Mogens Kilian

A Taxonomic Study of the Genus Haemophilus, with the Proposal of a New Species

A collection of 426 Haemophilus strains isolated from people with infectious diseases and from the normal flora of mucous membranes in humans and various animal species was studied in an attempt to revise and improve the taxonomy of the genus Haemophilus. The examinations included the determination of a number of biochemical and physiological properties, of which several had not previously been applied to the taxonomy of haemophili. The resulting data reavealed many hitherto unrecognized characters of taxonomic significance and several of the species can now be more accurately defined. The classification presented is supported by the DNA base composition of a large number of representative strains. A diagnostic key to the different taxa is presented. Haemophilus influenzae and H. parainfluenzae have been subdivided into a number of biotypes. It is possible to demonstrate a relationship between the individual biotypes of H. influenzae and the origin of the strains assigned to them. The results indicate that H. aegyptius, H. parahaemolyticus and H. paraphrohaemolyticus do not merit specific status. Four unnamed taxa of V-factor-dependent haemophili have been recognized. The name Haemophilus segnis is proposed for one of these taxa, which consists mainly of strains isolated from the human oral cavity. It is demonstrated that the name H. Ducreyi has been used for different groups of bacteria, and that only one of these groups can legitimately be assigned to the genus Haemophilus. Haemolytic V-factor-dependent strains from swine, previously included in H. parahaemolyticus, are significantly different from strains of human origin and should be named H. pleuropneumoniae. None of the strains from swine and fowls were haemin-dependent. The relationships of these strains to the species H. suis and H. gallinarum, and to H. parasuis and H. paragallinarum are discussed. Haemophilus piscium is shown not to belong to the genus Haemophilus. The taxonomic position of H. aphrophilus is uncertain and its possible relationship to Actinobacillus actinomycetemcomitans requires further study. The positive correlation found between the ecology of the strains studied and their affiliation with the different taxa is discussed.

Taxonomic Study of Viridans Streptococci: Description of Streptococcus gordonii sp. nov. and Emended Descriptions of Streptococcus sanguis (White and Niven 1946), Streptococcus oralis (Bridge and Sneath 1982), and Streptococcus mitis (Andrewes and Horder 1906)

We examined a collection of 151 strains of the viridans type of streptococci, which were isolated mainly from human oral cavities and included several reference strains, in an attempt to revise and improve the taxonomy of this group of bacteria. Our examinations included determinations of a high number of biochemical and physiological characteristics and serological reactivity. The resulting data revealed several hitherto unrecognized characters of taxonomic significance, and several of the species can now be more accurately defined. A diagnostic key to the taxa is presented. Strains previously identified as Streptococcus sanguis could be divided into two clearly distinct species, Streptococcus sanguis sensu stricto (type strain, ATCC 10556) and a new species, Streptococcus gordonii (type strain, ATCC 10558). Streptococcus mitis was divided into two biovars, consisting of strains possessing group O antigens and strains possessing group K antigen. The group of strains assigned to Streptococcus anginosus was biochemically and serologically heterogeneous, but the data did not allow natural subdivisions. Based on the results of this study, emended descriptions of the species Streptococcus oralis, S. mitis, and S. sanguis are provided. The classification resulting from this study is in complete agreement with previously published genetic data.

Comparison of the Initial Streptococcal Microflora on Dental Enamel in Caries-Active and in Caries-Inactive Individuals

This study compared the initial (4 h) microflora on enamel in 7 caries-active and in 7 caries-inactive adolescents. In both groups the microflora was dominated by streptococci which comprised 61 and 78% (median values) of the total viable counts in caries-active and caries-inactive individuals, respectively (p less than 0.01). Identification of a total of 700 streptococcal isolates according to a recently revised classification showed that the predominant streptococci belonged to the species Streptococcus oralis, Streptococcus mitis biovar 1, and Streptococcus sanguis. Early plaque from caries-inactive individuals differed from that of caries-active individuals by significantly higher proportions of S. sanguis (p less than 0.05) and IgA1 protease producing streptococci (p less than 0.05). In caries-active individuals, there was a tendency to elevated levels of S. mitis biovar 1 (p less than 0.10). In addition, caries-active individuals were colonized by significantly higher numbers of mutans streptococci on the enamel surfaces (p less than 0.01). However, in both groups Streptococcus mutans (serotype c) comprised less than or equal to 2% of the early streptococcal flora. Streptococcus gordonii, S. mitis biovar 2, and Streptococcus salivarius were present in low proportions and did not show differences in distribution that could be related to caries activity. The observed differences in the composition of the early streptococcal microflora may be a factor that governs the eventual cariogenic potential of dental plaque.

Biological significance of IgA1 proteases in bacterial colonization and pathogenesis: critical evaluation of experimental evidence*

IgA1 protease activity, which allows bacteria to cleave human IgA1 in the hinge region, represents a striking example of convergent evolution of a specific property in bacteria. Although it has been known since 1979 that IgA1 protease is produced by the three leading causes of bacterial meningitis in addition to important urogenital pathogens and some members of the oropharyngeal flora, the exact role of this enzyme in bacterial pathogenesis is still incompletely understood owing to lack of a satisfactory animal model. Cleavage of IgA1 by these post‐proline endopeptidases efficiently separates the monomeric antigen‐binding fragments from the secondary effector functions of the IgA1 antibody molecule. Several in vivo and in vitro observations indicate that the enzymes are important for the ability of bacteria to colonize mucosal membranes in the presence of S‐IgA antibodies. Furthermore, the extensive cleavage of IgA sometimes observed in vivo, suggests that IgA1 protease activity results in a local functional IgA deficiency that may facilitate colonization of other microorganisms and the penetration of potential allergens. It has been hypothesized that IgA1 protease activity of Haemophilus influenzae, Neisseria meningitidis, and Streptococcus pneumoniae, under special immunological circumstances, allows these bacteria to take advantage of specific IgA1 antibodies in a strategy to evade other immune factors of the human body. The decisive factor is the balance between IgA antibodies against surface antigens of the respective bacteria and their IgA1 protease. Recent studies have shown that serine‐type IgA1 proteases of H. influenzae, meningococci, and gonococci belong to a family of proteins used by a diverse group of Gramnegative bacteria for colonization and invasion.

Risk of aggressive periodontitis in adolescent carriers of the JP2 clone of Aggregatibacter (Actinobacillus) actinomycetemcomitans in Morocco: a prospective longitudinal cohort study.

BACKGROUND Periodontitis is a loss of supporting connective tissue and alveolar bone around teeth, and if it occurs in an aggressive form it can lead to tooth loss before the age of 20 years. Although the cause of periodontitis in general remains elusive, a particular clone (JP2) of the gram-negative rod Aggregatibacter (Actinobacillus) actinomycetemcomitans is considered a possible aetiological agent of the aggressive form in adolescents living in or originating from north and west Africa, where the disease is highly prevalent. We did a population-based longitudinal study of adolescents to assess the role of the JP2 clone in the initiation of aggressive periodontitis. METHODS A total of 700 adolescents from public schools in Rabat, Morocco, were enrolled in the study. We used PCR to detect A actinomycetemcomitans in plaque samples (taken from molar and incisor sites) and to differentiate between the JP2 clone and other non-JP2 genotypes of the bacterium. 18 individuals were found to already have periodontitis and were excluded. The 682 periodontally healthy adolescents (mean age 12.5 years; SD 1.0) were classified according to their A actinomycetemcomitans carrier status at baseline. After 2 years, 428 (62.8%) individuals returned for re-examination, which included recording of periodontal attachment loss measured from the cemento-enamel junction to the bottom of the periodontal pockets of all teeth present. FINDINGS Individuals who carried the JP2 clone of A actinomycetemcomitans alone (relative risk 18.0; 95% CI 7.8-41.2, p<0.0001) or together with non-JP2 clones of A actinomycetemcomitans (12.4; 5.2-29.9, p<0.0001) had a significantly increased risk of periodontal attachment loss. A much less pronounced disease risk was found in those carrying non-JP2 clones only (3.0; 1.3-7.1, p=0.012). INTERPRETATION The JP2 clone of A actinomycetemcomitans is likely to be an important aetiological agent in initiation of periodontal attachment loss in children and adolescents. Co-occurrence of non-JP2 clones of A actinomycetemcomitans reduces the risk of development of periodontitis, suggesting competition for the ecological niche between the JP2 and non-JP2 clones of this species.

Evolution of Streptococcus pneumoniae and its close commensal relatives.

Streptococcus pneumoniae is a member of the Mitis group of streptococci which, according to 16S rRNA-sequence based phylogenetic reconstruction, includes 12 species. While other species of this group are considered prototypes of commensal bacteria, S. pneumoniae is among the most frequent microbial killers worldwide. Population genetic analysis of 118 strains, supported by demonstration of a distinct cell wall carbohydrate structure and competence pheromone sequence signature, shows that S. pneumoniae is one of several hundred evolutionary lineages forming a cluster separate from Streptococcus oralis and Streptococcus infantis. The remaining lineages of this distinct cluster are commensals previously collectively referred to as Streptococcus mitis and each represent separate species by traditional taxonomic standard. Virulence genes including the operon for capsule polysaccharide synthesis and genes encoding IgA1 protease, pneumolysin, and autolysin were randomly distributed among S. mitis lineages. Estimates of the evolutionary age of the lineages, the identical location of remnants of virulence genes in the genomes of commensal strains, the pattern of genome reductions, and the proportion of unique genes and their origin support the model that the entire cluster of S. pneumoniae, S. pseudopneumoniae, and S. mitis lineages evolved from pneumococcus-like bacteria presumably pathogenic to the common immediate ancestor of hominoids. During their adaptation to a commensal life style, most of the lineages gradually lost the majority of genes determining virulence and became genetically distinct due to sexual isolation in their respective hosts.

Haemophilus haemolyticus: a human respiratory tract commensal to be distinguished from Haemophilus influenzae.

BACKGROUND Haemophilus influenzae is a common pathogen in adults with chronic obstructive pulmonary disease (COPD). In a prospective study, selected isolates of apparent H. influenzae had an altered phenotype. We tested the hypothesis that these variant strains were genetically different from typical H. influenzae. METHODS A prospective study of adults with COPD was conducted. Strains of apparent H. influenzae obtained from a range of clinical sources were evaluated by ribosomal DNA sequence analysis, multilocus sequence analysis, DNA-DNA hybridization, and sequencing of the conserved P6 gene. RESULTS Variant strains were determined to be Haemophilus haemolyticus by means of 4 independent methods. Analysis of 490 apparent H. influenzae strains, identified by standard methods, revealed that 39.5% of sputum isolates and 27.3% of nasopharyngeal isolates were H. haemolyticus. Isolates obtained from normally sterile sites were all H. influenzae. In a prospective study, acquisitions of new strains of H. haemolyticus were not associated with exacerbations of COPD, whereas 45% of acquisitions of new strains of H. influenzae were associated with exacerbations. CONCLUSIONS Standard methods do not reliably distinguish H. haemolyticus from H. influenzae. H. haemolyticus is a respiratory tract commensal. The recognition that some strains of apparent H. influenzae are H. haemolyticus substantially strengthens the association of true H. influenzae with clinical infection.

Dynamics of Streptococcus agalactiae Colonization in Women during and after Pregnancy and in Their Infants

ABSTRACT The population dynamics of Streptococcus agalactiae (group B streptococci [GBS]) colonization of the vagina and anorectal area was investigated in a cohort of 77 Danish women during and after their pregnancy by a new sensitive method. The mean carriage rate among individual observations was 36%, and the cumulative carriage rate over the entire observation period was 54%. Examination of more than 1,500 GBS isolates by pulsed-field gel electrophoresis demonstrated that the GBS population was remarkably homogeneous and stable in each carrier. Virtually all carriers were colonized by a single GBS clone on all occasions spanning up to 2 years. Repeated detection of the same clone even in women who were recorded as intermittent carriers suggests that the actual carrier rate exceeds 50% but that fluctuations in the GBS proportions of the flora occasionally preclude their detection. Newborns and young infants usually carried the same GBS clone as their mothers. However, only twice were identical clones of GBS detected in different women in contrast to the observed clonal relationships of clinical isolates. These observations strongly suggest differences in the properties and epidemiology of virulent GBS clones compared to clones commonly carried by healthy individuals.

Assigning strains to bacterial species via the internet

BackgroundMethods for assigning strains to bacterial species are cumbersome and no longer fit for purpose. The concatenated sequences of multiple house-keeping genes have been shown to be able to define and circumscribe bacterial species as sequence clusters. The advantage of this approach (multilocus sequence analysis; MLSA) is that, for any group of related species, a strain database can be produced and combined with software that allows query strains to be assigned to species via the internet. As an exemplar of this approach, we have studied a group of species, the viridans streptococci, which are very difficult to assign to species using standard taxonomic procedures, and have developed a website that allows species assignment via the internet.ResultsSeven house-keeping gene sequences were obtained from 420 streptococcal strains to produce a viridans group database. The reference tree produced using the concatenated sequences identified sequence clusters which, by examining the position on the tree of the type strain of each viridans group species, could be equated with species clusters. MLSA also identified clusters that may correspond to new species, and previously described species whose status needs to be re-examined. A generic website and software for electronic taxonomy was developed. This site http://www.eMLSA.net allows the sequences of the seven gene fragments of a query strain to be entered and for the species assignment to be returned, according to its position within an assigned species cluster on the reference tree.ConclusionThe MLSA approach resulted in the identification of well-resolved species clusters within this taxonomically challenging group and, using the software we have developed, allows unknown strains to be assigned to viridans species via the internet. Submission of new strains will provide a growing resource for the taxonomy of viridans group streptococci, allowing the recognition of potential new species and taxonomic anomalies. More generally, as the software at the MLSA website is generic, MLSA schemes and strain databases for other groups of related species can be hosted at this website, providing a portal for microbial electronic taxonomy.

Live Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria meningitidis activate the inflammatory response through Toll-like receptors 2, 4, and 9 in species-specific patterns

Toll‐like receptors (TLRs) are pattern recognition receptors (PRR) that recognize molecular structures on pathogens and activate host defenses. Although much is known about specific bacterial components that activate TLRs, few studies have addressed the question of which TLRs are involved in immune activation by live bacteria. Here, we demonstrate that live Streptococcus pneumoniae, Haemophilus influenzae type b, and Neisseria meningitidis, the three principal causes of bacterial meningitis, use distinct sets of TLRs to trigger the inflammatory response. Using human embryonic kidney 293 cell lines, each overexpressing one type of TLR, we found that S. pneumoniae triggered activation of the transcription factor nuclear factor‐κB and expression of interleukin‐8, only in cells expressing TLR2 or ‐9. The same response was evoked by H. influenzae in cells expressing TLR2 or ‐4 and by N. meningitidis in cells expressing TLR2, ‐4, or ‐9. It is interesting that the ability of S. pneumoniae and N. meningitidis to activate TLR9 was severely attenuated when bacteria had been heat‐inactivated prior to stimulation of the cells. In human peripheral blood mononuclear cells, we blocked TLR2, ‐4, or ‐9 and confirmed the essential role of these TLRs and also identified differential functions of TLRs in activation of the inflammatory response. Collectively, we here demonstrate that S. pneumoniae, H. influenzae, and N. meningitidis each activate several TLRs in species‐specific patterns and show that infection with live pathogens may lead to activation of PRR not targeted by inactivated bacteria.