was seen after complete primary immunization with two vaccine doses. Our findings indicate that PCV-7 provides highly effective protection against vaccine serotype IPD when administered in a 2+1 dose schedule.