Efficacy of Tea Tree Oil in the Treatment of Equine Streptothricosis.

Streptothricosis is a dermatitis characterized by matted tufts of hair and coalescing, pustular crusts that affects many livestock species, including horses. It results from cutaneous infection by the actinobacterium Dermatophilus congolensis. For economic reasons, the ailment is often treated with commercially available over-the-counter (OTC) products or home remedies rather than prescribed medications. This study aimed to determine the efficacy of tea tree oil (TTO), an essential oil of Melaleuca alternifolia, as an OTC treatment for streptothricosis. Bacteria were isolated from presumptive streptothricosis lesions on horses at a farm in Forest, Virginia. These isolates were microbiologically and genetically confirmed to be D. congolensis. The antimicrobial activity of TTO against D. congolensis isolates was determined by minimum inhibitory concentration and disc diffusion assays and compared with three OTC products advertised specifically for the treatment of "rain rot," a colloquial term for streptothricosis. A 1% TTO solution (v/v, in baby oil) and the three selected OTC products were applied to equine streptothricosis lesions to evaluate in vivo resolution of the lesions. Tea tree oil exhibited antimicrobial behavior against D. congolensis in vitro and produced marked improvement of streptothricosis lesions in vivo. These results have implications for development of TTO as a possible treatment for streptothricosis.

Water-soluble exudates from seeds of Kochia scoparia exhibit antifungal activity against Colletotrichum graminicola.

Plant seed exudates are composed of complex mixtures of chemicals with potential for bioactive compounds with antimicrobial properties. This study focused on kochia (Kochia scoparia), one of many weedy plant species considered invasive in many agricultural systems. Extraction of compounds in water yielded an exudate mass equivalent to 7% of the original seed mass used. Water-soluble exudates were tested against 16 known plant pathogens in disk diffusion assays and kochia exudates were found to inhibit Colletotrichum graminicola, the fungal causative agent of anthracnose and stalk rot in maize. The narrow range of fungi found as targets suggested the mechanism of inhibition may be specific rather than broadly antifungal. A decline in viability of cells over four orders of magnitude occurred within six hours of exposure to exudate. The minimum inhibitory concentration was 3.125 mg L-1. Hyphae formation in C. graminicola appeared inhibited following exposure to the exudate. Small molecular weight compounds as determined by GC/MS analysis showed high relative amounts of the sugars fructose, galactopyranose, glucose, and sorbitol, along with moderate proportions of organic acids and amino acids. Protein content averaged 0.7% in the standard concentration (100 mg mL-1) used for inhibition assays. Size fractionation of the exudate and subsequent disk diffusion assays revealed bioactive fractions with compounds in the MW range <5 kDa. To the best of our knowledge, this study is the first to show promising bioactivity against C. graminicola that was associated with water-extractable compounds from a common weed species. The results suggest that seeds of persistent plant species with long-lived seed banks like kochia may have potential for use in the discovery of compounds active in inhibiting fungal pathogens.

The activity and stability of cell-associated activity of bovicin HC5, a bacteriocin from Streptococcus bovis HC5.

Streptococcus bovis HC5 cultures released a broad spectrum lantibiotic, bovicin HC5, into the cell-fee culture supernatant after they reached stationary phase, but most of the antibacterial activity remained cell-associated. Cell-associated bovicin HC5 was more resistant to degradation by Pronase E than the cell-free activity. Acidic NaCl (pH 2.0, 100 mM) did not release all of the cell-associated activity, and cells that were sequentially treated with acidic NaCl and Pronase E still had antibacterial activity. Cell-associated activity retained after acidic NaCl treatment was still able to catalyze potassium efflux from S. bovis JB1, a sensitive strain. These results indicate that cell-associated bovicin HC5 is more active and stable than cell-free bovicin HC5.

The effect of calcium and magnesium on the activity of bovicin HC5 and nisin.

Some Gram-positive bacteria produce small peptides (bacteriocins) that have antimicrobial activity, but many bacteria can become bacteriocin resistant. Bovicin HC5, a lantibiotic produced by Streptococcus bovis HC5, has the ability to inhibit nisin-resistant bacteria. Because nisin resistance has in many cases been correlated with an alteration of lipoteichoic acids or the polar head groups of membrane phospholipids, we decided to examine the effect of divalent cations on nisin and bovicin HC5 activity. Both bacteriocins catalyzed potassium efflux from S. bovis JB1, a non-bacteriocin-producing strain. The addition of large amounts (100 mM) of calcium or magnesium increased the ability of S. bovis JB1 to bind Congo red (an anionic dye) and counteracted bacteriocin-mediated potassium loss. Calcium was more effective than magnesium in decreasing nisin activity, but the reverse was observed with bovicin HC5. Nisin-resistant S. bovis JB1 cells bound three times as much Congo red as nisin-sensitive cells, and this result is consistent with the idea that changes in cell surface charge can be a mechanism of bacteriocin resistance. The nisin-resistant cells were less susceptible to bovicin HC5, but bovicin HC5 still caused a 50% depletion of intracellular potassium. These results indicate that nisin and bovicin HC5 react differently with the cell surfaces of Gram-positive bacteria.

Effect of pH on the activity of bovicin HC5, a bacteriocin from Streptococcus bovis HC5.

The bacteriocin, bovicin HC5, catalyzed potassium efflux from Streptococcus bovis JB1, and this activity was highly pH dependent. When the pH was near neutral, glucose-energized cells were not affected by bovicin HC5, but the intracellular steady-state concentration of potassium decreased at acidic pH values. The idea that pH was affecting bovicin HC5 binding was supported by the observation that acidic pH also enhanced the efflux of potassium from non-energized cells that had been loaded with potassium. The relationship between bovicin HC5 concentration and potassium depletion was a saturation function, but cooperativity plots indicated that the binding of one bovicin molecule to the cell membrane facilitated the binding of another.

The susceptibility of ionophore-resistant Clostridium aminophilum F to other antibiotics.

OBJECTIVE: To determine if ionophore-resistant ruminal bacteria are cross-resistant to other classes of antibiotics. Clostridium aminophilum was used as a model organism because this Gram-positive ruminal bacterium can adapt to ionophores (monensin and lasalocid). Non-adapted cultures lagged for at least 12 h with 1 microM monensin or lasalocid, but initiated no growth if the concentration was 10 microM. Adapted cultures did not lag with 1 microM monensin or lasalocid, grew well even if the ionophore concentration was 10 microM and contained cells at least 100,000-fold more resistant than those in non-adapted cultures. METHODS: Ionophore-adapted and non-adapted cultures were assayed for their susceptibility to other classes of antibiotics (penicillin G, ampicillin, cephalosporin C, vancomycin, carbenicillin, tetracycline, chloramphenicol, erythromycin, streptomycin, linocomycin, rifampicin, trimethoprim, novobiocin, polymyxin B and bacitracin) using a broth microdilution method. RESULTS: Adapted cultures retained their resistance phenotype for at least 28 generations even if ionophore was no longer present. Monensin-adapted cultures were as resistant to lasalocid as those adapted to lasalocid, but lasalocid-adapted cultures lagged with 1 microM monensin. Monensin- and lasalocid-resistant C. aminophilum F cultures were as susceptible to most antibiotics as non-adapted cultures. The only antibiotic that seemed to have a common mechanism of resistance was bacitracin, and the ionophore-adapted cultures had a 32-fold greater MIC. CONCLUSION: The use of ionophores in cattle feed and the selection of ionophore-resistant ruminal bacteria does not necessarily lead to other types of antibiotic resistance.

Ionophore resistance of ruminal bacteria and its potential impact on human health.

In recent years, there has been a debate concerning the causes of antibiotic resistance and the steps that should be taken. Beef cattle in feedlots are routinely fed a class of antibiotics known as ionophores, and these compounds increase feed efficiency by as much as 10%. Some groups have argued that ionophore resistance poses the same public health threat as conventional antibiotics, but humans are not given ionophores to combat bacterial infection. Many ruminal bacteria are ionophore-resistant, but until recently the mechanism of this resistance was not well defined. Ionophores are highly lipophilic polyethers that accumulate in cell membranes and catalyze rapid ion movement. When sensitive bacteria counteract futile ion flux with membrane ATPases and transporters, they are eventually de-energized. Aerobic bacteria and mammalian enzymes can degrade ionophores, but these pathways are oxygen-dependent and not functional in anaerobic environments like the rumen or lower GI tract. Gram-positive ruminal bacteria are in many cases more sensitive to ionophores than Gram-negative species, but this model of resistance is not always clear-cut. Some Gram-negative ruminal bacteria are initially ionophore-sensitive, and even Gram-positive bacteria can adapt. Ionophore resistance appears to be mediated by extracellular polysaccharides (glycocalyx) that exclude ionophores from the cell membrane. Because cattle not receiving ionophores have large populations of resistant bacteria, it appears that this trait is due to a physiological selection rather than a mutation per se. Genes responsible for ionophore resistance in ruminal bacteria have not been identified, but there is little evidence that ionophore resistance can be spread from one bacterium to another. Given these observations, use of ionophores in animal feed is not likely to have a significant impact on the transfer of antibiotic resistance from animals to man.