Reinier Mutters

Reclassification of the genus Pasteurella Trevisan 1887 on the basis of deoxyribonucleic acid homology, with proposals for the new species Pasteurella dagmatis, Pasteurella canis, Pasteurella stomatis, Pasteurella anatis, and Pasteurella langaa

Deoxyribonucleic acid (DNA)-DNA hybridization was used to determine the genetic relationships among a variety of previously established or proposed species of Pasteurella and their positions within the family Pasteurellaceae Pohl 1981. Our results indicated that the genus Pasteurella sensu stricto, which can be separated from the Actinobacillus group, consists of at least the following 11 species: Pasteurella multocida, with three subspecies (P. multocida subsp. multocida, P. multocida subsp. septica, and P. multocida subsp. gallicida); Pasteurella dagmatis sp. nov., containing organisms previously labeled Pasteurella “gas,” Pasteurella new species 1, or Pasteurella pneumotropica type Henriksen; Pasteurella gallinarum; Pasteurella canis sp. nov., previously labeled P. multocida biotype 6 or “dog type” strains; Pasteurella stomatis sp. nov., which contains Pasteurella strains isolated from dogs and cats; Pasteurella avium (Hinz and Kunjara) Mutters et al. 1985; Pasteurella volantium Mutters et al. 1985, a new species consisting of V-factor-requiring strains that occur in humans and birds; Pasteurella anatis sp. nov.; Pasteurella langaa sp. nov., containing strains previously designated taxa 1 and 4 of Bisgaard; and two new species, which were provisionally designated Pasteurella species A and Pasteurella species B. The previously recognized taxa Pasteurella ureae, Pasteurella haemolytica biotypes A and T, Pasteurella testudinis, and P. pneumotropica biotypes Jawetz and Heyl do not belong to the genus Pasteurella but are more closely related to the Actinobacillus group. The exact taxonomic positions of Pasteurella aerogenes, P. multocida biotype 1, group HB-5, “Pasteurella piscicida,” the SP group, and the bovine lymphangitis group are still unknown, but these organisms do not belong to the genus Pasteurella.

DNA-DNA hybridization determined in micro-wells using covalent attachment of DNA.

The present study was aimed at reducing the time and labour used to perform DNA-DNA hybridizations for classification of bacteria at the species level. A micro-well-format DNA hybridization method was developed and validated. DNA extractions were performed by a small-scale method and DNA was sheared mechanically into fragments of between 400 and 700 bases. The hybridization conditions were calibrated according to DNA similarities obtained by the spectrophotometric method using strains within the family Pasteurellaceae. Optimal conditions were obtained with 300 ng DNA added per well and bound by covalent attachment to NucleoLink. Hybridization was performed with 500 ng DNA, 5% (w/w) of which was labelled with photo-activatable biotin (competitive hybridization) for 2.5 h at 65 degrees C in 2 x SSC followed by stringent washing with 2 x SSC at the same temperature. The criteria for acceptance of results were a maximum of 15% standard deviation, calculated as a percentage of the mean for four replicate micro-wells, and that DNA similarities were not significantly different in at least two independent experiments. The relationship between DNA similarities obtained by the micro-well method (y) and by the spectrophotometric method (x) was y = 0.534x+30.6, when these criteria had been applied to 23 pairs of strains of Actinobacillus species, avian [Pasteurella] haemolytica-like bacteria and Mannheimia species. The correlation (Pearson) between DNA similarities obtained by interchange of strains used for covalent binding and hybridization was 0.794. Significantly lower DNA similarities were observed by the spectrophotometric compared with the micro-well method for three pairs of hybridizations. After removal of these data, the relationship between DNA similarities obtained by the micro-well and spectrophotometric methods improved to y = 0.855x + 11.0. It was found that the accuracy and precision of the micro-well method was at the same level as that of the spectrophotometric method, but the labour and analysis time were reduced significantly. The use of hybridization in the micro-well format will allow DNA-DNA hybridizations to be carried out between all strains selected for a particular taxonomic study, in order to construct complete data matrices and improve species definition.

Reclassification of German, British and Dutch isolates of so-called Pasteurella multocida obtained from pneumonic calf lungs.

The taxonomic relationship of 131 strains previously identified as Pasteurella multocida obtained from calf pneumonia in West Germany, United Kingdom and Netherlands was investigated by extended phenotypic and limited genotypic characterization. Twenty-four strains were classified as P. multocida ssp. multocida, 15 strains as P. avium biovar 2 and 13 strains as P. canis biovar 2. Sixty-five and five strains were tentatively classified as ornithine negative P. multocida ssp. multocida and P. multocida ssp. septica, respectively. Genetic investigations showed that ornithine negative strains of P. multocida were related on species level. Less genomic binding was found between an ornithine negative strain of P. multocida ssp. septica and the type strains of the three subspecies of P. multocida. The taxonomic position of ornithine negative strains of P. multocida is still under investigation. The taxonomic position of the remaining nine strains is uncertain underlining the need for genotypic characterization within the genus Pasteurella to aid in defining single species by phenotypic tests.

Pasteurella avium (Hinz and Kunjara 1977) comb. nov. and Pasteurella volantium sp. nov.

A deoxyribonucleic acid-deoxyribonucleic acid hybridization study (spectrophotometric method) showed that the species Haemophilus avium Hinz and Kunjara 1977 is composed of three deoxyribonucleic acid homology groups; these three groups are genetically closer to Pasteurella multocida, the type species of the genus Pasteurella, than to Haemophilus influenzae, the type species of the genus Haemophilus. We propose to transfer the species Haemophilus avium to the genus Pasteurella as Pasteurella avium comb. nov. (type strain, ATCC 29546) and to change the circumscription of this taxon to include only strains that are unable to ferment L-arabinose, maltose, and mannitol and unable to hydrolyze o-nitrophenyl-β-D-galactopyranoside. The name Pasteurella volantium sp. nov. (type strain, NCTC 3438) is proposed for strains (formerly identified as H. avium) that are unable to ferment L-arabinose and are able to ferment maltose and mannitol and to hydrolyze o-nitrophenyl-β-D-galactopyranoside. A third unnamed species consists of former H. avium strains that are able to ferment L-arabinose.

DNA from periodontopathogenic bacteria is immunostimulatory for mouse and human immune cells

ABSTRACT Although bacterial DNA (bDNA) containing unmethylated CpG motifs stimulates innate immune cells through Toll-like receptor 9 (TLR-9), its precise role in the pathophysiology of diseases is still equivocal. Here we examined the immunostimulatory effects of DNA extracted from periodontopathogenic bacteria. A major role in the etiology of periodontal diseases has been attributed to Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Peptostreptococcus micros. We therefore isolated DNA from these bacteria and stimulated murine macrophages and human gingival fibroblasts (HGF) in vitro. Furthermore, HEK 293 cells transfected with human TLR-9 were also stimulated with these DNA preparations. We observed that DNA from these pathogens stimulates macrophages and gingival fibroblasts to produce tumor necrosis factor alpha and interleukin-6 in a dose-dependent manner. Methylation of the CpG motifs abolished the observed effects. Activation of HEK 293 cells expressing TLR-9 which were responsive to bDNA but not to lipopolysaccharide confirmed that immunostimulation was achieved by bDNA. In addition, the examined bDNA differed in the ability to stimulate murine macrophages, HGF, and TLR-9-transfected cells. DNA from A. actinomycetemcomitans elicited a potent cytokine response, while DNA from P. gingivalis and P. micros showed lower immunostimulatory activity. Taken together, the results strongly suggest that DNA from A. actinomycetemcomitans, P. gingivalis, and P. micros possesses immunostimulatory properties in regard to cytokine secretion by macrophages and fibroblasts. These stimulatory effects are due to unmethylated CpG motifs within bDNA and differ between distinct periodontopathogenic bacteria strains. Hence, immunostimulation by DNA from A. actinomycetemcomitans, P. gingivalis, and P. micros could contribute to the pathogenesis of periodontal diseases.

Comparison of two full-mouth approaches in the treatment of peri-implant mucositis: a pilot study.

OBJECTIVES The aim of the present study was to test the hypothesis that an additional full-mouth disinfection results in a greater clinical and microbiological improvement compared with sole mechanical debridement within one session in patients with peri-implant mucositis and treated chronic periodontitis. MATERIAL AND METHODS The study included 13 partially edentulous patients (mean age 51.5 years) with treated chronic periodontitis and 36 dental implants with mucositis (bleeding on probing and/or a gingival index > or =1 at least at one site at baseline, absence of peri-implant bone loss during the last 2 years before baseline). After randomized assignment to a test and a control group, patients received a one-stage full-mouth scaling with or without chlorhexidine. Clinical and microbiological examination was performed at baseline, after 1, 2, 4 and 8 months. Additional microbial samples were taken 24 h after treatment. Microbiological analysis was performed by real-time polymerase chain reaction. RESULTS Both treatment modalities resulted in significant reductions of probing depth at implant sites after 8 months, with no significant group differences. The bacteria at implants and teeth could be reduced in every group 24 h after treatment; however, this reduction was not significant after 8 months. CONCLUSIONS Both treatment modalities led to an improvement of the clinical parameters and a temporary reduction of the microflora at implants with mucositis, but without significant inter-group differences after 8 months.

Nicoletella semolina gen. nov., sp. nov., a New Member of Pasteurellaceae Isolated from Horses with Airway Disease

ABSTRACT Gram-negative, nonmotile bacteria that are catalase, oxidase, and urease positive are regularly isolated from the airways of horses with clinical signs of respiratory disease. On the basis of the findings by a polyphasic approach, we propose that these strains be classified as Nicoletella semolina gen. nov, sp. nov., a new member of the family Pasteurellaceae. N. semolina reduces nitrate to nitrite but is otherwise biochemically inert; this includes the lack of an ability to ferment glucose and other sugars. Growth is fastidious, and the isolates have a distinctive colony morphology, with the colonies being dry and waxy and looking like a semolina particle that can be moved around on an agar plate without losing their shape. DNA-DNA hybridization data and multilocus phylogenetic analysis, including 16S rRNA gene (rDNA), rpoB, and infB sequencing, clearly placed N. semolina as a new genus in the family Pasteurellaceae. In all the phylogenetic trees constructed, N. semolina is on a distinct branch displaying ∼5% 16S rDNA, ∼16% rpoB, and ∼20% infB sequence divergence from its nearest relative within the family Pasteurellaceae. High degrees of conservation of the 16S rDNA (99.8%), rpoB (99.6%), and infB (99.7%) sequences exist within the species, indicating that N. semolina isolates not only are phenotypically homogeneous but also are genetically homogeneous. The type strain of N. semolina is CCUG43639T (DSM16380T).

Occurrence of V-factor (NAD) independent strains of Haemophilus paragallinarum

Strains of Bisgaard taxon 31, isolated from chickens in South Africa suffering from a respiratory disease with clinical symptoms and gross lesions similar to infectious coryza, showed great phenotypical similarities with Haemophilus paragallinarum infection except for NAD requirement, beta-galactosidase activity and maltose fermentation. Deoxyribonucleic acid-deoxyribonucleic acid hybridization confirmed a high level of genetic relatedness (DNA binding value, 89%) with Haemophilus paragallinarum. Guanine + cytosine content and genome size data also support the classification of taxon 31 strains within the species Haemophilus paragallinarum.

Fusobacterium canifelinum sp. nov., from the Oral Cavity of Cats and Dogs

Fourteen strains of Gram-negative, anaerobic, fluoroquinolone-resistant, non-sporulating rods were isolated from various infections in cats and dogs, as well as from wounds in humans after cat- or dog-bites. These strains were characterized by sequencing of the 16S-23S rDNA internal transcribed spacer (ITS) regions, 16S rDNA, DNA-DNA hybridization, phylogenetic analysis, and phenotypic tests. The results indicate that the novel strains belong to a distinct species, closely related to Fusobacterium nucleatum. The species Fusobacterium canifelinum sp. nov. is proposed, with strain ATCC BAA 689T as the type strain.

Transfer of Pasteurella ureae Jones 1962 to the Genus Actinobacillus Brumpt 1910: Actinobacillus ureae comb. nov.

Because deoxyribonucleic acid-deoxyribonucleic acid hybridization has shown that the species Pasteurella ureae belongs to the genus Actinobacillus Brumpt 1910, we propose the transfer of P. ureae to the genus Actinobacillus under the new combination Actinobacillus ureae. The type strain of A. ureae is strain S. D. Henriksen 3520/59 (= ATCC 25976 = NCTC 10219).