Robert A. Whiley

Genetic approaches to the identification of the mitis group within the genus Streptococcus

The usefulness and reliability of partial sequence analysis of the manganese-dependent superoxide dismutase gene (sodA), autolysin (lytA) gene amplification and species-specific PCR based on the D-alanine:D-alanine ligase (ddl) gene for differentiating each member of the mitis group of the genus Streptococcus was investigated. On the phylogenetic tree based on sodA partial sequences (366 bp) from 96 strains, including all species currently within the mitis group isolated in different geographic areas (mainly Japan and the UK), eight well separated clusters were generated corresponding to recognized species, and all strains fell into those clusters to which they had also been assigned by DNA-DNA hybridization. The Streptococcus pneumoniae sub-cluster was located within the Streptococcus mitis cluster, but the sodA gene of S. pneumoniae was very conserved and therefore could be separated from all other species examined. Furthermore, the lytA gene amplification approach could also be used to differentiate S. pneumoniae from other species. The species-specific amplification product of the ddl gene was successfully detected in Streptococcus sanguinis and Streptococcus gordonii, but failed to be detected in some strains of Streptococcus oralis including the type strain and S. mitis. We conclude that the partial sequence analysis of the sodA gene could be applied globally as a reliable and easy method for the accurate identification of all species currently within the mitis group.

Streptococcus downei sp. nov. for Strains Previously Described as Streptococcus mutans Serotype h

Strains of streptococci originally isolated from the dental plaque of monkeys (Macaca fascicularis) and designated as Streptococcus mutans serotype h were compared with the other species of the mutans streptococcus group. Despite the close resemblance noted previously between these strains and Streptococcus sobrinus, closer examination revealed several important differences. Strains of serotype h ferment mannitol but not sorbitol, melibiose, inulin, or raffinose, do not produce hydrogen peroxide, and are unable to grow in the presence of bacitracin at 2 units per ml. They exhibit a distinct polypeptide pattern by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and possess several antigens absent from S. sobrinus as revealed by Western blotting (immunoblotting). Virtually identical polar lipid patterns were observed by two-dimensional thin-layer chromatography for both serotype h strains and S. sobrinus, although on the basis of long-chain fatty acid analysis by capillary gas-liquid chromatography, the former could be distinguished by the presence of a peak tentatively identified as cyclopropane acid (cis-9, 10-methyleneoctadecanoate (ΔC 19:0). Deoxyribonucleic acid (DNA)-DNA hybridization studies by both the S1 nuclease and renaturation rate methods showed that serotype h strains differ from S. mutans, S. sobrinus, Streptococcus cricetus, Streptococcus rattus, Streptococcus ferus, and Streptococcus macacae. On the basis of these data, we believe that S. mutans serotype h strains represent a distinct species for which the name Streptococcus downei is proposed. The DNA base composition is 41 to 42 moles percent guanine plus cytosine. The type strain is strain MFe28 (NCTC 11391T), which is cariogenic in monoassociated germfree rats.

Use of microwave energy to disinfect a long-term soft lining material contaminated with Candida albicans or Staphylococcus aureus

STATEMENT OF PROBLEM Soft lining materials have been found to be more susceptible to microbial adhesion than acrylic resin base materials. Denture hygiene is essential to maintain the serviceability of the denture, and microwave energy has been suggested for denture disinfection. PURPOSE The purpose of this study was to determine the effectiveness of microwave energy in the disinfection of a long-term soft lining material. MATERIAL AND METHODS A long-term soft lining material was contaminated with known microorganisms and the reduction of organism counts after test disinfection regimes calculated. The disinfection regimes were microwaving for 5 minutes, leaving dry overnight, and soaking overnight in a dilute sodium hypochlorite solution. The test microorganisms were Candida albicans or Staphylococcus aureus. RESULTS For both organisms, soaking in sodium hypochlorite reduced the number of viable adherent microorganisms recovered significantly more than exposure to microwave energy, which led to greater reduction than leaving the lining material dry overnight (p < 0.001, Wilcoxon nonparametric signed rank test). CONCLUSION With reference to the tested microorganisms, disinfection of Molloplast-b soft lining material in dilute sodium hypochlorite solution proved to be more effective than exposure to microwave energy, which in turn was more effective than leaving the lining dry overnight.

A study of small-colony, β-haemolytic, Lancefield group C streptococci within the anginosus group : Description of Streptococcus constellatus subsp. pharyngis subsp. nov., associated with the human throat and pharyngitis

beta-Haemolytic, Lancefield group C streptococci within the anginosus-species group were shown by genetic and phenotypic criteria to be heterogeneous and to constitute two distinct taxa related at subspecies level to Streptococcus constellatus and Streptococcus anginosus, respectively. The first group, referred to here as DNA group 1, comprised six strains with 86-100% intragroup overall genomic DNA relatedness; five of the strains were originally isolated from the human throat and one was from an abdominal mass. They shared 61-77% DNA relatedness (delta Tm values = 1.2-1.5 degrees C) with reference strains of S. constellatus and were clearly differentiated from S. constellatus (now named Streptococcus constellatus subsp. constellatus) by the ability to produce beta-N-acetylgalactosaminidase, beta-N-acetylglucosaminidase, beta-D-fucosidase, beta-D-galactosidase and beta-D-glucosidase. The name S. constellatus subsp. pharyngis is proposed for these strains on the grounds that they are genetically and phenotypically distinct and exhibit a predeliction for the human throat, being isolated also from cases of pharyngitis. The DNA G + C content is 35-37 mol%. The type strain is MM9889aT (= NCTC 13122T). The second group (DNA group 2) was formed by five beta-haemolytic, Lancefield group C strains originally isolated from various human infections. DNA group 2 strains (81-100% intragroup DNA relatedness) shared 60-72% DNA relatedness (delta Tm values = 2.1-4.1 degrees C) with S. anginosus strains NCTC 10713T and MAS 283 but were not clearly differentiated phenotypically from S. anginosus, showed no clear pattern of clinical association, and therefore are not formally proposed as a new subspecies here.

Intermedilysin Is Essential for the Invasion of Hepatoma HepG2 Cells by Streptococcus intermedius

Streptococcus intermedius causes endogenous infections leading to abscesses. This species produces intermedilysin (ILY), a human‐specific cytolysin. Because of the significant correlation between higher ILY production levels by S. intermedius and deep‐seated abscesses, we constructed ily knockout mutant UNS38 B3 and complementation strain UNS38 B3R1 in order to investigate the role of ILY in deep‐seated infections. Strain UNS38 reduced the viability of human liver cell line HepG2 at infection but not of rat liver cell line BRL3A. Isogenic mutant strain UNS38 B3 was not cytotoxic in either cell line. Quantification of S. intermedius revealed that in infected HepG2 cells UNS38 but not UNS38 B3 increased intracellularly concomitantly with increasing cell damage. This difference between UNS38 and UNS38 B3 was not observed with UNS38 B3R1. Invasion and proliferation in BRL3A cells was not observed. Masking UNS38 or UNS38 B3R1 with ILY antibody drastically decreased adherence and invasion of HepG2. Moreover, coating strain UNS38 B3 with ILY partially restored adherence to HepG2 but without subsequent bacterial growth. At 1 day post‐infection, many intact UNS38 were detected in the damaged phagosomes of HepG2 with bacterial proliferation observed in the cytoplasm of dead HepG2 after an additional 2 day incubation. These results indicate that surface‐bound ILY on S. intermedius is an important factor for invasion of human cells by this bacterium and that secretion of ILY within host cells is essential for subsequent host cell death. These data strongly implicate ILY as an important factor in the pathogenesis of abscesses in vivo by this streptococcus.

Sequence Diversity and Antigenic Variation at the rag Locus of Porphyromonas gingivalis

ABSTRACT The rag locus of Porphyromonas gingivalis W50 encodes RagA, a predicted tonB-dependent receptor protein, and RagB, a lipoprotein that constitutes an immunodominant outer membrane antigen. The low G+C content of the locus, an association with mobility elements, and an apparent restricted distribution in the species suggested that the locus had arisen by horizontal gene transfer. In the present study, we have demonstrated that there are four divergent alleles of the rag locus. The original rag allele found in W50 was renamed rag-1, while three novel alleles, rag-2 to rag-4, were found in isolates lacking rag-1. The three novel alleles encoded variants of RagA with 63 to 71% amino acid identity to RagA1 and each other and variants of RagB with 43 to 56% amino acid identity. The RagA/B proteins have homology to numerous Bacteroides proteins, including SusC/D, implicated in polysaccharide uptake. Monoclonal and polyclonal antibodies raised against RagB1 of P. gingivalis W50 did not cross-react with proteins from isolates carrying different alleles. In a laboratory collection of 168 isolates, 26% carried rag-1, 36% carried rag-2, 25% carried rag-3, and 14% carried rag-4 (including the type strain, ATCC 33277). Restriction profiles of the locus in different isolates demonstrated polymorphism within each allele, some of which is accounted for by the presence or absence of insertion sequence elements. By reference to a previously published study on virulence in a mouse model (M. L. Laine and A. J. van Winkelhoff, Oral Microbiol. Immunol. 13:322-325, 1998), isolates that caused serious disease in mice were significantly more likely to carry rag-1 than other rag alleles.

Effect on polymorphonuclear cell function of a human-specific cytotoxin, intermedilysin, expressed by Streptococcus intermedius

ABSTRACT Streptococcus intermedius is a member of the normal flora of the mouth but is also an opportunistic pathogen associated with purulent infections at oral and nonoral sites. Intermedilysin (ILY) has been shown to be a cytolysin capable of generating pores in the cell membrane of erythrocytes demonstrable by electron microscopy. This effect has been shown to be specific for human cells. Since polymorphonuclear cells (PMNs) are the main cell involved in innate immunity we investigated the effect of purified intermedilysin fromStreptococcus intermedius on PMN function. Active ILY at a concentration of 40 ng/μl caused a significant decrease in the number of intact PMNs after 60 min. The active cytolysin, when compared with heat-inactivated ILY, did not appear to be chemotactic for the PMNs but did cause an increase in intracellular calcium, with increased cell surface CD11b expression, metabolic burst, and phagocytosis ofStaphylococcus aureus. These findings may have implications for the role of ILY in deep-seated abscesses.

Differential potentiation of the virulence of the Pseudomonas aeruginosa cystic fibrosis liverpool epidemic strain by oral commensal Streptococci.

BACKGROUND The Pseudomonas aeruginosa Liverpool epidemic strain (LES) is an important cystic fibrosis (CF) pathogen and is associated with increased morbidity and a worsened prognosis, compared with other CF-associated strains. However, interactions of common LES phenotypic variants with other members of the polymicrobial biofilms associated with chronic CF respiratory disease, such as oral commensal streptococci, have not been investigated. METHODS Biofilm population dynamics, virulence factor production, and pathogenicity in Galleria mellonella larvae of common LES phenotypes (ie, low production, intermediate production, and overproduction of pyocyanin) in the presence or absence of anginosus group streptococci (AGS) were compared. RESULTS AGS populations isolated from biofilm cocultures were P. aeruginosa phenotypic variant dependent, with higher AGS cell densities than those in monoculture frequently observed. Coexistence of AGS with a producer of low or intermediate levels of pyocyanin was found to result in enhancement of virulence factor production. In addition, the LES formed pathogenic partnerships with AGS in the G. mellonella infection model, with killing dependent on LES phenotype and AGS species. CONCLUSIONS The pathogenic potential of LES phenotypic variants can be enhanced by the presence of oral commensal streptococci. As adaptive mutations leading to reduced virulence factor production are commonplace, the observations made are relevant in the general context of the biology of P. aeruginosa infection during CF.

Interactions between Eikenella corrodens and ‘Streptococcus milleri-group’ organisms: possible mechanisms of pathogenicity in mixed infections

Interactions between the ‘Streptococcus milleri-group’ organisms (SMG; S. intermedius, S. constellatus and S. anginosus) and Eikenella corrodens were investigated. Coaggregation reactions occurred frequently between S. anginosus (83% of strain combinations) or S. constellatus (87%) and E. corrodens isolates, but were infrequent between S. intermedius and E. corrodens (28%). No enhancement of enzyme activities against lipid, phosphate, peptide and saccharide substrates tested were detected with combinations of species in comparison to the species alone. Exponential growth of S. constellatus and S. intermedius, in mixed culture with E. corrodens, occurred within 6h post inoculation, in comparison to 25h without E. corrodens. No growth stimulation of S. anginosus was observed. It is concluded that both coaggregation and growth stimulation occur between E. corrodens and SMG isolates, and may be important mechanisms in the establishment of mixed infections involving these bacteria.

The rag Locus of Porphyromonas gingivalis Contributes to Virulence in a Murine Model of Soft Tissue Destruction

ABSTRACT The rag locus of Porphyromonas gingivalis encodes a putative TonB-dependent outer membrane receptor, RagA, and a 55-kDa immunodominant antigen, RagB. Inactivation of either ragA or ragB prevented expression of both RagA and RagB. Both the ragA and ragB mutants were significantly less virulent than wild-type strains in a murine model of infection.