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Bacteria of the genus Streptococcus , earlier considered typically animal, currently have also been causing infections in humans. It is necessary to make clinicians aware of the emergence of new species that may cause the development of human diseases. There is an increasing frequency of isolation of streptococci such as S. suis , S. dysgalactiae , S. iniae and S. equi from people. Isolation of Streptococcus bovis/Streptococcus equinus complex bacteria has also been reported. The streptococcal species described in this review are gaining new properties and virulence factors by which they can thrive in new environments. It shows the potential of these bacteria to changes in the genome and the settlement of new hosts. Information is presented on clinical cases that concern streptococcus species belonging to the groups Bovis, Pyogenic and Suis. We also present the antibiotic resistance profiles of these bacteria. The emerging resistance to β-lactams has been reported. In this review, the classification, clinical characteristics and antibiotic resistance of groups and species of streptococci considered as animal pathogens are summarized.

Purpose: Oesophageal squamous cell carcinoma (ESCC) is the dominant type of oesophageal cancer among the East Asian population. The role of ESCC tissue bacteria in neoplastic progression has not been fully elucidated. Our goal was to uncover different bacterial communities in pathological staging grouping of ESCC and to identify microorganisms that could predict the likelihood of prognosis., Methodology: Tissue samples were obtained from 45 patients and assessed using 16S ribosomal RNA gene sequencing. Significant bacteria were selected to perform survival analysis and evaluate prognostic biomarker.Results/Key findings. We observed variations in the abundance of oesophageal flora among different pathological characteristics of ESCC. Phylum Bacteroidetes, Firmicutes and Spirochaetes showed significantly higher relative abundances among N+ (positive lymph node) patients when compared to N- (negative lymph node) controls, whereas Proteobacteria showed lower abundances in N+ patients. Both genera Prevotella and Treponema were more abundant in the N+ group. In regard to T stage, the abundance of only Streptococcus in T3-4 was significantly higher than that in T1-2, while the other genera showed no significance. On multivariable analysis adjusted for the effects of standard clinicopathological features, combined Streptococcus and Prevotella abundance retained its association with unfavourable survival (hazard ratio, 6.094; 95 % confidence interval, 1.072-34.646; P=0.042), suggesting that this may be an independent prognostic indicator for ESCC., Conclusion: Combined Streptococcus and Prevotella abundance is regarded as an independent species prognostic biomarker in ESCC patients.

Daptomycin is a lipopeptide antibiotic with activity against Gram-positive bacteria. We showed previously that Staphylococcus aureus can survive daptomycin exposure by releasing membrane phospholipids that inactivate the antibiotic. To determine whether other pathogens possess this defence mechanism, phospholipid release and daptomycin activity were measured after incubation of Staphylococcus epidermidis, group A or B streptococci, Streptococcus gordonii or Enterococcus faecalis with the antibiotic. All bacteria released phospholipids in response to daptomycin, which resulted in at least partial inactivation of the antibiotic. However, E. faecalis showed the highest levels of lipid release and daptomycin inactivation. As shown previously for S. aureus, phospholipid release by E. faecalis was inhibited by the lipid biosynthesis inhibitor platensimycin. In conclusion, several pathogenic Gram-positive bacteria, including E. faecalis, inactivate daptomycin by releasing phospholipids, which may contribute to the failure of daptomycin to resolve infections caused by these pathogens.

Platelets have been reported to become activated in response to bacteria and this is proposed to contribute to the acute response to bacterial infection. In the present study, we investigated platelet aggregation in response to group G streptococci (GGS) in vitro in healthy human donors and in vivo in a mouse model of streptococcal sepsis. Platelet aggregation by GGS was dependent on the bacterial surface protein FOG and engagement of the platelet fibrinogen receptor; however, it was independent of IgG and the platelet Fc receptor. Platelets exerted no antibacterial effects on the bacteria, and aggregates formed were markedly unstable, allowing bacteria to rapidly return to the plasma and grow post-aggregation. Thrombocytopenia and platelet activation occurred during invasive infection with GGS, and platelets were demonstrated to contribute to bacterial dissemination during infection. These findings reveal an important role for bacteria-platelet interactions during the pathogenesis of streptococcal infection.

Understanding the mechanisms of augmented bacterial pathogenicity in post-viral infections is the first step in the development of an effective therapy. This study assessed the effect of human coronavirus NL63 (HCoV-NL63) on the adherence of bacterial pathogens associated with respiratory tract illnesses. It was shown that HCoV-NL63 infection resulted in an increased adherence of Streptococcus pneumoniae to virus-infected cell lines and fully differentiated primary human airway epithelium cultures. The enhanced binding of bacteria correlated with an increased expression level of the platelet-activating factor receptor (PAF-R), but detailed evaluation of the bacterium-PAF-R interaction revealed a limited relevance of this process.

Three Gram-positive, catalase-negative, coccus-shaped organisms were isolated from the oral cavities of bears. The isolates were tentatively identified as a streptococcal species based on the results of biochemical tests. Comparative 16S rRNA gene sequencing studies confirmed that the organisms were members of the genus Streptococcus, but they did not correspond to any recognized species of the genus. The nearest phylogenetic relative of the new isolates was Streptococcus ratti ATCC 19645(T) (98.6 %), however, DNA-DNA hybridization analysis showed that the isolates displayed less than 15 % DNA-DNA relatedness with the type strain of S. ratti. Colonies of the novel strains grown on mitis salivarius agar showed an extracellular polysaccharide-producing colony morphology. Based on phenotypic and phylogenetic evidence, it is proposed that the novel isolates are classified in the genus Streptococcus as Streptococcus ursoris sp. nov. The type strain of S. ursoris is NUM 1615(T) (=JCM 16316(T)=DSM 22768(T)).

Four Gram-stain-positive, catalase-negative, coccoid-shaped isolates were obtained from the oral cavities of wild boars and characterized by phenotypic and phylogenetic studies. On the results of biochemical tests, the organisms were tentatively identified as a streptococcal species. Comparative 16S rRNA gene sequencing studies confirmed that the organisms are members of the genus Streptococcus, with Streptococcus equi subsp. equi ATCC 33398(T) as their closest phylogenetic relative (94.7 % similarity). DNA-DNA hybridization analysis showed that the isolates displayed less than 10 % relatedness to Streptococcus equi subsp. equi DSM 20561(T). From the phylogenetic and phenotypic evidence, the four isolates represent a novel species of the genus Streptococcus, for which the name Streptococcus dentapri sp. nov. (type strain NUM 1529(T) =JCM 15752(T) =DSM 21999(T)) is proposed.

Biofilms provide a reservoir of potentially infectious micro-organisms that are resistant to antimicrobial agents, and their importance in the failure of medical devices and chronic inflammatory conditions is increasingly being recognized. Particular research interest exists in the association of biofilms with wound infection and non-healing, i.e. chronic wounds. In this study, fluorescent in situ hybridization (FISH) was used in combination with confocal laser scanning microscopy (CLSM) to detect and characterize the spatial distribution of biofilm-forming bacteria which predominate within human chronic skin wounds (Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus sp. and Micrococcus sp.). In vitro biofilms were prepared using a constant-depth film fermenter and a reconstituted human epidermis model. In vivo biofilms were also studied using biopsy samples from non-infected chronic venous leg ulcers. The specificity of peptide nucleic acid (PNA) probes for the target organisms was confirmed using mixed preparations of planktonic bacteria and multiplex PNA probing. Identification and location of individual bacterial species within multi-species biofilms demonstrated that P. aeruginosa was predominant. CLSM revealed clustering of individual species within mixed-species biofilms. FISH analysis of archive chronic wound biopsy sections showed bacterial presence and allowed bacterial load to be determined. The application of this standardized procedure makes available an assay for identification of single- or multi-species bacterial populations in tissue biopsies. The technique provides a reliable tool to study bacterial biofilm formation and offers an approach to assess targeted biofilm disruption strategies in vivo.

The combined use of confocal laser scanning microscopy (CLSM) and fluorescent in situ hybridization (FISH) offers new opportunities for analysis of the spatial relationships and temporal changes of specific members of the microbiota of intact dental biofilms. The purpose of this study was to analyse the patterns of colonization and population dynamics of Actinomyces naeslundii compared to streptococci and other bacteria during the initial 48 h of biofilm formation in the oral cavity. Biofilms developed on standardized glass slabs mounted in intra-oral appliances worn by ten individuals for 6, 12, 24 and 48 h. The biofilms were subsequently labelled with probes against A. naeslundii (ACT476), streptococci (STR405) or all bacteria (EUB338), and were analysed by CLSM. Labelled bacteria were quantified by stereological tools. The results showed a notable increase in the number of streptococci and A. naeslundii over time, with a tendency towards a slower growth rate for A. naeslundii compared with streptococci. A. naeslundii was located mainly in the inner part of the multilayered biofilm, indicating that it is one of the species that attaches directly to the acquired pellicle. The participation of A. naeslundii in the initial stages of dental biofilm formation may have important ecological consequences.

Gram-positive, catalase-negative, chain-forming, coccus-shaped organisms were isolated both from intraperitoneally grown vesicles of the fox tapeworm Echinococcus multilocularis and the oropharynges of laboratory-kept Mongolian jirds (Meriones unguiculatus). The strains displayed no haemolytic activity on Columbia sheep blood agar, pyrrolidonyl arylamidase activity was negative and the organisms reacted weakly with Lancefield group D antiserum. On the basis of phenotypic characteristics, the strains were tentatively identified as members of the genus Streptococcus. Comparative 16S rRNA gene sequencing studies confirmed their assignment to the genus Streptococcus and revealed that Streptococcus hyointestinalis DSM 20770(T) was their closest phylogenetic neighbour (96.5 % sequence similarity). The levels of 16S rRNA gene sequence similarity between the isolates and representatives of species of the genus Streptococcus were only 95.7-96.2 %. On the basis of the phenotypic and molecular data presented, the isolates from Mongolian jirds represent a novel species of the genus Streptococcus, for which the name Streptococcus merionis sp. nov. is proposed. The type strain is WUE3771(T) (=DSM 19192(T)=CCUG 54871(T)).

Biochemical and molecular genetic studies were performed on an unknown Gram-positive, catalase-negative, coccus-shaped organism isolated from clinical samples from pigs. On the basis of the results of cellular morphological and biochemical tests, the organism was identified as a streptococcal species. 16S rRNA gene sequence comparisons confirmed its identification as a member of the genus Streptococcus, but the organism was distinct from any recognized species of this genus. The closest phylogenetic relative of the unknown organism corresponded to Streptococcus suis NCTC 10234(T) (97.2 % 16S rRNA gene sequence similarity) and this phylogenetic position was confirmed by analysis of rpoB and sodA sequences. DNA-DNA hybridization studies showed that the unidentified organism produced a DNA reassociation value of 36.6 % with respect to S. suis NCTC 10234(T). The novel bacterium was distinguished from S. suis and other Streptococcus species using biochemical tests. On the basis of phenotypic and phylogenetic data, the unidentified organism represents a novel species of the genus Streptococcus, for which the name Streptococcus plurextorum sp. nov. is proposed. The type strain is 1956-02(T) (=CECT 7308(T)=CCUG 52972(T)).

The development of complex multispecies communities such as biofilms is controlled by interbacterial communication systems. We have previously reported an intergeneric communication between two oral bacteria, Streptococcus cristatus and Porphyromonas gingivalis, that results in inhibition of fimA expression. Here, we demonstrate that a surface protein, arginine deiminase (ArcA), of S. cristatus serves as a signal that initiates intergeneric communication. An ArcA-deficient mutant of S. cristatus is unable to communicate with P. gingivalis. Furthermore, arginase activity is not essential for the communication, and ArcA retains the ability to repress expression of fimA in the presence of arginine deiminase inhibitors. These results present a novel mechanism by which intergeneric communication in dental biofilms is accomplished.

Phenotypic and phylogenetic studies were performed on six unidentified, Gram-positive, catalase-negative, chain-forming Streptococcus-like organisms recovered from grey seals. Biochemically the six strains were highly related to each other, but they did not appear to correspond to any recognized species of the genus Streptococcus. Comparative 16S rRNA gene sequencing studies confirmed that phylogenetically the strains were members of the genus Streptococcus, but sequence divergence values of greater than 3 % compared with reference streptococcal species demonstrated that the organisms from seals represent a novel species. SDS-PAGE analysis of whole-cell proteins confirmed the phenotypic distinctiveness of the seal organisms. Based on biochemical criteria and molecular chemical and genetic evidence, it is proposed that the unknown organism from seals be classified as a novel species, Streptococcus halichoeri sp. nov., the type strain of which is CCUG 48324T (= CIP 108195T).

Poly(ethylene oxide) (PEO)-brushes are generally recognized as protein-repellent surfaces, and although a role in discouraging microbial adhesion has been established for some strains and species, no study exists on the effects of PEO-brushes on a large variety of bacterial and yeast strains. In this paper, a PEO-brush has been covalently attached to glass and silica by reaction in a polymer melt. Subsequently, the presence of a PEO-brush was demonstrated using contact angle measurements, X-ray photoelectron spectroscopy and ellipsometry. For five bacterial (Staphylococcus epidermidis, Staphylococcus aureus, Streptococcus salivarius, Escherichia coli and Pseudomonas aeruginosa) and two yeast strains (Candida albicans and Candida tropicalis), adhesion to PEO-brushes was compared with adhesion to bare glass in a parallel plate flow chamber. The initial deposition rates of Sta. epidermidis, Sta. aureus and Str. salivarius to glass were relatively high, between 2400 and 2600 cm(-2) s(-1), while E. coli and P. aeruginosa deposited much more slowly. The initial deposition rates of the yeasts to glass were 144 and 444 cm(-2) s(-1) for C. albicans GB 1/2 and C. tropicalis GB 9/9, respectively. Coating of the glass surface with a PEO-brush yielded more than 98 % reduction in bacterial adhesion, although for the more hydrophobic P. aeruginosa a smaller reduction was observed. For both yeast species adhesion suppression was less effective than for the bacteria and here too the more hydrophobic C. tropicalis showed less reduction than the more hydrophilic C. albicans. The PEO-brush had a thickness of 22 nm in water, as inferred from ellipsometry. Assuming that on bare glass the adhered micro-organisms are positioned only a few nanometers away from the surface and that the brush keeps them at a distance of 22 nm, it is calculated that the brush yields a sevenfold attenuation of the Lifshitz-Van der Waals attraction to the surface between the micro-organisms and the surface. Decreased Lifshitz-van der Waals attraction may be responsible for the suppression of the microbial adhesion observed.

Streptococcus gordonii produces two alpha-amylase-binding proteins, AbpA and AbpB, that have been extensively studied in vitro. Little is known, however, about their significance in oral colonization and cariogenicity (virulence). To clarify these issues, weanling specific pathogen-free Osborne-Mendel rats, TAN : SPFOM(OM)BR, were inoculated either with wild-type strains FAS4-S or Challis-S or with strains having isogenic mutations of abpA, abpB, or both, to compare their colonization abilities and persistence on the teeth. Experiments were done with rats fed a sucrose-rich diet containing low amounts of starch or containing only starch. The mutants and wild-types were quantified in vivo and carious lesions were scored. In 11 experiments, S. gordonii was a prolific colonizer of the teeth when rats were fed the sucrose (with low starch)-supplemented diet, often dominating the flora. Sucrose-fed rats had several-fold higher recoveries of inoculants than those eating the sucrose-free, starch-supplemented diet, regardless of inoculant type. The strain defective in AbpB could not colonize teeth of starch-only-eating rats, but could colonize rats if sucrose was added to the diet. Strains defective in AbpA surprisingly colonized better than their wild-types. A double mutant deficient in both AbpA and AbpB (abpA/abpB) colonized like its wild-type. Wild-types FAS4-S and Challis-S had no more than marginal cariogenicity. Notably, in the absence of AbpA, cariogenicity was slightly augmented. Both the rescue of colonization by the AbpB- mutant and the augmentation of colonization by AbpA- mutant in the presence of dietary sucrose suggested additional amylase-binding protein interactions relevant to colonization. Glucosyltransferase activity was greater in mutants defective in abpA and modestly increased in the abpB mutant. It was concluded that AbpB is required for colonization of teeth of starch-eating rats and its deletion is partially masked if rats eat a sucrose-starch diet. AbpA appears to inhibit colonization of the plaque biofilm in vivo. This unexpected effect in vivo may be associated with interaction of AbpA with glucosyltransferase or with other colonization factors of these cells. These data illustrate that the complex nature of the oral environment may not be adequately modelled by in vitro systems.

'Streptococcus bovis/Streptococcus equinus' is a large bacterial complex including different species frequently isolated from infections of humans (Streptococcus gallolyticus, Streptococcus infantarius) or animals (S. bovis, S. equinus, Streptococcus alactolyticus). The separation of S. bovis into three different biotypes has been partially correlated with genetic differentiation. In addition, recent advances in bacterial phylogeny have led to the inclusion of Streptococcus macedonicus and Streptococcus waius in this complex. The aim of this study was to improve physiological differentiation between species related to the complex and to clarify their respective phylogenetic positions. In this study, physiological, genetic and phylogenetic analyses of a set of 88 streptococcal strains were performed. The diversity of strains of S. bovis biotype II was analysed, and it was confirmed that they belong to different species, either S. equinus or S. infantarius. It was demonstrated that S. gallolyticus, S. bovis biotype II.2, S. macedonicus and S. waius form a single DNA cluster separated into three different subspecies. They are delineated by different biochemical traits, limited DNA-DNA relatedness and noticeable divergence in 16S rDNA sequences. According to the current definition of species, the names S. gallolyticus subsp. gallolyticus subsp. nov., S. gallolyticus subsp. pasteurianus subsp. nov. and S. gallolyticus subsp. macedonicus subsp. nov. are proposed for these three subspecies.

The manufacture of yoghurt relies on the simultaneous utilization of two starters: Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus (Lb. bulgaricus). A protocooperation usually takes place between the two species, which often results in enhanced milk acidification and aroma formation compared to pure cultures. Cell-wall proteinases of Lactococcus lactis and lactobacilli have been shown to be essential to growth in milk in pure cultures. In this study, the role of proteinases PrtS from S. thermophilus and PrtB from Lb. bulgaricus in bacterial growth in milk was evaluated; a negative mutant for the prtS gene of S. thermophilus CNRZ 385 was constructed for this purpose. Pure cultures of S. thermophilus CNRZ 385 and its PrtS-negative mutant were made in milk as well as mixed cultures of S. thermophilus and Lb. bulgaricus: S. thermophilus CNRZ 385 or its PrtS-negative mutant was associated with several strains of Lb. bulgaricus, including a PrtB-negative strain. The pH and growth of bacterial populations of the resulting mixed cultures were followed, and the Lactobacillus strain was found to influence both the extent of the benefit of Lb. bulgaricus/S. thermophilus association on milk acidification and the magnitude of S. thermophilus population dominance at the end of fermentation. In all mixed cultures, the sequential growth of S. thermophilus then of Lb. bulgarius and finally of both bacteria was observed. Although proteinase PrtS was essential to S. thermophilus growth in milk in pure culture, it had no effect on bacterial growth and thus on the final pH of mixed cultures in the presence of PrtB. In contrast, proteinase PrtB was necessary for the growth of S. thermophilus, and its absence resulted in a higher final pH. From these results, a model of growth of both bacteria in mixed cultures in milk is proposed.

The Sca permease in the oral bacterium Streptococcus gordonii is a member of a family of ATP-binding cassette (ABC)-type transporters for manganese (Mn(2+)) and related cations that are associated with streptococcal virulence in a number of infection models. Since Mn(2+) has a protective function against oxidative damage in a variety of bacteria, we have investigated the role of Sca permease in oxidative stress tolerance in Streptococcus gordonii. A single Mn(2+)-dependent superoxide dismutase (SOD), encoded by sodA, is expressed by S. gordonii and was >10-fold up-regulated under oxidative stress conditions. Inactivation of sodA resulted in increased susceptibility of S. gordonii cells to growth inhibition by dioxygen (O(2)), and to killing by paraquat (a superoxide anion generator) and by hydrogen peroxide (H(2)O(2)). Expression of thiol peroxidase, encoded by the tpx gene located immediately downstream of the scaCBA operon, was also up-regulated under oxidative conditions. Inactivation of tpx led to increased susceptibility of cells to H(2)O(2), but not to O(2) or paraquat. In low-Mn(2+) medium (0.01 micro M Mn(2+)) sodA and tpx genes were transcriptionally down-regulated, SOD activity was reduced and cells were more sensitive to growth inhibition by O(2). A Sca permease-deficient (scaC) mutant showed further reduced SOD activity and hypersensitivity to O(2) in medium containing <0.1 micro M Mn(2+). These results demonstrate that the Sca (Mn(2+)) permease in S. gordonii is essential for protection against oxidative stress.

Fibronectin binding is considered to be an important virulence factor in streptococcal infections. Adhesion of the oral bacterium Streptococcus gordonii to immobilized forms of fibronectin is mediated, in part, by a high molecular mass wall-anchored protein designated CshA. In this study, a second fibronectin-binding protein of S. gordonii is described that has been designated as FbpA (62.7 kDa). This protein, which is encoded by a gene located immediately downstream of the cshA gene, shows 85 and 81% identity to the fibronectin-binding proteins PavA, of Streptococcus pneumoniae, and FBP54, of Streptococcus pyogenes, respectively. Purified recombinant FbpA bound to immobilized human fibronectin in a dose-dependant manner, and isogenic mutants in which the fbpA gene was inactivated were impaired in their binding to fibronectin. This effect was apparent only for cells in the exponential phase of growth, and was associated with reduced surface hydrophobicity and the surface expression of CshA. Cells in the stationary phase of growth were unaffected in their ability to bind to fibronectin. By utilizing gene promoter fusions with cat (encoding chloramphenicol O-acetyltransferase), it was demonstrated that cshA expression was down-regulated during the exponential phase of growth in the fbpA mutant. Expression of fbpA, but not cshA, was sensitive to atmospheric O2 levels, and was found to be up-regulated in the presence of elevated O2 levels. The results suggest that FbpA plays a regulatory role in the modulation of CshA expression and, thus, affects the adhesion of S. gordonii to fibronectin.

Two species of dairy streptococci, Streptococcus waius and Streptococcus macedonicus, were originally characterized by 16S-23S intergenic spacer sequence analysis, random amplified polymorphic DNA fingerprinting, PFGE analysis and DNA-DNA reassociation experiments. All genetic data suggested that S. waius strains belong to the previously described species S. macedonicus. Likewise, the phenotypic characterization showed that strains of S. macedonicus and S. waius were highly related and easily differentiated from the closest phylogenetic neighbour, Streptococcus bovis, principally by their failure to produce a blackening reaction in medium containing aesculin. The utilization of maltose and cellobiose by S. macedonicus/S. waius strains allowed their differentiation from the most studied dairy species, Streptococcus thermophilus. On the basis of genetic and phenotypic data S. macedonicus and S. waius species should be considered synonyms and S. macedonicus has the priority.

Previous research with Streptococcus mutans and other oral streptococci has demonstrated that the acid shock of exponential-phase cells (pH 7.5 to 5.5) resulted in the induction of an acid tolerance response (ATR) increasing survival at low pH (3.5-3.0). The current study was designed to determine whether two fresh isolates, H7 and BM71, and two laboratory strains, Ingbritt and LT11, were capable of a stationary-phase ATR as estimated by a survival test at pH 3.5 for 3 h. All four strains were unable to generate a stationary-phase ATR under control conditions at pH 7.5, with the exception of a burst of survivors in the transition between the exponential and stationary phases when the carbon source (glucose) was depleted. Adaptation at pH 5.5 resulted in the expected pH-dependent exponential-phase ATR, but only the fresh isolates exhibited a stationary-phase ATR at this pH. Glucose starvation of cells in complex medium was shown to enhance acid tolerance for the fresh isolates, but not the laboratory strains. This tolerance was, however, greatly diminished for all strains in a defined medium with a low concentration of amino acids. Growth of strain H7 in complex medium resulted in the formation of at least 56 extracellular proteins, nine of which were degraded in the early stationary phase following the induction of proteolytic activity during the transition period. No proteolytic activity was observed with strain LT11 and only 19 extracellular proteins/peptides were apparent in the medium with only one being degraded in the early stationary phase. Strain H7 was also shown to have two- to fourfold higher levels of intracellular glycogen in the stationary phase than strain LT11. These results suggest that S. mutans H7 possessed the required endogenous metabolism to support amino acid/peptide uptake in the early-stationary phase, which resulted in the formation of basic end products that, in turn, contributed to enhanced intracellular pH homeostasis.

Use of green fluorescent protein (GFP) as a molecular reporter is restricted by several environmental factors, such as its requirement for oxygen in the development of the fluorophore, and its poor fluorescence at low pH. There are conflicting data on these limitations, however, and systematic studies to assess the importance of these factors for growing bacterial cultures are lacking. In the present study, homogeneous expression of the gfpmut3* gene directed by a synthetic constitutive lactococcal promoter was demonstrated in batch cultures and in biofilms of Streptococcus gordonii DL1. A lower limit of oxygen concentration for maturation of the GFP fluorophore was determined: fluorescence was emitted at 0.1 p.p.m. dissolved oxygen (in conventionally prepared anaerobic media lacking reducing agents), whereas no fluorescence was detected in the presence of 0.025 p.p.m. dissolved oxygen (obtained by addition of L-cysteine as reducing agent). When an anaerobically grown (non-fluorescent) >50 microm thick biofilm was shifted to aerobic conditions, fluorescence could be detected within 4 min, reaching a maximum over the next 16 min. It was not possible to detect any fluorescence gradients (lateral or vertical) within the >50 microm thick biofilm, and fluorescence development after the shift to aerobic conditions occurred throughout the biofilm (even at the substratum). This suggests that oxygen gradients, which might result in reduced GFP fluorescence, did not exist in the >50 microm thick biofilm of this organism. Production of lactic acid and the subsequent acidification in batch cultures of S. gordonii DL1 led to a decrease in fluorescence intensity. However, severe pH reduction was prevented when the bacterium was grown as a biofilm in a flowcell, and a homogeneous distribution of a strong fluorescence signal was observed. These findings show that GFP can be applied to studies of oxygen-tolerant anaerobic bacteria, that densely packed, flowcell-grown biofilms of S. gordonii do not develop oxygen gradients inhibitory to GFP fluorescence development, and that the often transient nature of GFP fluorescence in acid-producing bacteria can be overcome in flowcells, probably by the elimination of metabolic by-product accumulation.

The proliferation of yeasts in the mixed bacterial and fungal biofilms colonising silicone rubber voice prostheses in laryngectomised patients is the main cause of malfunctioning of the valve mechanism on the oesophageal side of the prostheses. Indwelling voice prostheses usually have to be replaced every 3-4 months. The consumption of probiotic bacteria is largely motivated by health claims related to the urogenital and lower digestive tract, but not to the upper digestive tract. The present study examined the influence of probiotic bacteria on the prevalence of yeasts in oropharyngeal biofilms on silicone rubber voice prostheses, as formed in a modified Robbins device. Exposure of oropharyngeal biofilms on voice prostheses to suspensions of Bifidobacterium infantis 420 or Enterococcus faecium 603 did not significantly reduce the number of yeasts in the biofilm. However, suspensions of Lactobacillus fermentum B54, L. rhamnosus 744 or L. lactis cremoris SK11 led to a reduction in the number of yeasts harvested from the voice prostheses. Suspensions of L. casei Shirota and Streptococcus thermophilus B significantly reduced the number of yeasts in the biofilm to 39% and 33%, respectively. The reduction brought about in yeast prevalence in the mixed biofilm was greatest by exposure to a suspension of L. lactis 53, with yeast prevalence only 4% of the control. In conclusion, the study demonstrated that the prevalence of yeasts in oropharyngeal biofilms on silicone rubber voice prostheses might be controlled by consumption of probiotic bacteria.

A collection of 267 consecutively isolated Streptococcus anginosus strains was screened for the prevalence of previously described 'motile' strains by hybridization with oligonucleotide probes in a reverse line blot assay. The motile strains represented 101 (37.8%) of the S. anginosus strains. The vast majority of these strains fermented mannitol and raffinose, whereas most other S. anginosus strains did not (P<0.001). Most (83/101) of the motile strains were recovered from the urogenital tract (including five strains from neonatal surveillance cultures) and only a minority (36) of them were associated with infection-related samples (P<0.001). Strains that hybridized with the same oligonucleotide probes as the type strain S. anginosus ATCC 33397T (= NCTC 10713T) were designated ATCC-like strains. They accounted for 74 (27.7%) of the strains examined, were commonly distributed among the different body parts and were significantly more associated with infection-related samples. Three other hybridization patterns were recognized in the reverse line blot assay, ribogroup I (n = 51), ribogroup II (n = 21) and ribogroup III (n = 19). Ribogroup II strains were significantly more frequently recovered from the abdominal cavity and were associated with infection-related samples. Ribogroup I included the majority of the S. anginosus strains that carried Lancefield group C. Comparison of the nearly complete 16S rRNA sequence of two representative strains of each ribogroup revealed that all five ribogroups were closely related (>97% sequence similarities), and that most sequence divergences between the ribogroups occurred in the 1024-1064 bp region of the 16S rRNA gene. The present data confirm the heterogeneity within the S. anginosus species.

Adhesion of Candida albicans to saliva-coated surfaces is an important early step in the colonization of the oral cavity. C. albicans cells also adhere to several species of oral streptococci including Streptococcus gordonii, Streptococcus oralis and Streptococcus sanguinis in what are believed to be multi-modal interactions. It is now demonstrated that incubation of streptococcal cells of these species with human parotid saliva further promotes the adhesion of C. albicans cells by up to 2-3-fold. Various species of streptococci were shown to adsorb different protein components of parotid saliva to their cell surfaces. The basic proline-rich proteins (bPRPs), to which C. albicans cells bind on nitrocellulose blot overlay, were strongly adsorbed to the surface of S. gordonii cells but not to S. oralis cells. Parotid saliva that was pre-adsorbed with S. gordonii cells and then applied to hydroxylapatite beads was <50% effective at supporting adhesion of C. albicans compared with control (non-adsorbed) saliva, demonstrating that bPRPs are major pellicle receptors. C. albicans cells did not adsorb bPRPs from fluid-phase parotid saliva. Following size-exclusion chromatography of parotid saliva samples, pooled fractions enriched in bPRPs promoted maximal adhesion of C. albicans to S. gordonii cells. The results demonstrate that C. albicans cells recognize only surface-bound forms of bPRPs and suggest that these proteins adsorbed to enamel or to streptococcal surfaces promote C. albicans adhesion and oral colonization.

The 6.5 kb cryptic plasmid pCI65st from Streptococcus thermophilus NDI-6, a strain isolated from the Indian fermented milk dahi, was subcloned and sequenced. Five putative ORFs were identified. ORF1 could encode a 315 aa polypeptide almost identical to the RepA protein of previously sequenced S. thermophilus plasmids, indicating that pCI65st is one of the pC194 group of small gram-positive rolling-circle plasmids. ORFs 2 and 4 were virtually identical and could specify proteins of approximately 150 aa with significant similarity to the small heat-shock proteins described from a variety of gram-positive bacteria. ORF3 could encode a 415 aa protein similar to enolase, an enzyme involved in glycolysis and gluconeogenesis. ORF5 could encode a 412 aa protein which had high similarity to the HsdS (specificity) proteins of type I restriction-modification systems. Variants of strain NDI-6 which lacked pCI65st were readily isolated after subculture of the parent strain at 32 degrees C. The plasmid-bearing parent culture was significantly more resistant to a temperature shift from 42 degrees C to 62 degrees C than its plasmid-free variant and expressed proteins which corresponded with the predicted translation products from ORF2 and ORF4. In addition, plasmid-free mutants were lysed in broth by bacteriophages to which the parent culture was resistant.

Cell-surface polypeptide CshA (259 kDa) mediates multiple adherence interactions of Streptococcus gordonii. By generating a chromosomal cshA promoter (p-cshA)-cat gene fusion and measuring both CAT enzyme activity and cat mRNA levels, it was shown that cshA is expressed maximally in cells in the late exponential phase of growth in batch culture. The expression of CAT enzyme activity from the p-cshA-cat promoter fusion was 28% decreased in early stationary phase cell extracts of mutant strain OB528 in which the hppA (oligopeptide-binding lipoprotein) gene was insertionally inactivated. This effect was correlated with proportionally reduced cell-surface expression of CshA protein and with impaired adherence of hppA mutant cells to cells of an oral Actinomyces naeslundii strain. cshA promoter activity was enhanced in streptococcal cells that were incubated in conditioned culture medium as opposed to fresh medium, but this did not occur in an hppA genetic background. It is suggested that HppA is necessary for the response of cells to an extracellular factor that modulates cshA transcription, and hence affects cell-surface CshA expression and streptococcal cell adherence properties.

The effect of Streptococcus sanguis, S. salivarius, Escherichia coli and Porphyromonas gingivalis on the adhesion of Candida albicans and C. krusei to human buccal epithelial cells (BEC) was investigated with a modified membrane filter system. The filters (12 microns diameter pores) acted as a support for the BEC which were pre-exposed to known concentrations of bacterial suspensions (for 45 min-1 h), and then re-incubated with standardised concentrations of yeast suspensions for various periods. The BEC with adherent yeasts were then transferred on to a glass slide, gram-stained and counted by light microscopy. Three of the four bacterial species significantly suppressed adhesion of C. albicans to BEC; S. sanguis had no effect. Both S. sanguis and S. salivarius suppressed adhesion of C. krusei to BEC pre-exposed to three different bacterial concentrations, although variable results were obtained with P. gingivalis and E. coli. Significant differences in the relative adhesion of C. albicans and C. krusei to BEC were also recorded. These results indicate that the adhesion of yeasts to BEC is modulated both by the composition and the quantity of the pre-existing bacterial flora on the BEC.

Streptococcus gordonii gave rise to beta-haemolytic variants (Bhp+ for beta-haemolysin production) at frequencies of 10(-4)-10(-3) on agar medium containing washed horse erythrocytes. Bhp+ variants reverted to the wild-type alpha-haemolytic phenotype (Bhp-) at the same frequencies. There was a significant probability (> or = 0.1) that phase variation in Bhp and phase variation in the previously described Spp (sucrose promoted phenotype) would occur concomitantly, but there was no correlation between these phenotypes. There was evidence also of independent phase variation in adhesion to saliva-coated hydroxyapatite (Asp for adhesion to salivary pellicles), in lactose-sensitive coaggregation (Cls for coaggregation, lactose-sensitive) and in the concentrations of particular cell surface antigens (Cap for cell antigen profile) in strains that had undergone phase changes in Spp and/or Bhp. Phase variation in all these phenotypes were transitions between high and low levels of activity and each appeared to occur as an independent event. Significant associations (P << 0.0001 by contingency table analysis) between particular phenotypes such as Bhp and Asp and between Asp, Cls and Cap phenotypes, however, were apparent. The results suggest that S. gordonii cells become predisposed to phase variation and that the resulting independent phenotypic changes may give rise to phenotypically diverse streptococcal populations able to accommodate rapid and transient environmental changes in the mouth.

The "Streptococcus milleri" (SMG) group have been shown to possess factors in vitro that may be involved in pathogenesis. All SMG strains are able to bind fibronectin via a cell-surface protein; the binding ranged from 12 to 198 mol/cell. Strains also bound to platelet-fibrin or fibrin clots and fibrinogen, giving maximum adhesion values of 16.5%, 21.8% and 151 mol/cell respectively. Members of the species S. constellatus produced thrombin-like activity. Lancefield group C SMG aggregated rat platelets, a bacterial cell-surface protein acting as mediator in the reaction. Most of the in-vitro factors did not correlate with each other, an indication that SMG strains possess a wide variety of pathogenic properties that may be involved in the production of abscesses or endocarditis. However, there was a correlation between the binding of large amounts of fibrinogen ( > 100 mol/cell) and the ability to aggregate platelets. This suggests that fibrinogen binding may aid in platelet aggregation.

Thirty strains from the Streptococcus milleri (anginosus) group (SMG) obtained from various sources were tested for a range of characters that could be associated with pathogenicity and the results were compared with those for type strains of S. sanguis, S. mutans and S. pyogenes. The SMG strains were heterogeneous in all tests. Most (18) belonged to one of the Lancefield groups with group F predominating. Adherence of strains isolated from abscesses to buccal epithelial cells was greater than that of other strains (p = 0.033). Compared with strains of S. sanguis, SMG strains were generally not aggregated by human saliva. They differed from the type strain of S. pyogenes in their relative ability to bind fibrinogen and fibronectin; they were less effective in binding fibrinogen (0.33-4.28% cf. 22% for S. pyogenes) and generally more effective in binding fibronectin (0.49-12.37% cf. 0.95%). Strains isolated from infections were statistically better at binding fibronectin than other strains (p less than 0.001). The ability of strains to adhere to saliva-coated hydroxyapatite (SHA) varied 10-fold, from 0.16-16.35%. The amount of fibronectin bound by SMG strains correlated with their ability to adhere to SHA (p less than 0.001). The hydrophobicity of the strains, as measured in the hexadecane partition assay, ranged from 0.0% to 99.0%. Some strains carried both positive and negative cell-surface charges and some strains with a highly hydrophobic cell surface also possessed a relatively high cell-surface charge. A minority of strains possessed a net positive cell-surface charge. Neither hydrophobicity nor cell-surface charge was related to the capacity of strains to adhere to SHA. Strains of SMG co-aggregated weakly with strains of Veillonella parvula, V. dispar, Actinomyces viscosus and A. naeslundii.

Forty-four strains of non-haemolytic streptococci, from a variety of sites, that required CO2 for aerobic growth were identified as Streptococcus milleri. Of these strains, 40 (90%) possessed the Lancefield group-F antigen, the remainder being non-groupable with antisera to the group antigens A, C, F and G.

The inhibitory activity of seven strains of faecal streptococci against 34 strains of Clostridium difficile was examined in vitro after growth of the streptococci for 24 and 48 h. All strains of C. difficile were inhibited at 48 h but at 24 h the inhibition was variable. Streptococcus faecium, a group D streptococcus and an ungroupable streptococcus exhibited the most striking inhibitory activity. Lowering of pH of the medium occurred at the site of inhibition, but the pH change alone did not explain the inhibition of C. difficile. This antagonism may be related in vivo to the resistance to intestinal colonisation by C. difficile exhibited by the normal bowel flora, and in vitro to failures to isolate C. difficile from faecal specimens.

An agglutination assay for detecting intermicrobial adherence between the cells of Candida albicans and various oral bacteria is described. Strains of Streptococcus sanguis, S. salivarius, S. mutans, S. mitis, Fusobacterium nucleatum and Actinomyces viscosus all coagglutinated with C. albicans. No interaction could be demonstrated between the cells of Bacteroides melaninogenicus and those of C. albicans. Preliminary investigations of these interactions suggest that binding of F. nucleatum and A. viscosus to C. albicans is mediated by bacterial proteins, possibly lectins. Other mechanisms must account for the binding of oral streptococci to C. albicans. The possible implications of these findings in relation to oral mucosal colonisation and oral candidal clearance are discussed.

Coaggregation of Bacteroides gingivalis and other black-pigmented bacteroides with several oral bacteria was studied with "reagent" strains specially prepared by methods that have been described previously. B. gingivalis coaggregated with Veillonella, Capnocytophaga and Actinomyces spp., but not with any Streptococcus spp. Coaggregation of B. gingivalis with other bacteria was inhibited and reversed by lactose. Of the asaccharolytic black-pigmented bacteroides, only B. gingivalis demonstrated any coaggregation with other bacteria, whereas within the saccharolytic species, B. loescheii showed a marked ability to coaggregate with several species of oral bacteria. This property of coaggregation by B. gingivalis may be an important factor in the pathogenesis of periodontal infections.

Eleven strains of Mycobacterium tuberculosis and 13 other species of mycobacteria were examined for susceptibility to 26 strains of viridans streptococci. Susceptibility was detected by the simultaneous antagonism technique, but not by tests of deferred antagonism. It is concluded that in-vitro inhibition of mycobacterial growth by actively growing streptococci is due to a peroxide-mediated antagonism which is too variable to be of value for identification. However, a standard bactericidal test with hydrogen peroxide is worth investigation because this might provide a simple inexpensive aid for the identification of M. avium-intracellulare serotypes.