In vitro evaluation of surface roughness, adhesion of periodontal ligament fibroblasts, and Streptococcus gordonii following root instrumentation with Gracey curettes and subsequent polishing with diamond-coated curettes

OBJECTIVES: The objective of the study was to evaluate the efficacy of an additional usage of a diamond-coated curette on surface roughness, adhesion of periodontal ligament (PDL) fibroblasts, and of Streptococcus gordonii in vitro. MATERIALS AND METHODS: Test specimens were prepared from extracted teeth and exposed to instrumentation with conventional Gracey curettes with or without additional use of diamond-coated curettes. Surface roughness (Ra and Rz) was measured before and following treatment. In addition, the adhesion of PDL fibroblasts for 72 h and adhesion of S. gordonii ATCC 10558 for 2 h have been determined. RESULTS: Instrumentation with conventional Gracey curettes reduced surface roughness (median Ra before: 0.36 μm/after: 0.25 μm; p < 0.001; median Rz before: 2.34 μm/after: 1.61 μm; p < 0.001). The subsequent instrumentation with the diamond-coated curettes resulted in a median Ra of 0.31 μm/Rz of 2.06 μm (no significance in comparison to controls). The number of attached PDL fibroblasts did not change following scaling with Gracey curettes. The additional instrumentation with the diamond-coated curettes resulted in a two-fold increase in the number of attached PDL fibroblasts but not in the numbers of adhered bacteria. CONCLUSIONS: Treatment of root surfaces with conventional Gracey curettes followed by subsequent polishing with diamond-coated curettes may result in a root surface which provides favorable conditions for the attachment of PDL fibroblasts without enhancing microbial adhesion. CLINICAL RELEVANCE: The improved attachment of PDL fibroblasts and the limited microbial adhesion on root surfaces treated with scaling with conventional Gracey curettes followed by subsequent polishing with diamond-coated curettes may favor periodontal wound healing.

Glycan-binding specificities of Streptococcus mutans and Streptococcus sobrinus lectin-like adhesins

Since the adhesion of bacteria to the tooth surface is a prerequisite for dental plaque and subsequent caries development, a promising caries preventive strategy could be to block the lectin-glycan-mediated adherence of cariogenic bacteria. The aim of the study was to evaluate potential differences in glycan-binding specificities of two Streptococcus mutans strains (DSM 20523 and DSM 6178) and Streptococcus sobrinus (DSM 20381). A competitive enzyme-linked lectin-binding assay was used to identify the binding specificities of isolated bacterial surface lectins. Blotting of the microbial proteins on neoglycoprotein-coated PVP membranes enabled a qualitative protein analysis of all specific bacterial lectins. Different glycan-binding sites could be identified for the S. mutans strains in comparison to S. sobrinus. An earlier reported glycan-binding specificity for terminal galactose residues could be confirmed for the S. mutans strains. For the S. sobrinus strain, more than one glycan-binding specificity could be found (oligomannose and terminal sialyl residues). Each of the tested strains showed more than one surface lectin responsible for the specific lectin-binding with varying molecular weight (S. mutans, 90/155 kDa and S. sobrinus, 35/45 kDa). The established experimental setup could be used as future standard procedure for the identification of bacterial lectin-derived binding specificities. The findings from this study might serve as basis for the design of an individual 'glycan cocktail' for the competitive inhibition of lectin-mediated adhesion of mutans streptococci to oral surfaces.

Capsule type of Streptococcus pneumoniae determines growth phenotype

The polysaccharide capsule of Streptococcus pneumoniae defines over ninety serotypes, which differ in their carriage prevalence and invasiveness for poorly understood reasons. Recently, an inverse correlation between carriage prevalence and oligosaccharide structure of a given capsule has been described. Our previous work suggested a link between serotype and growth in vitro. Here we investigate whether capsule production interferes with growth in vitro and whether this predicts carriage prevalence in vivo. Eighty-one capsule switch mutants were constructed representing nine different serotypes, five of low (4, 7F, 14, 15, 18C) and four of high carriage prevalence (6B, 9V, 19F, 23F). Growth (length of lag phase, maximum optical density) of wildtype strains, nontypeable mutants and capsule switch mutants was studied in nutrient-restricted Lacks medium (MLM) and in rich undefined brain heart infusion broth supplemented with 5% foetal calf serum (BHI+FCS). In MLM growth phenotype depended on, and was transferred with, capsule operon type. Colonization efficiency of mouse nasopharynx also depended on, and was transferred with, capsule operon type. Capsule production interfered with growth, which correlated inversely with serotype-specific carriage prevalence. Serotypes with better growth and higher carriage prevalence produced thicker capsules (by electron microscopy, FITC-dextran exclusion assays and HPLC) than serotypes with delayed growth and low carriage prevalence. However, expression of cpsA, the first capsule gene, (by quantitative RT-PCR) correlated inversely with capsule thickness. Energy spent for capsule production (incorporation of H3-glucose) relative to amount of capsule produced was higher for serotypes with low carriage prevalence. Experiments in BHI+FCS showed overall better bacterial growth and more capsule production than growth in MLM and differences between serotypes were no longer apparent. Production of polysaccharide capsule in S. pneumoniae interferes with growth in nutrient-limiting conditions probably by competition for energy against the central metabolism. Serotype-specific nasopharyngeal carriage prevalence in vivo is predicted by the growth phenotype.

Cross-sectional study of Streptococcus species in quarter milk samples of dairy cows in the canton of Bern, Switzerland

A total of 2538 quarter milk samples from 638 lactating dairy cows from 47 farms in the canton of Bern, Switzerland, were investigated for streptococci. A novel, simple and inexpensive laboratory method was used for the differentiation of Streptococcus species, and a risk factor analysis was carried out. The prevalence in the quarter milk samples was 0.2 per cent for Streptococcus agalactiae, 1.3 per cent for Streptococcus uberis, 1.3 per cent for Streptococcus dysgalactiae, 0.1 per cent for Enterococcus species and 2.9 per cent for minor Streptococcus species (designated Streptococcus-Lactococcus-Enterococcus [SLE] group). Based on the somatic cell count (SCC), S uberis and S dysgalactiae were classified as 'major' pathogens and the bacteria in the SLE group as 'minor' pathogens. For S uberis, S dysgalactiae and bacteria in the SLE group, the most significant risk factor was an intramammary infection (IMI) of a neighbouring quarter by the same pathogen. Other significant risk factors for S uberis infection were a positive California Mastitis Test (CMT) result and a SCC of more than 100,000 cells/ml. Significant risk factors for IMI with S dysgalactiae were a positive CMT result, teat injury and palpable abnormalities in the udder. Infection with bacteria in the SLE group was significantly associated with a SCC of more than 100,000 cells/ml, a lactation number of more than 2, the right rear quarter (as the location of infection) and a positive CMT result.

Intramammary infections with the contagious Staphylococcus aureus genotype B in Swiss dairy cows are associated with low prevalence of coagulase-negative staphylococci and Streptococcus spp

The association between the contagious Staphylococcus aureus genotype B (GTB) and the presence of coagulase-negative staphylococci (CNS) and Streptococcus spp. (non-agalactiae streptococci), was investigated, and the identification of problem herds without genotyping was evaluated. Milk samples from 10 herds with Staph. aureus GTB herd problems (PH cases) were compared with samples from 19 herds with at least one Staph. aureus isolate of non-B genotype (CH cases). All samples were bacteriologically analysed and Staph. aureus genotyping carried out using a ribosomal spacer-PCR. Cow and quarter prevalences of Staph. aureus, CNS and Streptococcus spp. differed significantly between PH and CH groups. PH cases were highly associated with decreased cow prevalences of CNS and Streptococcus spp. These altered prevalences also contributed significantly to the identification of problem herds without resorting to genotyping. Common herd-level risk factors did not explain the difference between the prevalences in PH and CH cases.

Streptococcus spp. and related bacteria: Their identification and their pathogenic potential for chronic mastitis - A molecular approach

Streptococcus spp. and related bacteria form a large group of organisms which are associated with bovine intramammary Infections (IMI). Some of them are the well-known mastitis pathogens Streptococcus uberis and Streptococcus agalactiae. In addition, there are a considerable number of these gram-positive, catalase-negative cocci (PNC) with unclear mastitic pathogenicity such as Aerococcus viridans which make the conventional diagnostics of PNC difficult. One diagnostic, API 20 Strep (API, Biomerieux) is recommended which, as a phenotypic assay, involves a series of miniaturized biochemical tests. Recently, preference is given to genotypic identification methods. In particular, sequencing of the 16S rRNA gene allows highly reproducible and accurate identification of bacteria and permits discovery of novel, clinically relevant bacteria. As a consequence, the aim of the present study was to compare identification of IMI-associated PNC by the API method as well as by sequencing of their 16S rRNA gene (16S). Furthermore, the correlation of these bacteria to bovine chronic mastitis and their phylogeny was investigated. 102 PNC isolated from single quarter milk samples were identified by API and 16S sequencing. Considering Streptococcus uberis, Streptococcus dysgalactiae subsp. dysgalactiae and Streptococcus agalactiae, both methods generated fully concordant results. In contrast, a very high disconcordance was observed for most of the other PNC, in particular Enterococcus spp., Aerococcus viridans and the viridans streptococci were shown as apathogenic. Lactococcus garvieae was found to be an opportunistic pathogen causing IMI during late lactation. In addition, PNC isolated from milk were frequently observed together with other bacteria, in particular with Staphylococcus spp. In these cases, the levels of somatic cell counts (SCC) were determined by the specific PNC present in the sample. Considering PNC phylogeny based on 16S sequencing, 3 major clusters were observed. They included all the common mastitis pathogens (cluster I), the Lactococcus spp., Enterococcus spp. and Aerococcus spp. (cluster II) and all the viridans streptococci (cluster III).

Inflammasome-dependent IFN-γ drives pathogenesis in Streptococcus pneumoniae meningitis

The pathology associated with Streptococcus pneumoniae meningitis results largely from activation of immune-associated pathways. We systematically investigated the production of IFN subtypes, as well as their influence on pathology, in a mouse model of S. pneumoniae meningitis. Despite the occurrence of a mixed IFN type I/II gene signature, no evidence for production or involvement of type I IFNs in disease progression was found. In contrast, type II IFN (IFN-γ) was strongly induced, and IFN-γ(-/-) mice were significantly protected from severe disease. Using intracellular cytokine staining and targeted cell-depletion approaches, NK cells were found to be the dominant source of IFN-γ. Furthermore, production of IFN-γ was found to be dependent upon ASC and IL-18, indicating that an ASC-dependent inflammasome pathway was responsible for mediating IFN-γ induction. The influence of IFN-γ gene deletion on a range of processes known to be involved in bacterial meningitis pathogenesis was examined. Although neutrophil numbers in the brain were similar in infected wild-type and IFN-γ(-/-) mice, both monocyte recruitment and CCL2 production were less in infected IFN-γ(-/-) mice compared with infected wild-type controls. Additionally, gene expression of NO synthase was strongly diminished in infected IFN-γ(-/-) mice compared with infected controls. Finally, bacterial clearance was enhanced in IFN-γ(-/-) mice, although the underlying mechanism remains unclear. Together, these data suggest that inflammasome-dependent IFN-γ contributes via multiple pathways to pathology during S. pneumoniae meningitis.

Genome sequence of Enterococcus hirae (Streptococcus faecalis) ATCC 9790, a model organism for the study of ion transport, bioenergetics, and copper homeostasis

Enterococcus hirae ATCC 9790 is a Gram-positive lactic acid bacterium that has been used in basic research for over 4 decades. Here we report the sequence and annotation of the 2.8-Mb genome of E. hirae and its endemic 29-kb plasmid pTG9790.

Heteroresistance to Fosfomycin Is Predominant in Streptococcus pneumoniae and Depends on the murA1 Gene

Fosfomycin targets the first step of peptidoglycan biosynthesis in Streptococcus pneumoniae catalyzed by UDP-N-acetylglucosamine enolpyruvyltransferase (MurA1). We investigated whether heteroresistance to fosfomycin occurs in S. pneumoniae. We found that of 11 strains tested, all but 1 (Hungary(19A)) displayed heteroresistance and that deletion of murA1 abolished heteroresistance. Hungary(19A) differs from the other strains by a single amino acid substitution in MurA1 (Ala364Thr). To test whether this substitution is responsible for the lack of heteroresistance, it was introduced into strain D39. The heteroresistance phenotype of strain D39 was not changed. Furthermore, no relevant structural differences between the MurA1 crystal structures of heteroresistant strain D39 and nonheteroresistant strain Hungary(19A) were found. Our results reveal that heteroresistance to fosfomycin is the predominant phenotype of S. pneumoniae and that MurA1 is required for heteroresistance to fosfomycin but is not the only factor involved. The findings provide a caveat for any future use of fosfomycin in the treatment of pneumococcal infections.

Serotype-specific invasiveness and colonization prevalence in Streptococcus pneumoniae correlate with the lag phase during in vitro growth

Serotypes of Streptococcus pneumoniae differ in colonization prevalence and the likelihood of causing disease. In vitro growth in brain heart infusion broth with or without 5% fetal calf serum (FCS) was compared for 47 clinical isolates representing 15 pneumococcal serotypes. Serotype-specific colonization prevalence and odds ratios for the invasive potential were obtained from an international and a local epidemiological study. The duration of the lag phase increased with the invasiveness and was inversely associated with the colonization prevalence of a serotype. Supplementation with FCS shortened the lag phase preferentially in serotypes associated with invasive disease (P=0.007). Reduction of oxidative stress by addition of manganese (Mn(2+)), Tiron, mannitol or catalase did not influence the duration of the lag phase significantly. Serotype specific invasiveness and colonization prevalence of S. pneumoniae are associated with the length of the lag phase during in vitro growth. This may correlate with serotype specific selection in vivo.

The prevalence of penicillin-non-susceptible Streptococcus pneumoniae among children aged < 5 years correlates with the biannual epidemic activity of respiratory syncytial virus

This study investigated whether the epidemiology of penicillin-non-susceptible pneumococci (PNSP) colonising small children correlated with the biannual epidemic activity of respiratory syncytial virus (RSV). Colonisation rates and the prevalence of PNSP among paediatric outpatients aged < 5 years was analysed between January 1998 and September 2003 using an established national surveillance network. Resistance trends were investigated using time-series analysis to assess the correlation with the biannual pattern of RSV infections and national sales of oral paediatric formulations of antibiotics and antibiotic prescriptions to children aged < 5 years for acute respiratory tract infections. PNSP rates exhibited a biannual cycle in phase with the biannual seasonal RSV epidemics (p < 0.05). Resistance rates were higher during the winter seasons of 1998-1999 (20.1%), 2000-2001 (16.0%) and 2002-2003 (19.1%), compared with the winter seasons of 1997-1998 (8.2%), 1999-2000 (11.6%) and 2001-2002 (9.5%). Antibiotic sales and prescriptions showed regular peaks during each winter, with no significant correlation with the biannual pattern of RSV activity and seasonal trends of PNSP. RSV is an important determinant of the spread of PNSP and must be considered in strategies aimed at antimicrobial resistance control.

Distribution and invasiveness of Streptococcus pneumoniae serotypes in Switzerland, a country with low antibiotic selection pressure, from 2001 to 2004

To describe the serotype-specific epidemiology of colonizing and invasive Streptococcus pneumoniae isolates, which is important for vaccination strategies, we analyzed a total of 2,388 invasive and 1,540 colonizing S. pneumoniae isolates collected between January 2001 and December 2004 within two nationwide surveillance programs. We found that the relative rank orders of the most frequent serotypes (serotypes 1, 3, 4, 6B, 7F, 14, 19F, and 23F) differed among invasive and colonizing isolates. Serotypes 1, 4, 5, 7F, 8, 9V, and 14 had increased invasive potential, and serotypes/serogroups 3, 6A, 7, 10, 11, 19F, and 23F were associated with colonization. The proportion of pediatric serotypes was higher among children < 5 years old (48.5%) and persons > 64 years old (34.1%) than among other age groups (29.1%); it was also higher in West Switzerland (40.2%) than in other geographic regions (34.7%). Likewise, serotype-specific proportions of penicillin-resistant isolates for types 6B, 9V, 14, and 19F were significantly higher in West Switzerland. The relative frequency of pediatric serotypes corresponded with antibiotic consumption patterns. We conclude that the epidemiology of invasive and colonizing S. pneumoniae isolates is influenced by the serotype-specific potential for invasiveness, and therefore, surveillance programs should include colonizing and invasive S. pneumoniae isolates. Antibiotic selection pressure determines the serotype distribution in different age groups and geographic regions and therefore the expected direct and indirect effects of the 7-valent conjugate vaccine.