Storage-related stability and antimicrobial efficacy of bottled, neutral-pH Electrolysed Oxidising Water.

OBJECTIVES: Denture stomatitis is prevalent in older people and poses serious health risks. Ready-to-use (RTU) neutral-pH Electrolysed Oxidizing Water (EOW) is an effective environmental disinfectant used in residential care settings and geriatric wards. However, the influence of storage on stability and effectiveness for denture disinfection has not been established. This research investigated the storage-related stability and antimicrobial activity of RTU EOW, and its efficacy against Candida albicans biofilms formed on denture resin. METHODS: The pH, oxidation/reduction potential (mV), available chlorine content (mg/L) and [HOCl] (mM) of RTU EOW (Envirolyte, New Zealand) solutions (n = 22) were measured from bottle opening to 28 days following storage at 4 °C, room temperature (RT) or 37 °C. Staphylococcus aureus and C. albicans cells were incubated in 80% EOW for contact times (CTs) up to 15 min and colony-forming units (cfu) determined. Minimum inhibitory concentrations (MIC90 EOW-HOCl) after CTs up to five minutes were determined for S. aureus and C. albicans reference strains and clinical isolates. C. albicans-denture resin disc biofilms were assessed after a five-minute CT with undiluted EOW by XTT-metabolic activity assay. RESULTS: [HOCl] remained stable when RTU EOW was stored at 4 °C or RT for five months after manufacture. One-minute CT resulted in log10 cfu reductions of >6 for S. aureus and >5 for C. albicans. Mean MIC90 for five-minute CT was 37 µM (S. aureus) and 54 µM (C. albicans). Undiluted EOW reduced C. albicans biofilm metabolic activity by 86%. CONCLUSIONS: RTU neutral-pH EOW is stable over five-months storage and is an effective denture disinfectant. CLINICAL SIGNIFICANCE: The efficacy of the RTU neutral EOW against C. albicans isolates and biofilms formed on denture resin surfaces supports its use as a denture disinfectant and can inform future research to assess its potential for preventing denture-related oral Candida infections in the older population, especially in resource-limited communities.

Anti-biofilm activity of silver nanoparticle-containing glass ionomer cements.

OBJECTIVE: To develop a silver nanoparticle (AgNP) formulation for incorporation into glass ionomer cements (GICs) which minimises biofilm growth on restoration surfaces. METHODS: GICs, Fuji IX, Ketac Molar, and Riva Selfcure were modified with 6, 10 and 24 µg per GIC capsule of α-lipoic acid-capped AgNPs. Monoculture biofilms of Streptococcus mutans were cultured (72 h) on GIC specimens (n = 3) and biofilm accumulation was quantified using a viability stain with confocal laser scanning microscopy. Compression strength and flexural strength (CS & FS) were measured according to ISO 9917-1:2007 (n = 8, n = 25). GIC colour was measured at 0, 1, and 14 days following AgNP incorporation using a digital spectrophotometer. Silver release from AgNP-modified GIC specimens was monitored at 1, 3, 7 and 14 days using inductively coupled plasma-mass spectrometry. RESULTS: AgNP-modified Fuji IX demonstrated the greatest reduction in biofilm accumulation, with 10 µg Ag/capsule inhibiting biofilm formation by 99%. Ketac Molar and Riva Selfcure required 24 µg Ag/capsule to achieve 78% biofilm reduction. AgNP-modified GICs demonstrated significantly higher CS and FS than sintered silver-containing GICs, and possessed equivalent or higher strength values when compared to unmodified GICs. The colour shades of AgNP-modified GICs were more comparable to VITA shades of non-modified GICs than were sintered silver-containing GICs. The silver (≥99.6%) remained within the GIC for at least two weeks following incorporation. SIGNIFICANCE: AgNP-modified GICs exhibited significant antibiofilm activity and retained mechanical properties equivalent or superior to non-modified GICs. AgNP-modified GICs could reduce bacterial colonisation on and around restorations thereby reducing restoration failure caused by secondary caries.

Antimicrobial Activity of a Colloidal AgNP Suspension Demonstrated In Vitro against Monoculture Biofilms: Toward a Novel Tooth Disinfectant for Treating Dental Caries.

A novel silver nanoparticle (AgNP) formulation was developed as a targeted application for the disinfection of carious dentine. Silver nitrate (AgNO3) was chemically reduced using sodium borohydrate (NaBH4) in the presence of sodium dodecyl sulfate (SDS) to form micelle aggregate structures containing monodisperse 6.7- to 9.2-nm stabilized AgNPs. AgNPs were characterized by measurement of electrical conductivity and dynamic light scattering, scanning electron microscopy, transmission electron microscopy, and inductively coupled plasma mass spectrometry. Antimicrobial activity of AgNPs was tested against planktonic cultures of representative gram-positive and gram-negative oral bacteria using well diffusion assays on tryptic soy broth media and monoculture biofilms grown with brain heart infusion ± sucrose anaerobically at 37°C on microtiter plates. Biofilm mass was measured by crystal violet assay. Effects were compared to silver diamine fluoride and chlorhexidine (negative controls) and 70% isopropanol (positive control) exposed cultures. In the presence of AgNPs, triplicate testing against Streptococcus gordonii DL1, C219, G102, and ATCC10558 strains; Streptococcus mutans UA159; Streptococcus mitis I18; and Enterococcus faecalis JH22 for planktonic bacteria, the minimum inhibitory concentrations were as low as 7.6 µg mL-1 and the minimum bacteriocidal concentrations as low as 19.2 µg mL-1 silver concentration. Microplate readings detecting crystal violet light absorption at 590 nm showed statistically significant differences between AgNP-exposed biofilms and where no antimicrobial agents were used. The presence of sucrose did not influence the sensitivity of any of the bacteria. By preventing in vitro biofilm formation for several Streptococcus spp. and E. faecalis, this AgNP formulation demonstrates potential for clinical application inhibiting biofilms.

Bacterial localization and viability assessment in human ex vivo dentinal tubules by fluorescence confocal laser scanning microscopy.

AIM: To develop a convenient method for the localization and quantification of live and dead bacteria in human ex vivo mineralized dentinal tubules. METHODOLOGY: The roots from human single-rooted teeth (n = 12) were infected with Enterococcus faecalis V583 and either treated with calcium hydroxide paste or left untreated; six control roots were uninoculated and untreated. Following further incubation, roots were stained with fluorescent DNA-binding reagents, washed thoroughly, sectioned and examined by confocal laser scanning microscopy. Computer-assisted determinations of fluorescence (bacterial viability) were compared statistically. RESULTS: Bacteria were distributed in the tubules throughout the length of the roots but tubule penetration distance was slightly reduced in the apical sections. There was no significant difference in bacterial tubule penetration between roots from different teeth and small standard deviations indicated reproducibility appropriate for experimental application. Following treatment with calcium hydroxide paste, live and dead bacteria were readily distinguishable by contrasting green and red fluorescence. Bacterial viability determinations amongst roots treated in the same way were not significantly different, and the small standard deviation is commensurate with experimental application. CONCLUSIONS: Fluorescent viability staining is a convenient, accurate and reproducible method for localizing and quantifying live and dead bacteria in human ex vivo mineralized dentinal tubules.

Genotypic comparison of bacteria recovered from human bite marks and teeth using arbitrarily primed PCR.

AIMS: This study assessed, for forensic purposes, the feasibility of genotypically matching oral streptococci recovered from recent human bite marks with those from the teeth of the biter. METHODS AND RESULTS: Streptococci were isolated from the incisors of eight volunteers. Arbitrarily primed PCR (AP-PCR) distinguished 106 streptococcal genotypes among the participants, each harbouring at least eight distinct strains. In a crime simulation, a sample from an experimental bite mark was analysed by an experimenter unaware of its origin. The bacteria were unambiguously matched to the biter by comparing the amplicon profiles with those from the eight participants. In contrast, bacteria from an additional bite mark (not generated by one of the original participants) could not be matched to any of the eight participants. Between 20 and 78% of catalogued bacterial genotypes were recovered 12 months later from each participant. Throughout the study period, none of the bacterial genotypes were shared between participants. CONCLUSIONS: Streptococci isolated from recent bite marks can be catalogued by AP-PCR and matched to the teeth responsible for the bite. SIGNIFICANCE AND IMPACT OF THE STUDY: The study provides 'proof of concept' that genotypic analysis of streptococci from bite marks may provide valuable forensic evidence in situations where the perpetrator's DNA cannot be recovered.

An in vivo study of the plaque control efficacy of Persica: a commercially available herbal mouthwash containing extracts of Salvadora persica.

AIM: This study compared the oral health efficacy of Persica mouthwash (containing an extract of Salvadora persica) with that of a placebo. DESIGN: In a double-blind, cross-over trial, participants were randomly allocated to use either the Persica mouthwash or a placebo for a three-week period. Plaque accumulation, gingival bleeding and the salivary concentrations of mutans streptococci (MS) were measured before and immediately following the experimental period. After an eight-week 'washout' period, the study was repeated with participants using the alternative mouthwash. PARTICIPANTS: Twenty-eight healthy students (aged between 18 and 42 years) volunteered to take part in this investigation. RESULTS: Compared with the pre-treatment values, both placebo and experimental groups demonstrated significantly reduced gingival bleeding (p < 0.01). Plaque scores were not significantly reduced following use of either Persica or the placebo. However, the use of Persica, but not the placebo, resulted in significant reduction in the carriage of MS (p < 0.05). CONCLUSION: Use of Persica mouthwash resulted in improved gingival health and lower carriage rate of cariogenic bacteria when compared with the pre-treatment values. The placebo (vehicle control) also improved gingival health significantly. Neither the Persica nor the placebo reduced the accumulation of dental plaque.

Isolation and genotypic comparison of oral streptococci from experimental bitemarks.

The feasibility of recovering and genotypically comparing oral bacteria from bitemarks for forensic purposes was assessed experimentally. Volunteers firmly bit their own upper arms and bitemarks were sampled at intervals to recover viable Streptococcus isolates. The recoverability of bacteria decreased over time but an average of more than one thousand viable organisms was recovered 24 hrs after biting, provided the site remained relatively undisturbed. Physical exertion, manual rubbing and application of moisturizing lotion all decreased bacterial recoverability compared to controls. Streptococci could also be recovered from bites inflicted on various fabrics. Genomic profiles (DNA "fingerprints") of bacteria recovered from bitemarks could be identified exclusively with those from the teeth of the individual responsible. These findings suggest that a bacterial genotyping approach to bitemark analysis could have forensic application in situations where the perpetrator's DNA cannot be recovered from an oral contact site.

IL-1 genotype and adult periodontitis among young New Zealanders.

Several recent studies have investigated the association between interleukin-1 genotype and periodontitis in clinical samples, where generalizability is an issue. The aim of this study was to investigate the association between adult periodontitis and IL-1 genotype in a population-based sample of 26-year-olds. Based on probing depth (PD) measurements, participants were divided into three disease groups: "Severe" (1+ teeth with 5+mm PD; N = 25), "Moderate" (2+ teeth with 4+mm PD; N = 36), and "Controls" (the remainder; N = 800). The "periodontitis-associated genotype" (PAG; Kornman et al., 1997) was present in 20.0% of the "Severe" group and in 34.8% of "Controls", whereas the IL-1A(+4845) [1,1]/IL-1B(+3953) [2,2] genotype was present in 12.0% and 0.9%, respectively. After controlling for sex, smoking status, and plaque levels, we found that those with IL-1B(+3953) [1,1]/IL-1A(+4845) [2,2] had 12.3 times the odds of being in the "Severe" group. Analysis of these data suggests that the IL-1A(+4845) [1,1]/IL-1B(+3953) [2,2] genotype is associated with periodontal disease in this young population. Future periodontal data collections as this cohort ages are required to confirm the predictive value of that genotype.

The effects of dietary ferric iron and iron deprivation on the bacterial composition of the mouse intestine.

The influence of dietary ferric iron on the intestinal microbiota of mice was investigated with a view to promoting benign lactic acid bacteria (which have minimal iron requirements) in order to enhance colonization-resistance potential. Three groups of eight mice received a diet differing only in iron content, for a period of 12 weeks. Dietary iron deprivation resulted in overall increased small intestinal bacterial populations, including lactic acid bacteria, but these differences were generally not significant (p > 0.05). With the exception of coliforms, all examined bacterial groups (anaerobes, micro-aerophiles, lactobacilli, and enterococci) were significantly (p < 0.05) elevated in the colons of iron-deprived mice. The relatively low numbers of total anaerobes in the colons of iron-replete and iron-overloaded mice suggested that, as well as promotion of bacteria under iron-deprived condition, provision of ferric iron suppressed bacteria, probably by oxidation of normally reduced environments.

Dental caries and the microbial ecology of dental plaque: a review of recent advances.

Our understanding of the microbial ecology of dental plaque has rapidly grown with recent developments in the techniques of molecular biology. In particular, knowledge of the mechanisms underlying the acquisition, establishment, pathogenicity, and evolution of the group of organisms responsible for dental caries--the mutans streptococci--has expanded to the point that we can now contemplate new opportunities for caries prevention. These advances reinforce developing concepts of dental plaque as an interdependent, interacting community of specialised organisms with an ability to rapidly adapt conferred by gene structures that facilitate the expeditious modular rearrangement of protein components.

Candida albicans: adherence, signaling and virulence.

The focus of this symposium was to present new information on the morphogenesis of Candida albicans, particularly how it relates to signal transduction pathways and other genes involved in the regulation of morphogenesis. In addition, we discuss the role of adherence and colonization of the oral cavity by the organism and discuss the role of mannan as an adhesin that recognizes the human red blood cell. C. albicans utilizes at least two signal pathways to regulate its conversion from a yeast form to filamentous growth (hyphae). One of these two pathways is similar to the Saccharomyces cerevisiae pseudohyphal/mating pathway, which utilizes the regulatory protein, Cphlp. The other pathway is not totally defined but requires a second regulatory protein, referred to as Efg1p. Other signal pathways may exist, which include a two-component histidine kinase and response regulator proteins. The latter pathway(s) may include proteins such as Chk1p, Ssk1p, Shi1p and Cos1p/Nik1p. Mutations in strains, which specifically target these proteins, result in morphogenesis defects and avirulence or attenuation of strains. A growth regulatory gene has also been recently defined whose expression is associated with growth cessation and which appears to be a necessary prerequisite in conversion of the organism to a filamentous growth form. Starvation of yeast cells induces exponentially grown cells (and usually non-germinative) to germinate. This phenomenon is also observed in cells that are transiently treated with metabolic inhibitors. During each of these treatments (starvation, metabolic inhibition), expression of a growth regulatory gene (CGRI) increases. Adherence of C. albicans to host cells and tissues is complex; several proteins, which appear to have host recognition functions, have been defined. In the oral cavity, C. albicans selectively adheres to salivary proteins, which are absorbed to many oral surfaces. This mechanism enables the cells to colonize surfaces of the oral cavity. An understanding of these interactions may lead to strategies to prevent oral disease. Mannan from C. albicans may provide a host recognition function for C. albicans. Recent experiments indicate that mannan binds to human red blood cells and causes hemolysis. Binding of mannan to the band 3 protein of human red blood cells has been established. This activity may be associated with the ability of the organism to utilize hemoglobin (and iron).

Bacteriocin production and sensitivity among coaggregating and noncoaggregating oral streptococci.

Twenty-one oral Streptococcus isolates of known interbacterial coaggregation groups were tested against one another (as both producers and indicators) to detect bacteriocin-like inhibitory activity. In agar-based antagonism tests, seven strains produced small inhibitory zones (< or = 3 mm diameter) but in liquid medium, only strain Streptococcus gordonii DL1 (Challis) produced a detectable antibacterial action (bacteriocin STH1). Five strains were sensitive to bacteriocin STH1, but neither the production of nor the sensitivity to any of the antagonistic agents correlated with coaggregation groupings. Four strains (C219, 903, 118 and Wicky) developed stable resistance in response to the bacteriocin, whereas one isolate (strain 34) remained sensitive following repeated bacteriocin exposure. With one exception (strain 903), bacteriocin STH1-sensitive strains were competent for genetic transformation, but not all competent strains were bacteriocin-sensitive. Bacteriocin-resistant derivatives of transformable strains exhibited decreased competence (80-90% reduction) compared with their parent strains.

Effect of treatment concentration on lipopolysaccharide affinity for two alloys.

OBJECTIVE: This study compared gram-negative bacterial lipopolysaccharide (LPS) adherence to and elution from a Type III gold and a Ni-Cr-Be alloy using Escherichia coli LPS. METHOD: One-half of the specimens of each alloy were pre-treated with 500 micrograms non-radiolabeled E. coli LPS for 24 h at 37 degrees C. All disks were then incubated with 0.15, 15 or 150 micrograms radiolabeled E. coli LPS for 24 h at 37 degrees C. To evaluate radiolabeled LPS elution, specimens were transferred to LPS-free water and incubated for 24 h at 37 degrees C. The elution scheme, which consisted of 24 h incubations and subsequent transfer to new LPS-free water, continued for up to 96 h total elution. Radiolabeled LPS adherence and elution was determined through liquid scintillation spectrometry. Control disks not treated with LPS were evaluated throughout the study with an enzymatic assay to ensure that extraneous LPS contamination did not occur. A multifactor ANOVA (p = 0.05) was used to evaluate differences in adherence to alloy specimens based upon alloy type, pretreatment status and [3H]LPS concentration. A repeated measures analysis ANOVA (p = 0.05) was used to evaluate differences in elution patterns among groups over time. Least square means were compared in case of significant effects. RESULTS: Toxin uptake at each treatment concentration was significantly different from the other treatment concentrations. In addition, significantly greater amounts of [3H]LPS eluted from the non-pretreated Ni-Cr-Be alloy following the 0.15 and 15 micrograms radiolabeled [3H]LPS treatment, whereas no difference in elution was found among experimental groups following the 150 micrograms [3H]LPS treatment. SIGNIFICANCE: E. coli LPS, an LPS type representative of enteric bacteria common to the gingival sulcus, has differing affinities for the alloys. This affinity difference could influence periodontal inflammatory processes, thereby resulting in differing tissue responses adjacent to dental restorations fabricated from these materials. The interaction of other LPS types with these alloys could differ.

Detection and comparison of specific hemin binding by Porphyromonas gingivalis and Prevotella intermedia.

A radioligand assay was designed to detect and compare specific hemin binding by the periodontal anaerobic black-pigmenting bacteria (BPB) Porphyromonas gingivalis and Prevotella intermedia. The assay included physiological concentrations of the hemin-binding protein rabbit serum albumin (RSA) to prevent self-aggregation and nonspecific interaction of hemin with cellular components. Under these conditions, heme-starved P. intermedia cells (two strains) expressed a single binding site species (4,100 to 4,600 sites/cell) with a dissociation constant (Kd) of 1.0 x 10(-9) M. Heme-starved P. gingivalis cells (two strains) expressed two binding site species; the higher-affinity site (1,000 to 1,500 sites/cell) displayed a Kd of between 3.6 x 10(-11) and 9.6 x 10(-11) M, whereas the estimated Kd of the lower-affinity site (1.9 x 10(5) to 6.3 x 10(5) sites/cell) ranged between 2.6 x 10(-7) and 6.5 x 10(-8) M. Specific binding was greatly diminished in heme-replete cells of either BPB species and was not displayed by iron-replete Escherichia coli cells, which bound as much hemin in the absence of RSA as did P. intermedia. Hemin binding by BPB was reduced following treatment with protein-modifying agents (heat, pronase, and N-bromosuccinimide) and was blocked by protoporphyrin IX and hemoglobin but not by Congo red. Hemopexin also inhibited bacterial hemin binding. These findings indicate that both P. gingivalis and P. intermedia express heme-repressible proteinaceous hemin-binding sites with affinities intermediate between those of serum albumin and hemopexin. P. gingivalis exhibited a 10-fold-greater specific binding affinity and greater heme storage capacity than did P. intermedia, suggesting that the former would be ecologically advantaged with respect to heme acquisition.

Effect of pH on Porphyromonas gingivalis endotoxin affinity for resins.

This study evaluated the effect of pH on Porphyromonas gingivalis lipopolysaccharide (LPS) affinity for polymethyl methacrylate, polyethyl methacrylate, and polyethyl and polyisobutyl methacrylate resins. Specimens were exposed to 1,010 endotoxin units LPS in potassium phosphate buffer at pH 6, pH 7, or pH 8. Control specimens were incubated in LPS-free water. Sequence I evaluated LPS uptake and release from resin when exposure and elution pH were identical, whereas Sequence II evaluated LPS release from resin when elution pH differed from exposure pH. A slightly acidic pH decreased LPS affinity for all resins compared to pH 7. A slightly alkaline pH increased LPS affinity for the polyethyl methacrylate resin but decreased LPS affinity for the others compared to pH 7. The pH may affect resin-LPS affinity by altering LPS molecular charge.

Porphyromonas gingivalis endotoxin affinity for dental ceramics.

This study evaluated the effects of chemical composition, surface treatment, and initial exposure dose on Porphyromonas gingivalis lipopolysaccharide adherence to and elution from dental ceramics. Lipopolysaccharide, commonly known as endotoxin, can initiate a variety of biologic responses. Opaque, body, and Dicor ceramic disks were individually exposed to 250, 1000, or 2500 EU/ml 3H-lipopolysaccharide and incubated for 24 hours at 37 degrees C. Disks were then transferred to fresh lipopolysaccharide-free water and incubated for up to 96 hours to evaluate elution. Mean initial lipopolysaccharide adherence ranged from 0.397 +/- 0.048 EU/mm2 to 5.056 +/- 0.117 EU/mm2. Greater initial exposure levels resulted in greater adherence, and at higher lipopolysaccharide exposure levels, lipopolysaccharide adherence differences were based on ceramic type. Mean lipopolysaccharide elution levels ranged from 0.063 +/- 0.02 EU/mm2 to 0.00 EU/mm2 at 96 hours for all groups. Greater initial adherence resulted in greater elution. Ceramic type did not affect elution. Surface finish affected elution at the 2500 EU exposure level. The affinity of lipopolysaccharide for dental ceramics could contribute to a periodontal inflammatory process.

Porphyromonas gingivalis lipopolysaccharide affinity for two casting alloys.

With the exception of plaque, the affinity of biologically active bacterial products for restorative materials and the influence of that affinity on periodontal health has not been detailed. This study recognized that Porphyromonas gingivalis endotoxin, which is cell envelope lipopolysaccharide (LPS) produced by a bacterium that is common to the crevicular microbial flora, has an affinity for dental casting alloys. Regardless of surface finish, no difference in LPS initial adherence or elution was recorded between a type III gold or nickel-chromium-beryllium alloy (p > 0.05), but LPS readily adhered and remained attached to both alloys. LPS affinity could contribute to periodontal inflammation in tissues that approximate restorations fabricated from either alloy.

Endotoxin affinity for provisional restorative resins.

PURPOSE: This study compared the relative affinity of Porphyromonas gingivalis and Escherichia coli endotoxin, bacterial cell envelope lipopolysaccharide (LPS), for three provisional resins. MATERIALS AND METHODS: As-polymerized and pumiced polymethyl methacrylate and polyethyl methacrylate resin discs were exposed to 1,000 endotoxin U/mL P. gingivalis or E. coli LPS in water for 24 hours at 37 degrees C, whereas control discs were placed in LPS-free water. LPS-treated discs were transferred at 24-hour intervals to fresh, LPS-free water for up to 96 hours, and the incubated eluates were tested for the presence of LPS. RESULTS: Initial adherence of P. gingivalis LPS to as-polymerized and pumiced-finish resin was a function of resin type, but surface characteristics modified adherence levels. When steady rates of elution were reached at 72 to 96 hours, as-polymerized specimens released significantly greater LPS levels than pumiced samples. Comparison of initial adherence of P. gingivalis and E. coli LPS with pumiced resins showed that adherence was based on a combination of LPS and resin type. P. gingivalis LPS had a greater relative affinity for polyethyl methacrylate, and E. coli LPS has a greater relative affinity for polymethyl methacrylate. Regardless of resin type, P. gingivalis LPS eluted at levels greater than E. coli LPS. CONCLUSIONS: The affinity of LPS for provisional resins seems to be a function of selective interactions based on the chemical nature of the resin, the surface finish of the resin, and the molecular structure of the LPS.

Inhibition of bactericidal and bacteriolytic activities of poly-D-lysine and lysozyme by chitotriose and ferric iron.

In a previous report from this laboratory (N. J. Laible and G. R. Germaine, Infect. Immun. 48:720-728, 1985), evidence was presented to suggest that the bactericidal actions of both reduced (i.e., muramidase-inactive) human placental lysozyme and the synthetic cationic homopolymer poly-D-lysine involved the activation of a bacterial endogenous activity that was inhibitable by N,N',N"-triacetylchitotriose (chitotriose). In the present investigation however, we found that the bactericidal and bacteriolytic action of poly-D-lysine could be prevented only by some commercially available chitotriose preparations and not by others. Analysis by physical and chemical methods failed to distinguish protective chitotriose (CTa) and nonprotective chitotriose (CTi) preparations. CTi and CTa preparations displayed equal capacities to competitively inhibit binding of [3H]chitotriose by immobilized lysozyme and were indistinguishable in their abilities to block the lytic activity of lysozyme against Micrococcus lysodeikticus cells. Elemental analysis revealed significantly higher levels of phosphorus, calcium, iron, sodium, manganese, and copper in CTa. Removal of metals from CTa by chelate chromatography completely abolished the poly-D-lysine-protective capacity. Of the metals detected, only ferric iron (5 to 10 microM) mimicked the protective action of CTa. A Fe(III) concentration of 50 microM was required to inhibit lysozyme (5 micrograms/ml). Both Fe(III) and CTa (but not CTi) quantitatively blocked the labeling of poly-D-lysine by fluorescamine, suggesting that the primary amino groups of the lysine residues participate in iron binding. Thus, it appears that the poly-D-lysine-protective capacity of certain chitotriose preparations was due not to the chitotriose itself but to contaminating metal ions which interact directly with the polycationic agent. In contrast, Fe(III) cannot account for inhibition of either the bactericidal or bacteriolytic activity of lysozyme by chitotriose.

Bacteriocin-like inhibitory activity associated with beta-hemolytic strains of Streptococcus salivarius.

Seven beta-hemolytic Streptococcus salivarius isolates produced bacteriocin-like inhibitory activity in deferred antagonism tests using a set of nine indicator bacteria (I1-I9). Five of these S. salivarius strains (KWF, TOVE-R, K17, K21, and K26) were inhibitory to indicators I2, I5, I6, and I7. Mutated non-hemolytic derivatives showed concomitant loss of inhibitory activity against I2, I5, and I6, but retained activity against I7. Inhibitory activity against I2, I5, and I6 was restored in beta-hemolytic revertants of such mutants. Strain 3638 was inhibitory to all of the indicator organisms except I3, and this pattern of inhibitory activity was retained by non-hemolytic derivatives. It appeared that strain 3638 produced an additional broadly-active inhibitory agent, since a mutant (strain 3638A), which was apparently defective in the production of this inhibitor, retained both the beta-hemolytic and I2-, I5-, I6-, and I7-inhibitory activities. Non-hemolytic derivatives of strain 3638A were inhibitory only to I7. Strain 3638, therefore, appeared to produce at least three inhibitory agents: one active only on I7; another acting on I2, I5, and I6 (and associated with beta-hemolytic activity); and a third apparently active on all of the indicators other than I3. S. salivarius strain JH inhibited all nine indicator strains and possessed a beta-hemolytic character which differed from that of the other strains in being readily eliminated on treatment with the plasmid-curing agent novobiocin. Non-hemolytic derivatives of JH retained inhibitory activity against the complete set of indicators.(ABSTRACT TRUNCATED AT 250 WORDS)