Jerry J. Pollock

A comparison of the inhibition of blastospore viability and germ-tube development in Candida albicans by histidine peptides and ketoconazole

Synthetic homologous peptides of L-histidine, ranging in length from 3 to 64 amino-acid residues, suppressed blastospore viability. Killing activity was dependent upon the peptide molecular size and concentration, and the time of cell exposure to the agent, but was independent of cell concentration in the range 10(5)-10(7) colony-forming units (c.f.u.) per ml. A 25 amino-acid residue polypeptide, similar to the human parotid salivary histidine-rich peptide (HRP-5), also affected yeast viability. Its killing effect was dependent upon the number of c.f.u. in the assay, as well as contact time with the blastospores and the final peptide concentration. HRP-5 inhibition increased with rising pH in the range 5-7.4, in contrast to poly-L-histidine and ketoconazole, which had optimal candidacidal activity at about pH 6. Poly-L-histidine, HRP-5, and ketoconazole each prevented conversion of blastospores to germ tubes, but their rank order of effectiveness varied with the assay selected. In N-acetylglucosamine-supplemented fetal calf serum, poly-L-histidine and HRP-5 were more effective inhibitors than ketoconazole, but the reverse was true in amino-acid-supplemented glucose beef-extract medium. Reduction of both germ-tube numbers and germ-tube size by HRP-5 was concentration-dependent.

Lysozyme-Protease-Inorganic Monovalent Anion Lysis of Oral Bacterial Strains in Buffers and Stimulated Whole Saliva

Similar to Streptococcus mutans, buffer suspensions of Lactobacillus casei, Lactobacillus plantarum, and Fusobacterium nucleatum all undergo cell lysis when treated with the lysozyme-protease-inorganic monovalent anion antibacterial system. For Lactobacillus species treated with lysozyme and proteases at pHs of 4 and 5.3, lysis resulted when a lytic activating concentration of bicarbonate anion followed enzyme treatment. Furthermore, synergistic lysis of these bacteria was noted when lysozyme-protease treatment was followed by bicarbonate anion used in combination with chloride or fluoride anions. Noteworthy, the halides were not active in promoting lysis when used by themselves in the absence of bicarbonate. For F. nucleatum suspended at pH 6.9, lysis was dependent upon the ionic strength of the buffer and resulted when lysozyme-protease treatment of the organism was followed by 100 mmol/L bicarbonate activation. When lysozyme and proteases were omitted from the incubation mixtures and replaced by stimulated whole saliva, pH 5.3, lysis was observed only with L. plantarum and S. mutans, but not with L. casei. The latter could be lysed, however, if suspended in saliva which was diluted several-fold with distilled water. In experiments where lysozyme was selectively depleted from whole saliva by immunoadsorption affinity chromatography, the great majority of the lysis capability of the saliva for L. plantarum was lost, although a significant degree of lysis appeared to be due to salivary factors other than lysozyme. F. nucleatum was also found to lyse in saliva at neutral pH, suggesting that both Gram-positive and Gram-negative oral bacteria may be susceptible to this antibacterial system in vivo.

Assessment of antimicrobial treatment of denture stomatitis using an in vivo replica model system: Therapeutic efficacy of an oral rinse

Five denture stomatitis patients demonstrating Candida albicans on both maxillary dentures and palates volunteered to test the effects of Peridex oral rinse in treating their oral disease. They used Peridex rinse both as a mouthrinse and as a denture soak for a period of 24 days. Agar replicas of the tissue-fitting surfaces of the maxillary dentures revealed elimination of C. albicans. Significant decreases in palatal inflammation were also noted, although some inflammation was still evident. Several weeks after the termination of Peridex oral rinses, inflammation increased as concentrations of C. albicans on the denture surface returned to pretreatment levels. A marked similarity in the site-specific localization of this yeast species on the denture was noted before and after Peridex rinse treatment.

The use of capillary electrophoresis to identify cationic proteins in human parotid saliva

Eight proteins, HRPs 1, 2, 3, 4, 5 and 6, lysozyme and histatin 6, are the major cationic components of the parotid salivas of normal healthy individuals. Histatins 2 and 4 appear to be further degradation products of the HRPs. Capillary electrophoresis separates all of these eight components, thus allowing future studies to correlate protein concentration with antimicrobial activity in health and disease.

An in vivo replica method for the site-specific detection ofCandida albicans on the denture surface in denture stomatitis patients: Correlation with clinical disease

A site-specific agar replica technique for detecting Candida albicans on the acrylic resin denture surface of denture stomatitis patients has been developed. The method is selective for C. albicans during a finite incubation period with a specific synthetic growth medium. C. albicans colonies can be geographically observed on the replica and their presence can be correlated with inflammatory lesions visible on the mucosa of the maxillary and mandibular residual ridges. In 12 denture stomatitis patients studied, a close clinical correlation of Newton type III patients was noted but this clinical correlation could not be observed in Newton type I and II patients. In general, the number of C. albicans colonies increased with the severity of the inflammation. The findings are discussed in light of lack of knowledge of the etiology of the stomatitis. The importance of the replica method is also discussed.

Synergism of lysozyme, proteases and inorganic monovalent anions in the bacteriolysis of oral Streptococcus mutans GS5

Streptococcus mutans GS5 was grown in synthetic medium containing radioactive thymidine to monitor deoxyribonucleic acid release. At neutral pH, cell lysis of hen egg-white lysozyme- or lysozyme-protease-treated cells was dependent upon the nature and concentration of the additive inorganic anions, HCO-3, SCN-, Cl- or F-. At acidic pH, NaHCO3, but not NaSCN, NaCl or NaF, was effective in promoting cell lysis which was due not only to the change in pH but also to the new HCO-3 anion concentration at the new pH. In both pH 4 and 5.2 reaction mixtures, the lysozyme and trypsin acted synergistically with NaHCO3 and the amount of lysis produced was markedly greater than in reaction mixtures containing lysozyme and bicarbonate but no protease. At apparent sub-lytic concentrations of NaHCO3, lysis was achieved by adding an appropriate concentration of one of NaSCN, NaCl or NaF to the lysozyme-protease-damaged cells. Thiocyanate proved to be most effective among the anions requiring lower concentrations to elicit lysis compared to chloride or fluoride for a fixed sub-lytic concentration of bicarbonate. As the NaHCO3 concentration increased, the lysis in the presence of these other anions increased until maximum levels of released deoxyribonucleic acid (DNA) were attained. In addition, the higher the NaHCO3 concentration, the more marked was the change in the degree of cell lysis. At a selected concentration at which NaHCO3 was not effective with any one salt, lysis could be achieved by combining all four inorganic anions at this concentration. The results suggest that the various anions present in oral fluids may together be sufficient to trigger lysis of oral microorganisms.

Lysozyme-mediated De-chaining of Streptococcus mutans and Its Antibacterial Significance in an Acidic Environment

The ability of physiological amounts of lysozyme to de-chain two serotype c strains of Streptococcus mutans was determined. Both human and hen lysozymes were equally effective in chain breakage of S. mutans DPR and S. mutans DJR. De-chaining did not affect growth of cultures, but resulted in finely dispersed suspensions, at stationary phase, which were visibly different from untreated cultures. Less than 50 μg lysozyme per ml culture medium reduced chain length to virtually all diplococci and single cells, and this chain disruption increased total viable cell count. De-chaining required an active enzyme indicating that a degree of hydrolysis of the peptidoglycan occurred at the septae of the streptococci. Dechained S. mutans did not survive as well as streptococci of normal chain length when incubated under acidic conditions (pH 5.5), but gross cellular lysis was not apparent. The reduced aciduric property of the disrupted chains may have been due to a participation of autolysins or to a lethal reaction triggered by the lysozyme-damaged peptidoglycan. De-chaining may be a mechanism by which lysozyme could regulate the levels of S. mutans in acidogenic plaque samples.

In vivo antifungal efficacy of salivary histidine-rich polypeptides: Preliminary findings in a denture stomatitis model system☆

Six denture stomatitis patients, all found to have Candida albicans on their maxillary denture and palatal tissue surfaces, volunteered in this preliminary study to test the in vivo efficacy of human salivary antifungal histidine-rich polypeptides (HRPs) in treating their oral disease. The patients were equally divided among the Newton types classification and, as expected, the severity of the inflammation was greatest in the Newton type III patients and least in the Newton type I patients. Patients received sterile solutions of either HRP-3 or HRP-4, which they used both as a mouthrinse and as a denture soak for a period of 1 week. Agar replicas of the tissue-fitting surface of the maxillary dentures revealed HRP reduction and/or elimination of C. albicans from the denture; in one Newton type II individual, this finding directly correlated with a site-specific reduction in palatal inflammation. In the Newton type II and type III individuals alike, there was a significant generalized decrease in inflammation suggesting the therapeutic efficacy of the HRPs. Killing of this yeast species by the HRPs, as determined by scanning electron microscopy (SEM), was probably responsible for the observed clinical benefits noted in this investigation. In the SEM, HRP-treated blastospores appeared severely deflated, as if they had been emptied of significant quantities of intracellular material.

Model system for the in vitro testing of a synthetic histidine peptide against Candida species grown directly on the denture surface of patients with denture stomatitis

The denture surface provides a nidus for the growth of microbial species that act to initiate, aggravate, and maintain clinical disease. The present investigation describes the development of a model system for the testing of the effectiveness of agents against these microbial species inhabiting the denture surface. It was observed through in vitro growth patterns that the model permitted the testing of representative samples of the microbial flora. Poly-L-histidine was observed to inhibit both Candida albicans and C. glabrata from growing from the denture surface into nutrient broth. Scanning electron microscopy of control and treated denture disks revealed that poly-L-histidine had either eliminated most microbial flora from the denture surface or had effected a noticeable distortion of those Candida blastospores still present on the surface. From microbiologic studies, it appeared that poly-L-histidine had inflicted direct but not lethal damage to the still-attached distorted blastospores because the latter were still able to promote growth in agent-free broth. The antifungal effects of poly-L-histidine were observed to be dependent on the concentration of the polypeptide. The data obtained were consistent for all of the patients regardless of their denture stomatitis classification.

Microbial caries induction in the roots of human teeth in vitro

Streptococcus mutans GS5, Lactobacillus casei DSM20011 and Actinomyces viscosus T14 produce artificial caries in the roots of extracted teeth. Roots were coated with wax leaving an 8 mm2 window exposed on the buccal surfaces, and then incubated for 8 days in the presence of the test organism, the synthetic medium being changed each day. Samples were then examined by SEM, or microradiographs were obtained from 120 microns sections. The pH at the root surface at the end of the induction averaged 4.43, 5.00 and 5.20, and the lesion depths measured on the microradiographs averaged 121, 83 and 34 microns, for Strep. mutans, L. casei and A. viscosus respectively. This relationship between pH and lesion depth confirms earlier findings. As all of these organisms can produce lesions in tooth structure, elimination of one type would probably not eliminate caries.