The inhibition of streptococci by lactoperoxidase, thiocyanate and hydrogen peroxide. The oxidation of thiocyanate and the nature of the inhibitory compound

1. The growth of the lactoperoxidase-sensitive Streptococcus cremoris 972 in a synthetic medium was inhibited by lactoperoxidase and thiocyanate. The glycolysis and oxygen uptake of suspensions of Strep. cremoris 972 in glucose or lactose were also inhibited. The lactoperoxidase-resistant Strep. cremoris 803 was not inhibited under these conditions but was inhibited in the absence of a source of energy. 2. Lactoperoxidase (EC 1.11.1.7), thiocyanate and hydrogen peroxide completely inhibited the hexokinases of non-metabolizing suspensions of both strains. The inhibition was reversible, hexokinase and glycolytic activities of Strep. cremoris 972 being restored by washing the cells free from inhibitor. The aldolase and 6-phosphogluconate-dehydrogenase activities of Strep. cremoris 972 were partially inhibited but several other enzymes were unaffected. 3. The resistance of Strep. cremoris 803 to inhibition was not due to the lack of hydrogen peroxide formation, to the destruction of peroxide, to the inactivation of lactoperoxidase or to the operation of alternative pathways of carbohydrate metabolism. 4. A ;reversal factor', which was partially purified from extracts of Strep. cremoris 803, reversed the inhibition of glycolysis of Strep. cremoris 972. The ;reversal factor' also catalysed the oxidation of NADH(2) in the presence of an intermediate oxidation product of thiocyanate and was therefore termed the NADH(2)-oxidizing enzyme. 5. The NADH(2)-oxidizing enzyme was present in lactoperoxidase-resistant streptococci but was absent from lactoperoxidase-sensitive streptococci.     

Antistreptococcal Activity of Lactoperoxidase

A study of the inhibition of the growth of Streptococcus cremoris 972 by the enzyme lactoperoxidase has shown, in agreement with previous investigations, that the inhibition requires a source of both peroxide and thiocyanate. The thiocyanate may play more than one role. It stabilizes the very dilute solutions of lactoperoxidase employed in these studies, and its oxidation products may be involved in the inhibition. Binding of the enzyme by the microorganism is suggested by the fact that when the organism was preincubated with the enzyme and then in a medium free from the enzyme, but containing peroxide and thiocyanate, the growth of the organism was inhibited. This inhibition has all the properties of the enzyme-containing system. Although no dialyzable factor could be demonstrated to cause the inhibition, the inhibitory state involving peroxide, the enzyme, and thiocyanate survived for at least 60 min before cells were added to the medium. When catalase was present in the medium prior to the addition of the cells, the inhibition was completely reversed. It was only partially reversed if catalase was added a few moments after the addition of the cells. The data have been interpreted as indicating that the inhibition takes place rapidly and requires the formation of a quaternary complex of the cells, thiocyanate, peroxide, and the enzyme lactoperoxidase.     

Agar medium for differential enumeration of lactic streptococci

An agar medium containing arginine and calcium citrate as specific substrates, diffusible (K₂HPO₄) and undiffusible (CaCO₃) buffer systems, and bromocresol purple as the pH indicator was developed to differentiate among lactic streptococci in pure and mixed cultures. Milk was added as the sole source of carbohydrate (lactose) and to provide growth-stimulating factors. Production of acid from lactose caused developing bacterial colonies to seem yellow. Subsequent arginine utilization by Streptococcus lactis and S. diacetilactis liberated ammonia, resulting in a localized pH shift back toward neutrality and a return of the original purple indicator hue. The effects of production of acid from lactose and ammonia were fixed around individual colonies by the buffering capacity of CaCO₃. After 36 hr at 32 C in a candle oats jar, colonies of S. cremoris were yellow, whereas colonies of S. lactis and S. diacetilactis were white. S. diacetilactis, on further incubation, utilized suspended calcium citrate, and, after 6 days, the citrate-degrading colonies exhibited clear zoning against a turbid background, making them easily distinguishable from the colonies of the other two species. The medium proved suitable for quantitative differential enumeration when compared with another widely used general agar medium for lactic streptococci.     

Lactoperoxidase-catalyzed oxidation of thiocyanate by hydrogen peroxide: 15N nuclear magnetic resonance and optical spectral studies

To establish the agent(s) responsible for the activity of the lactoperoxidase (LPO)/SCN-/H2O2 system, the oxidation of thiocyanate with hydrogen peroxide, catalyzed by lactoperoxidase, has been studied by 15N NMR and optical spectroscopy at different concentrations of thiocyanate and hydrogen peroxide and at different pHs. The formation of hypothiocyanite ion (OSCN-) as one of the oxidation products correlated well with the activity of the LPO/SCN-/H2O2 system and was maximum when the concentrations of the H2O2 and SCN- were nearly the same and the pH was less than 6.0. At [H2O2]/[SCN-] = 1, OSCN- decomposed very slowly back to thiocyanate. When the ratio [H2O2]/[SCN-] was above 2, formation of CN- was observed, which was confirmed by 15N NMR and also by changes in the optical spectrum of LPO. The oxidation of thiocyanate by H2O2 in the presence of LPO does not take place at pH greater than 8.0. Since thiocyanate does not bind to LPO above this pH, the binding of thiocyanate to LPO is considered to be prerequisite for the oxidation of thiocyanate. Maximum inhibition of oxygen uptake by Streptococcus cremoris 972 bacteria was observed when hydrogen peroxide and thiocyanate were present in equimolar amounts and the pH was below 6.0.     

In-vitro bacteriocin-mediated antagonism by oral streptococci against human carrier strains of staphylococci

All strains of oral streptococci tested and specially those of Streptococcus mutans, Strep. sanguis and Strep. minor produced more than one distinct bacteriocin-like substance with variable inhibitory activity on 20 indicator staphylococci. Inhibitory activity was comparatively higher on nasal strains of Staph. aureus and Staph. epidermidis than on strains of both species isolated from the mouth. Nineteen of 20 staphylococcal indicators were inhibited by 1–12 of the 12 effector streptococci. Sensitivity of nasal staphylococci to bacteriocins (frequency of positive inhibitory tests and total inhibition zone diameters) was significantly higher ( P < 0·001, χ² test and P < 0·05, t test respectively) than that of oral ones. The sensitivity of nasal over oral Staph. aureus ( P < 0·001 and P < 0·01) and of oral Staph. epidermidis over oral Staph. aureus ( P < 0·01 and P < 0·05) was also significantly higher. The evaluation of variability of inhibitory patterns of bacteriocins produced by streptococci (p-typing), of sensitivity patterns of staphylococci to bacteriocins (s-typing) and of the significantly higher sensitivity of nasal over oral staphylococci to bacteriocins from the epidemiological and ecological veiwpoints are discussed.     

Improved medium for lactic streptococci and their bacteriophages

Incorporation of 1.9% β-disodium glycerophosphate (GP) into a complex medium resulted in improved growth by lactic streptococci at 30 C. The medium, called M17, contained: Phytone peptone, 5.0 g; polypeptone, 5.0 g; yeast extract, 2.5 g; beef extract, 5.0 g; lactose, 5.0 g; ascorbic acid, 0.5 g; GP, 19.0 g; 1.0 M MgSO₄·7H₂O, 1.0 ml; and glass-distilled water, 1,000 ml. Based on absorbance readings and total counts, all strains of Streptococcus cremoris, S. diacetilactis, and S. lactis grew better in M17 medium than in a similar medium lacking GP or in lactic broth. Enhanced growth was probably due to the increased buffering capacity of the medium, since pH values below 5.70 were not reached after 24 h of growth at 30 C by S. lactis or S. cremoris strains. The medium also proved useful for isolation of bacterial mutants lacking the ability to ferment lactose; such mutants formed minute colonies on M17 agar plates, whereas wild-type cells formed colonies 3 to 4 mm in diameter. Incorporation of sterile GP into skim milk at 1.9% final concentration resulted in enhanced acid-producing activity by lactic streptococci when cells were inoculated from GP milk into skim milk not containing GP. M17 medium also proved superior to other media in demonstrating and distinguishing between lactic streptococcal bacteriophages. Plaques larger than 6 mm in diameter developed with some phage-host combinations, and turbid plaques, indicative of lysogeny, were also easily demonstrated for some systems.     

Naphthalene oxygenase fromCorynebacterium renale: Characterisation and mechanism of oxygenation

The formation ofcis-l,2,-dihydroxy-l,2,-dihydronaphthalene from naphthalene by naphthalene oxygenase, purified fromCorynebacterium renale ATCC 15075, was demonstrated to involve oxidation of a mol NADH and consumption of one mol oxygen. The enzyme contains one g-atom Fe²⁺ and one FAD. Catalase inhibited product formation and H2O2 could substitute for NADH in the reaction. Superoxide dismutase inhibited enzyme activity when either NADH or H₂O₂ was present; the generation of superoxide anion on addition of NADH to the enzyme, in the absence of naphthalene, was detected by the nitro blue tetrazolium reduction method. Hydroxyl radical scavengers, ethanol, mannitol and sodium benzoate, inhibited product formation when either NADH or H₂O₂ was present. Electron spin resonance studies, under aerobic conditions, indicated that iron of the enzyme underwent valence changes during the course of the reaction     

The electron transport system of the anaerobic Propionibacterium shermanii

1. Electron transport particles obtained from cellfree extracts of Propionibacterium shermanii by centrifugation at 105000xg for 3 hrs oxidized NADH, d,l-lactate, l-glycerol-3-phosphate and succinate with oxygen and, except for succinate, with fumarate, too.
2. Spectral investigation of the electron transport particles revealed the presence of cytochromes b, d and o, and traces of cytochrome a ₁ and a c-type cytochrome. Cytochrome b was reduced by succinate to about 50%, and by NADH, lactate or glycerol-3-phosphate to 80–90.
3. The inhibitory effects of amytal and rotenone on NADH oxidation, but not on the oxidation of the other substrates, indicated the presence of the NADH dehydrogenase complex, or “site I region”, in the electron transport system of P. shermanii.
4. NQNO inhibited substrate oxidations by oxygen and fumarate, as well as equilibration of the flavoproteins of the substrate dehydrogenases by way of menaquinone. The inhibition occurred at low concentrations of the inhibitor, and reached 80–100%, depending on the substrate tested. The site of inhibition of the respiratory activity was located between menaquinone and cytochrome b. In addition, inhibition of flavoprotein equilibration suggested that NQNO acted upon the electron transfer directed from menaquinol towards the acceptor to be reduced, either cytochrome b or the flavoproteins, which would include fumarate reductase.
5. In NQNO-inhibited particles, cytochrome b was not oxidized by oxygen-free fumarate, but readily oxidized by oxygen. It was concluded from this and the above evidence that the branching-point of the electron transport chain towards fumarate reductase was located at the menaquinone in P. shermanii. It was further concluded that all cytochromes were situated in the oxygen-linked branch of the chain, which formed a dead end of the system under anaerobic conditions.
6. Antimycin A inhibited only oxygen-linked reactions of the particles to about 50% at high concentrations of the inhibitor. Inhibitors of terminal oxidases were inactive, except for carbon monoxide.

     

Uric Acid and Thiocyanate as Competing Substrates of Lactoperoxidase

The physiological function of urate is poorly understood. It may act as a danger signal, an antioxidant, or a substrate for heme peroxidases. Whether it reacts sufficiently rapidly with lactoperoxidase (LPO) to act as a physiological substrate remains unknown. LPO is a mammalian peroxidase that plays a key role in the innate immune defense by oxidizing thiocyanate to the bactericidal and fungicidal agent hypothiocyanite. We now demonstrate that urate is a good substrate for bovine LPO. Urate was oxidized by LPO to produce the electrophilic intermediates dehydrourate and 5-hydroxyisourate, which decayed to allantoin. In the presence of superoxide, high yields of hydroperoxides were formed by LPO and urate. Using stopped-flow spectroscopy, we determined rate constants for the reaction of urate with compound I (k₁ = 1.1 × 10⁷ m⁻¹ s⁻¹) and compound II (k₂ = 8.5 × 10³ m⁻¹ s⁻¹). During urate oxidation, LPO was diverted from its peroxidase cycle because hydrogen peroxide reacted with compound II to give compound III. At physiologically relevant concentrations, urate competed effectively with thiocyanate, the main substrate of LPO for oxidation, and inhibited production of hypothiocyanite. Similarly, hypothiocyanite-dependent killing of Pseudomonas aeruginosa was inhibited by urate. Allantoin was present in human saliva and associated with the concentration of LPO. When hydrogen peroxide was added to saliva, oxidation of urate was dependent on its concentration and peroxidase activity. Our findings establish urate as a likely physiological substrate for LPO that will influence host defense and give rise to reactive electrophilic metabolites.     

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 < 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.     

CorA Affects Tolerance of Escherichia coli and Salmonella enterica Serovar Typhimurium to the Lactoperoxidase Enzyme System but Not to Other Forms of Oxidative Stress

The enzyme lactoperoxidase is part of the innate immune system in vertebrates and owes its antimicrobial activity to the formation of oxidative reaction products from various substrates. In a previous study, we have reported that, with thiocyanate as a substrate, the lactoperoxidase system elicits a distinct stress response in Escherichia coli MG1655. This response is different from but partly overlapping with the stress responses to hydrogen peroxide and to superoxide. In the current work, we constructed knockouts in 10 lactoperoxidase system-inducible genes to investigate their role in the tolerance of E. coli MG1655 to this antimicrobial system. Five mutations resulted in a slightly increased sensitivity, but one mutation (corA) caused hypersensitivity to the lactoperoxidase system. This hypersensitive phenotype was specific to the lactoperoxidase system, since neither the sensitivity to hydrogen peroxide nor to the superoxide generator plumbagin was affected in the corA mutant. Salmonella enterica serovar Typhimurium corA had a similar phenotype. Although corA encodes an Mg²⁺ transporter and at least three other inducible open reading frames belonged to the Mg²⁺ regulon, repression of the Mg stimulon by Mg²⁺ did not change the lactoperoxidase sensitivity of either the wild-type or corA mutant. Prior exposure to 0.3 mM Ni²⁺, which is also transported by CorA, strongly sensitized MG1655 but not the corA mutant to the lactoperoxidase system. Furthermore, this Ni²⁺-dependent sensitization was suppressed by the CorA-specific inhibitor Co(III) hexaammine. These results indicate that CorA affects the lactoperoxidase sensitivity of E. coli by modulating the cytoplasmic concentrations of transition metals that enhance the toxicity of the lactoperoxidase system.     

Antibacterial Activity of the Lactoperoxidase System in Milk Against Pseudomonads and Other Gram-Negative Bacteria

Products of thiocyanate oxidation by lactoperoxidase inhibit gram-positive bacteria that produce peroxide. We found these products to be bactericidal for such gram-negative bacteria as Pseudomonas species and Escherichia coli, provided peroxide is supplied exogenously by glucose oxidase and glucose. By the use of immobilized glucose oxidase the bactericidal agent was shown to be dialyzable, destroyed by heat and counteracted, or destroyed by reducing agents. Because the system is active against a number of gram-negative bacteria isolated from milk, it may possibly be exploited to increase the keeping quality of raw milk.     

The site of inhibition of dibromothymoquinone in mitochondrial respiration

Dibromothymoquinone (2,5-dibromo, 6-isopropyl, 3-methyl benzoquinone, DBMIB) is a quinone analogue recently introduced as a specific inhibitor of chloroplast photosynthesis at the level of plastoquinone. In beef heart mitochondria DBMIB inhibits the oxidation of both succinate and NAD linked substrates; the apparent K_I is 6 μM for βhydroxybutyrate oxidation and 61μM for succinate oxidation respectively. In sonic fragments NADH oxidation is also inhibited; however, the rotenone block of respiration can be partially bypassed by the autooxidation of reduced DBMIB. Under the same conditions succinoxidase of ETP is inhibited, as in intact mitochondria; autoxidation of DBMIB reduced by succinate can however be obtained in presence of detergents. Hexahydrocoenzyme Q₄ reverses the DBMIB inhibition of succinate in sonic fragments. The site of inhibition by DBMIB is the oxygen side of CoQ, since DBMIB can function as electron acceptor in the NADH-CoQ assay for site I energization in submitochondrial particles, studied by measuring the quenching of atebrin fluorescence.     

Direct interactions with commensal streptococci modify intercellular communication behaviors of Streptococcus mutans

The formation of dental caries is a complex process that ultimately leads to damage of the tooth enamel from acids produced by microbes in attached biofilms. The bacterial interactions occurring within these biofilms between cariogenic bacteria, such as the mutans streptococci, and health-associated commensal streptococci, are thought to be critical determinants of health and disease. To better understand these interactions, a Streptococcus mutans reporter strain that actively monitors cell–cell communication via peptide signaling was cocultured with different commensal streptococci. Signaling by S. mutans, normally highly active in monoculture, was completely inhibited by several species of commensals, but only when the bacteria were in direct contact with S. mutans. We identified a novel gene expression pattern that occurred in S. mutans when cultured directly with these commensals. Finally, mutant derivatives of commensals lacking previously shown antagonistic gene products displayed wild-type levels of signal inhibition in cocultures. Collectively, these results reveal a novel pathway(s) in multiple health-associated commensal streptococci that blocks peptide signaling and induces a common contact-dependent pattern of differential gene expression in S. mutans. Understanding the molecular basis for this inhibition will assist in the rational design of new risk assessments, diagnostics, and treatments for the most pervasive oral infectious diseases.Subject terms: Biofilms, Microbial ecology, Bacteriology, Bacterial genetics     

Potential of Lactic Streptococci to Produce Bacteriocin

A survey was made on the bacteriocin-producing potential of lactic streptococci. Bacteriocin-like activities were isolated and partially purified from about 5% of the 280 strains investigated. The frequency of production varied from about 1% in Streptococcus lactis subsp. diacetylactis to 9 and 7.5% in S. lactis and Streptococcus cremoris, respectively. Eight strains of S. cremoris produced bacteriocins which, on the basis of heat stability at different pH values and inhibitory spectrum, could be divided into four types. From 54 S. lactis strains, 5 strains produced inhibitory substances, namely, three nisin-like antibiotics and two different bacteriocins. Only 1 of 93 S. lactis subsp. diacetylactis strains produced a bacteriocin which was very similar to bacteriocins of type I in S. cremoris. All of the bacteriocins that were partially purified by ammonium sulfate precipitation showed very limited inhibitory spectra. Most of the lactic streptococci and a few members of the genera Clostridium, Leuconostoc, and Pediococcus were inhibited. None of the bacteriocins acted on gram-negative bacteria. The bacteriocinogenic strains were also characterized on the basis of plasmid content. All strains possessed between one and nine plasmids ranging from 1 to 50 megadaltons.     

Casein as a necessary factor in the production of stimulatory material for associative growth of lactic streptococci

Strains of Streptococcus cremoris KH and HC produced material that was stimulatory for S. cremoris R₆ in milk and in the dialyzable fraction of milk, but not in the dialysate fraction of milk, lactic acid whey, or lactose broth. The addition of casein to these latter media permitted the production of this stimulatory material to occur. Tryptone, peptone, and yeast extract could not be substituted for casein in producing the stimulatory material or in initiating associative growth in the lactic acid whey. The minimum concentration of casein required appeared to be from 2.0 to 2.5%.     

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.     

Characterization of mitochondrial bioenergetic functions between two forms of Leishmania donovani – a comparative analysis

Leishmaniasis is a growing health problem in many parts of the world partly due to drug resistance of the parasite. This study reports on the fisibility of studying mitochondrial properties of two forms of wild-type L. donovani through the use of selective inhibitors. Amastigote forms of L. donovani exhibited a wide range of sensitivities to these inhibitors. Mitochondrial complex II inhibitor thenoyltrifluoroacetone and F_oF₁-ATP synthase inhibitors oligomycin and dicyclohexylcarbodiimide were refractory to growth inhibition of amastigote forms, whereas they strongly inhibited the growth of promastigote forms. This result indicated that complex II and F_oF₁-ATP synthase were not functional in amastigote forms suggesting the presence of attenuated oxidative phosphorylation in the mitochondria of amastigote forms. In contrast, mitochondrial complex I inhibitor rotenone and complex III inhibitor antimycin A inhibited cellular multiplication and substrate level phosphorylation in amastigote forms, suggesting the role of complex I and complex III for the survival of amastigote forms. Further we studied the mitochondrial activities of both forms by measuring oxygen consumption and ATP production. In amastigote form, substantial ATP formation by substrate level phosphorylation was observed in NADPH-fumarate, NADH-fumarate, NADPH-pyruvate and NADH-pyruvate redox couples. None of the redox couple generated ATP formation was inhibited by F_oF₁-ATP synthase inhibitor oligomycin. Therefore, we may conclude that there are significant differences between these two forms of L. donovani in respect of mitochondrial bioenergetics. Our results demonstrated bioenergetic disfunction of amastigote mitochondria. Therefore, these alterations of metabolic functions might be a potential chemotherapeutic target.     

Deoxyribonucleic Acid Homology Among Lactic Streptococci

A comparison was made by deoxyribonucleic acid homology of 45 strains of lactic streptococci, using two strains of Streptococcus cremoris and three strains of Streptococcus lactis as reference strains. All S. cremoris strains were grouped together by deoxyribonucleic acid homology. S. lactis strains formed a second group, except that three strains of S. lactis showed a high degree of homology with S. cremoris strains. The three Streptococcus diacetylactis strains could not be differentiated from S. lactis strains. In spite of these differences between S. lactis and S. cremoris strains, the majority of S. cremoris, S. lactis, and S. diacetylactis strains studied had at least 50% of their base sequences in common. In contrast, Streptococcus thermophilus strains generally showed little relationship with the other strains of lactic streptococci. The relevance of these findings to the selection of starter strains for cheese making is discussed.