Streptococci species of anginosus group isolated from oral and maxillofacial infections

The aim of the study was to isolate and identify at species level streptococci strains of anginosus group in pus samples collected from 110 patients with oral and maxillofacial (OMF) infections. Gram-stained smears and cultures on selective and nonselective media were done from each of the 111 pus samples (2 samples were collected from one of the patients, who presented 2 oral abscesses at the same time). The isolates were identified on the basis of cultural and biochemical characteristics. Speciation of the anginosus group isolates was performed using the Rapid ID 32 Strep system (Bio Mérieux, France). Fourty-four anginosus group strains were isolated from 42 patients. Fourty of these isolates were identified as Streptococcus anginosus (2 nonidentical isolates were found in 2 patients), 3 isolates as Streptococcus constellatus and only one as Streptococcus intermedius. The study confirmed that the anginosus group is often involved in OMF infections alone or in association with other aerobic and/or anaerobic bacteria. In the investigated cases, Streptococcus anginosus was by far the most frequently isolated species within the anginosus group.     

Identification of the anginosus group within the genus Streptococcus using polymerase chain reaction

The aim of this study was to establish an identification method for the anginosus group within the genus Streptococcus by polymerase chain reaction (PCR). Using a primer pair based on the group-specific sequences of penicillin-binding protein 2B (pbp2b) gene, a 275-bp fragment was amplified from each species in the group but no size-matched products were obtained in other streptococci. Further identification in the species or subspecies level was possible by a multiplex PCR with primers for the 16S ribosomal RNA gene of Streptococcus anginosus, the hyaluronate lyase genes both of Streptococcus intermedius and Streptococcus constellatus subsp. constellatus, and the intermedilysin (ily) gene of S. intermedius. In the case ofStreptococcus constellatus subsp. pharyngis, the amplified fragment from the S. intermedius-type hyaluronate lyase gene was obtained, while that from the ily gene was not. These results also indicate that two different hyaluronate lyase genes are distributed among the anginosus group.     

A qualitative and quantitative study of the cellular fatty acids of 'Streptococcus milleri' with capillary gas chromatography

Fatty acid analysis was done with GC and GC-MS on 21 strains of 'Streptococcus milleri', representative of the various proposed species. Although no qualitative differences were found in the fatty acid profiles, discriminant analysis of the quantitative data revealed three groups. Streptococcus anginosus and Streptococcus constellatus were indistinguishable but separated from the other two groups which comprised Streptococcus intermedius, with a wide fermentation pattern and Streptococcus intermedius with a narrow fermentation pattern. Three of the strains could be distinguished from the others by a 'fingerprint' of a particularly prominent fatty acid peak. The results support the suggestion that there is more than one species in this group of organisms and that the technique might be of value in epidemiological investigations of 'S. milleri'.     

Detection of Streptococcus anginosus from saliva by real-time polymerase chain reaction

AIMS: The purpose of the present investigation was to assess the salivary levels of Streptococcus anginosus in periodontitis patients. METHODS AND RESULTS: The salivary levels of Strep. anginosus were assessed using real-time polymerase chain reaction (PCR). Streptococcus anginosus was detected in 28 of 37 (75.6%) of periodontitis patients and in three of the 20 (15%) healthy subjects. The mean values for bleeding on probing and probing depth in positive patients were statistically higher than those in negative patients. A significant decrease in Strep. anginosus levels was observed after periodontal treatment. CONCLUSIONS: Although the levels of Strep. anginosus are extremely low, they may reflect the status of periodontal health. SIGNIFICANCE AND IMPACT OF THE STUDY: Real-time PCR is a useful method for obtaining the relative quantities of Strep. anginosus from saliva samples and for monitoring the effect of therapy.     

Cloning and expression of hyaluronate lyase genes of Streptococcus intermedius and Streptococcus constellatus subsp. constellatus(1)

Hyaluronate lyase (HAase) genes of Streptococcus intermedius and Streptococcus constellatus subsp. constellatus were isolated. In S. constellatus subsp. constellatus, the deduced amino acid sequence of HAase was most similar to that of S. intermedius (68%), whereas the enzyme of S. intermedius was most similar to that of S. pneumoniae (72%). Upstream of the HAase gene on the opposite strands, an open reading frame of a putative glutathione peroxidase started in S. intermedius, and this arrangement was similar to that in S. pneumoniae but unlike that in S. constellatus subsp. constellatus. Cell lysates of Escherichia coli carrying each streptococcal gene showed HAase activity, demonstrating that each cloned gene actually coded for HAase.     

DNA-DNA hybridization studies and phenotypic characteristics of strains within the 'Streptococcus milleri group'

Twenty-five strains resembling 'Streptococcus milleri' were compared by DNA-DNA hybridization, by whole-cell-derived polypeptide patterns on SDS-PAGE, and by biochemical tests. Four homology groups were revealed by DNA-DNA hybridization. DNA homology groups 1, 2 and 3 were closely related and contained the type strains NCDO 2226 (Streptococcus constellatus), NCDO 2227 (Streptococcus intermedius) and NCTC 10713 (Streptococcus anginosus), respectively. DNA homology group 4 consisted of four strains received as variants of Streptococcus intermedius which were found not to be closely related to strains in groups 1-3. The data from SDS-PAGE polypeptide patterns and biochemical tests supported the recognition of three centres of variation within the 'Streptococcus milleri group' corresponding to DNA homology groups 1-3 and indicated that strains of DNA homology group 4 are members of an as yet undescribed species within the viridans streptococci.     

Experimental abscess formation caused by human dental plaque

Human dental plaque consists of a wide variety of microorganisms, some of which are believed to cause systemic infections, including abscesses, at various sites in the body. To confirm this hypothesis experimentally, we examined the abscess-forming ability of native dental plaque in mice, the microbial features of the infectious locus produced by the plaque, and the anti-phagocytic property of microbial isolates. Aliquots of a suspension of supragingival dental plaque containing 6 x 10(6) colony-forming unit of bacteria were injected subcutaneously into the dorsa of mice. Abscess formation was induced in 76 of 85 mice using ten different plaque samples. Thirteen microorganisms were isolated from pus samples aspirated from abscess lesions. The microbial composition of pus, examined in 17 of 76 abscesses, was very simple compared to that of the plaque sample that had induced the abscess. The majority of the isolates belonged to the Streptococcus anginosus group, normally a minor component of plaque samples. S. anginosus was the most frequently detected organism and the most prevalent in seven abscesses, and Streptococcus intermedius and Streptococcus constellatus were predominant in one and three abscess samples, respectively. Each isolate of S. anginosus group produced abscesses in mice, and heat-treated supragingival dental plaque influenced the abscess-forming ability of S. anginosus isolate. These isolates possessed a high antiphagocytic capacity against human polymorphonuclear leukocytes. Our results suggest that human supragingival dental plaque itself is a source of the infectious pathogens that cause abscess formation.     

Serogrouping of oral Streptococcus intermedius

Employing twenty fresh oral isolates of Streptococcus intermedius, studies were carried out to characterize serological relations among the isolates and also between the isolates and the strains of bacterial species closely related to S. intermedius. The Rantz-Randall extracts from the cells were used as antigens. The anti-rabbit serum raised against S. intermedius ATCC 27335T reacted with the cell extracts from only three strains of the isolates, which were designated serogroup I strains. The other isolates were classified into four serogroups, I, III, IV, and V, which specifically reacted with the cell extracts from the homologous serogroup strains. However, the serogroup II antiserum formed in immunodiffusion a common precipitin line between the extracts from the cells of serogroups II and I. The serogroups I, III, IV, and V antisera reacted with none of the extracts from the bacterial cells closely related to S. intermedius, which included Streptococcus anginosus ATCC 33397T, Streptococcus constellatus ATCC 27823T, three NCTC strains of "Streptococcus milleri," and three ATCC strains of Streptococcus MG. The precipitin line formed by the homologous reaction of the serogroup II antiserum was found to be a reaction of identity with that formed by the extract from "S. milleri" NCTC 10708. Conversely, the antiserum against NCTC 10708 strain did not react with the cell extracts of serogroup II.     

The establishment of reproducible, complex communities of oral bacteria in the chemostat using defined inocula

Nine commonly isolated oral bacterial populations were inoculated into a glucose-limited and a glucose-excess (amino acid-limited) chemostat maintained at a constant pH 7.0 and a mean community generation time of 13.9 h. The bacterial populations were Streptococcus mutans ATCC 2-27351, Strep. sanguis NCTC 7865, Strep. mitior EF 186, Actinomyces viscosus WVU 627, Lactobacillus casei AC 413, Neisseria sp. A1078, Veillonella alkalescens ATCC 17745, Bacteroides intermedius T 588 and Fusobacterium nucleatum NCTC 10593. All nine populations became established in the glucose-limited chemostat although Strep. sanguis and Neisseria sp. were present only after a second and third inoculation, respectively. In contrast, even following repeated inoculations, Strep. mutans, B. intermedius and Neisseria sp. could not be maintained under glucose-excess conditions. A more extensive pattern of fermentation products and amino acid catabolism occurred under glucose-limited growth; this simultaneous utilization of mixed substrates also contributed to the higher yields (Y molar glucose) and greater species diversity of these communities. Microscopic and biochemical evidence suggested that cell-to-cell interactions and food chains were occurring among community members. To compare the reproductibility of this system, communities were established on three occasions under glucose-limitation and twice under glucose-excess conditions. The bacterial composition of the steady-state communities and their metabolic behaviour were similar when grown under identical conditions but varied in a consistent manner according to the nutrient responsible for limiting growth. Although a direct simulation of the oral cavity was not attempted, the results show that the chemostat could be used as an environmentally-related model to grow complex but reproducible communities of oral bacteria for long periods from a defined inoculum.     

Abscess forming ability of streptococcus milleri group: synergistic effect with Fusobacterium nucleatum.

The abscess forming abilities of "Streptococcus milleri" strains (Streptococcus constellatus, Streptococcus anginiosus, and Streptococcus intermedius) isolated from dentoalveolar abscesses and the synergistic effect of Fusobacterium nucleatum co-inoculated with the isolates were examined on a mouse subcutaneous abscess model. Five days after inoculation, all S. milleri strains formed abscesses, which showed less pathological spread to surrounding connective tissues than those formed by Staphylococcus aureus 209P strain and were similar to those by F. nucleatum ATCC25586. When each S. milleri strain and F. nucleatum were co-inoculated, abscess sizes and each bacterial number recovered from abscesses increased in comparison to those treated by bacterial mono-inoculation of each S. milleri strain or F. nucleatum alone. The strongest synergistic effect was observed in the combination of S. constellatus and F. nucleatum. In a time course experiment with this combination, the recovery of S. constellatus subsequently decreased after the decrement of F. nucleatum, and it appeared that the association with F. nucleatum maintained the bacterial number of S. constellatus in the abscess. The cell-free supernatant of F. nucleatum had a tendency to increase the abscess size caused by S. constellatus in this model. When S. constellatus was cultured with F. nucleatum culture supernatant in vitro, growth enhancement in the early phase was observed. Furthermore, the phagocytic killing of S. constellatus by human polymorphonuclear leukocytes (PMNs) was significantly suppressed and the PMN membranes appeared to be injured by addition of the F. nucleatum culture supernatant. These results suggest that the pathogenicity of S. milleri strains in odontogenic infections may be enhanced by the co-existence of F. nucleatum.     

Sialidase of Streptococcus intermedius: a putative virulence factor modifying sugar chains

A sialidase gene of Streptococcus intermedius was cloned. It was most similar to nanA, a major sialidase gene in Streptococcus pneumoniae, and was expressed in Escherichia coli. Since the gene-knockout S. intermedius strain lost detectable sialidase activity, the gene might code, either solely or mainly, the glycosidase in the bacterial genome. Polymerase chain reaction using the primers for the nanA homologue in S. intermedius (described as nanA below) showed that this sialidase gene was commonly distributed within the isolates of S. intermedius, but not found in the strains of other species among the anginosus group. In biofilm formation assay under cultivation with mucin, the nanA-deleted S. intermedius maintained the amount of biofilm for 72 hr, while that of the parent strain decreased during incubation from 24 to 72 hr. Since sialidase activity in the parent strain increased during that time period, sialidase might contribute to the degradation of biofilm under sialic acid-rich conditions. When S. intermedius was added into the HepG2 hepatoma culture, the calculated disassociation constant (K(d)) of EDTA-releasable bacterial adhesion to the cells was higher in the nanA-deleted strain than in the parent. Furthermore, the rate constant, assuming endocytosis of the bacterium mediated by ASGP-R in HepG2 cells, seemed to be increased by sialidase pretreatment of the bacterial cells before addition to the cell culture. According to the results, modification of sugar chains by sialidase on the bacterial surface and in the surrounding environment might influence both bacterial interaction and host-bacterial interaction in S. intermedius.     

Growth,photosynthetic and biochemical responses of tea cultivars infected with various diseases

Under natural and greenhouse conditions we found a significant reduction in the physiological and biochemical constituents in leaves of five disease types when compared to healthy ones. The growth characteristics such as height, dry mass, photosynthetic and transpiration rates, stomatal conductance, and water use efficiency were reduced significantly more in susceptible cv. TRI-2024 than in tolerant cv. TRI-2025. Also contents of total sugars, nitrogen, amino acids, proteins, polyphenols, and catechin were reduced in diseased plant leaves. However, the reduction was more prominent in susceptible than tolerant cultivar. Canker size and barker moisture content were larger in the susceptible cultivar than in the tolerant cultivar.     

新生儿颊粘膜定植菌株的拮抗性研究

本文分离在新生儿颊粘膜上皮细胞表面形成微菌落、且初代培养时菌落较纯的１５株ａ溶血细菌,进行对Ｂ链球菌、金黄色葡萄球菌、绿脓杆菌、大肠杆菌４种共７株菌的体外拮抗实验。１５株菌中包括８株ａＳｔｒｅｐ．,其中Ｓｔｒｅｐ．ｍｉｔｉｓ４株,Ｓｔｒｅｐ．ｏｒａｌｉｓ２株、Ｓｔｒｅｐ．Ｓａｌｉｖ．Ｓａｌｉｖａｒｉｕｓ１株、Ｓｔｒｅｐ．ｉｎｔｅｒｍｅｄｉｕｓ１株;ＬＣ．ｌａｃｔｉｓ．ｃｒｅｍｏｒｉｓ５株、Ｇｅｍｅｌａｍｏｒｂｉｌｏｒｕｍ１株、Ｇｅｍｅｌａｈａｅｍｏｌｙｓａｎｓ１株。结果约６０％（９／１５）的颊粘膜定植株对两株及两株以上的病原菌株或阴性杆菌株有拮抗作用,只对１株菌有损坏抗作用的ａ溶血菌４株,完全无拮抗作用的菌株２株;ａＳｔｒｅｐ．、ＬＣ．ｌａｃｔｉｓ．ｃｒｅｍｏｒｉｓ的拮抗作用较强;拮抗作用的有无、强弱有很强的菌株特异性     

Phosphatase Activity of Anaerobic Organisms

Anaerobic organisms were tested for phosphatase activity in different pH ranges. Several groups of organisms displayed characteristic patterns. Bacteroides fragilis, B. melaninogenicus, and B. ruminicola produced phosphatase with strongest activity at pH 8.6. Fusobacterium mortiferum was the only species of this genus to show strong hydrolysis. The enzyme was active in both acid and alkaline ranges. The activity of gram-positive organisms was variable, the most active groups being Clostridium perfringens, Peptostreptococcus intermedius, P. micros, and Peptococcus constellatus. The incorporation of phosphatase activity into the identification scheme of anaerobes seems feasible. There was a correlation of hydrolysis with several important pathogens.     

Two new loci affecting cell division identified as suppressors of an ftsQ-null mutation in Streptomyces coelicolor A3(2)

We have cloned and sequenced the gene encoding the surface protein antigen PAa (antigen I/II family) from Streptococcus cricetus E49 (serotype a) using degenerate PCR. The deduced amino acid sequence of PAa reveals two repeating regions (A region; alanine-rich region, P region; proline-rich region). Two additional tandem repeats were found in the A region and part of the P region was deleted compared to antigen I/II. Homology and phylogenetic analyses reveal that PAa is homologous to Streptococcus sobrinus PAg rather than Streptococcus mutans PAc. Using degenerate PCR a gene homologous to PAc was identified in Streptococcus intermedius, but not found in Streptococcus rattus or Streptococcus anginosus.     

The hydrophobicity of 'viridans' streptococci isolated from the human mouth

The hydrophobicity of human oral streptococci was measured with the hexadecane assay modified by the incorporation of polyethylene glycol 6000. Large variability in the hydrophobicity between cultures of some strains grown on different occasions was observed whereas other strains were less variable. The variation in hydrophobicity was significantly reduced by growing the cells in continuous culture in a chemostat under glucose-limiting conditions. The Streptococcus mutans strains used all had low hydrophobicity and the mean hydrophobicity of this species was significantly lower (P less than 0.05) than the mean hydrophobicity of Strep. salivarius, Strep. sanguis Type I and Strep. sanguis Type II strains. This finding supports the view that hydrophobicity is a contributing factor in the adhesion of viridans streptococci to oral surfaces.     

Acetylene reduction (nitrogen fixation) by enterobacteriaceae isolated from paper mill process waters

Using selective media containing galactitol, over 130 Enterobacteriaceae have been isolated from paper mill process waters collected from different localities. These bacteria were extensively characterized and tested for acetylene-reducing (nitrogen-fixing) activity under anaerobic conditions. High activity was found in representatives of Klebsiella pneumoniae, Enterobacter aerogenes, Enterobacter cloacae, Erwinia herbicola, Citrobacter freundii, Citrobacter intermedius, and Escherichia coli. Under argon, nitrogenase synthesis was generally not repressed by 5 mM l-glutamate, l-aspartate, l-leucine or Casamino Acids (0.5 g/liter). In many strains, both the specific activities (nanomoles of C(2)H(4) per minute per milligram of protein) and the activities (nanomoles of C(2)H(4) per minute) had considerably declined after 24 h. In three selected strains, activity in intact cells grown under nitrogen was unaffected by the presence during assay of 10 mM l-amino acids or ammonium acetate. All of the strains examined were tolerant towards inactivation of nitrogen-fixing activity by 1.8% (vol/vol) oxygen during assay, and inactivation by up to 10% oxygen was partly reversible. Representatives of the six taxa synthesized nitrogenase in stirred aerobic cultures, though the protein concentrations attained were lower than under anaerobic conditions. It seems reasonable to suggest that under natural conditions, nitrogen fixation is able to contribute significantly to the nitrogen economy of the cells.     

Effect of denture adhesive on the micro-organisms in vivo

doi: 10.1111/j.1741‐2358.2010.00381.x Effect of denture adhesive on the micro‐organisms in vivo Background: Denture adhesives increase the retention and stability of dentures in edentulous patients, especially in cases where salivary flow is impaired or in the management of traumatised oral mucosa. Objectives: The effect of a denture adhesive on the oral flora at different time intervals. Method: Thirty denture‐wearing patients were involved in this study. While half of the group received a denture adhesive, the other half did not. At baseline, 1 and 2 months after delivering the dentures, smear samples were obtained from the saliva, palate and the dentures. Candida albicans, Candida krusei, Candida glabrata, Candida spp., Staphylococcus aureus, Moraxella catarrhalis, α‐haemolytic streptococci, β‐haemolytic streptococci, Pneumococcus aureus, S. anginosus, S. intermedius, S. constellatus, S. sanguis, S. gordonii, S. mitis, S. mutans, S. salivarius, and yeasts were investigated. The data were statistically analysed using anova and repeated measures. Results: Most types of the micro‐organisms were not seen and could not be analysed statistically except α‐haemolytic streptococci and C. albicans. No statistically significant difference was found for α‐haemolytic streptococci and C. albicans in saliva, palate and the denture at all time intervals. Conclusions: Prolonged use of the denture adhesive tested up to 2 months did not yield to increase in micro‐organisms of the oral flora.