Effect of precursors on biosynthesis of monensins A and B

Precursors of monensins (acetate, propionate, butyrate, isobutyrate) affect the total production and the relative proportion of monensins A and B. Addition of propionate into the fermentation medium causes a prevalence of monensin B whereas butyrate and isobutyrate stimulate the production of monensin A and suppress the production of monensin B.     

Biosynthesis of monensins a and b: the role of isoleucine

Isoleucine added to the cultivation medium of Streptomyces cinnamonensis C-100-5 induced a relative increase of the production of monensin B at the expense of monensin A. U-14C-Isoleucine was found not to be a specific monensin B precursor. The incorporation of 1-13C-2-methylbutyrate into monensins A and B showed the label to be evenly incorporated in both products at carbon atoms originating from C(1) of propionate. In regulatory mutants insensitive to 2-amino-3-chlorobutyrate isoleucine influenced the production of monensins only slightly but strains resistant to 2-aminobutyrate and norleucine decreased their total production by 2-12% in the presence of isoleucine which was associated with a decrease of monensin A content by 14-52%. The inhibitory effect of isoleucine on the biosynthesis of valine, a specific precursor of the butyrate unit of monensin A, is discussed.     

The Effect of Nisin and Monensin on Ruminal Fermentations In Vitro

When mixed ruminal bacteria and alfalfa were incubated in vitro, monensin and nisin both inhibited methane production so long as the concentrations were greater than 1 μM. Monensin- and nisin-dependent methane depressions caused a decrease in the acetate to propionate ratio (4.5 to 3.0). Total volatile fatty acid production was decreased by both monensin and nisin addition at concentrations greater than 2 μM. Starch-digesting ruminal bacteria were initially inhibited by monensin and nisin, but this effect disappeared after two to four transfers. Nisin always inhibited cellulolytic bacteria, but the nisin-dependent inhibition of cellulose digestion was no greater than the inhibition caused by monensin. Monensin and nisin also inhibited amino acid degradation, and nisin was more effective than monensin in controlling the growth of Clostridium aminophilum, an obligate amino acid-fermenting ruminal bacterium that can tolerate low concentrations of monensin. Because nisin was as potent as monensin, bacteriocins such as nisin may have potential as feed additives. Received: 2 December 1996 / Accepted: 10 February 1997     

Monensin does not increase yields of propionic acid fermentations

Summary Monensin, a polyether antibiotic, inhibited growth and acid production by five species ofPropionibacterium. The ratio of propionate to acetate produced was not affected. Neither subinhibitory levels of monensin nor adaptation of two propionibacteria to growth in the presence of monensin resulted in increased yields of propionic acid. Monensin cannot be used to increase yields in propionic acid fermentations.     

A shift towards succinate-producing Prevotella in the ruminal microbiome challenged with monensin

The time-resolved impact of monensin on the active rumen microbiome was studied in a rumen-simulating technique (Rusitec) with metaproteomic and metabolomic approaches. Monensin treatment caused a decreased fibre degradation potential that was observed by the reduced abundance of proteins assigned to fibrolytic bacteria and glycoside hydrolases, sugar transporters and carbohydrate metabolism. Decreased proteolytic activities resulted in reduced amounts of ammonium as well as branched-chain fatty acids. The family Prevotellaceae exhibited increased resilience in the presence of monensin, with a switch of the metabolism from acetate to succinate production. Prevotella species harbour a membrane-bound electron transfer complex, which drives the reduction of fumarate to succinate, which is the substrate for propionate production in the rumen habitat. Besides the increased succinate production, a concomitant depletion of methane concentration was observed upon monensin exposure. Our study demonstrates that Prevotella sp. shifts its metabolism successfully in response to monensin exposure and Prevotellaceae represents the key bacterial family stabilizing the rumen microbiota during exposure to monensin.     

Effect of monensin and 2-bromoethanesulfonic acid on fatty acid metabolism and methane production from cattle manure

Summary The effect of monensin and 2-bromoethanesulfonic acid (BESA) on methane production from cattle manure and on volatile fatty acids metabolism was tested. At 10 days retention time 0.81 biogas per liter cattle manure and day were produced. Methanogenesis was inhibited 20% by 3 mM BESA per liter and 45% by 2–5 mg monensin per liter. When the digestion was inhibited with either of the both drugs, the acetate pool increased drastically. Like in untreated fermentations the propionate pool increased in BESA-inhibited fermentations for several hours after substrate addition. After 24 h however it did not decrease to the low level reached in non-inhibited fermentations. When monensin was the inhibitor, the propionate pool did not change for 15 h, but then decreased with the same rate as in the control experiment. Adaptation processes or detoxification may be responsible for the delayed degradation.The degradation of low concentrations of buty-rate to acetate and the turn over rates of the butyrate pool are almost identical in cattle manure containing BESA, monensin, or no inhibitor. The turn over of ¹⁴C-acetate from butyrate degradation is delayed in BESA and monensin inhibited fermentations.From the data presented it can be concluded, that BESA mainly inhibits the methanogens, while monensin seems to inhibit both, methanogenic and nonmethanogenic organisms. However, a fast adaptation to or detoxification of the antibiotic seems to occur.     

Effect of monensin on fermentation characteristics of the artificial rumen.

Addition of monensin (Rumensin, Eli Lilly and Co.) to an artificial rumen immediately depressed the digestion of roughage and of roughage/concentrate (50:50) feeds. Methane and propionate production were affected only with the roughage/concentrate feed.     

In vitro study of the effect of different ionophore antibiotics and of certain derivatives on rumen fermentation and on protein nitrogen degradation

An in vitro study was carried out to evaluate the effect of different ionophore antibiotics and some of their derivatives on rumen fermentation and on the degradation of peanut meal nitrogen. The increase in the production of propionic acid at the expense of acetic acid, observed with lonomycin, nigericin, cationomycin and lysocellin, was identical to that noted with monensin. The decrease in methanogenesis observed in the presence of monensin was also found with cationomycin and lysocellin. With the exception of lysocellin, which greatly reduced protein degradation of peanut meal, and of nigericin, which had no effect on this parameter, the 2 other molecules presented the same action as monensin. The negative effect of monensin on microbial ammonia uptake was demonstrated with the same intensity in the presence of cationomycin; it was slightly higher with nigericin and particularly accentuated with lonomycin and lysocellin. Three ester derivatives of monensin (monensin acetate, monensin propionate and monensin butyrate) had a similar action to that of monensin on the orientation of rumen fermentations. The monensin isobutyrate derivative appeared to be more active than monensin and only weakly altered microbial ammonia uptake. The oxolonomycin and hydroxolonomycin derivatives behaved identically to lonomycin with respect to microbial metabolism and protein nitrogen degradation. Unlike the molecules from which they derive, the deacylated cationomycin and nigericic acid had no effect on the orientation of rumen fermentations. Of the compounds tested and presenting a potential 'growth-promoting action' at least comparable to that of monensin, and which demonstrated lower toxicity on mice, three molecules (oxolonomycin, lysocellin and cationomycin) appeared to present a zootechnical interest as feed additives for growing cattle.     

Resistance of Streptomyces cinnamonensis to butyrate and isobutyrate: production and properties of a new anti-isobutyrate (AIB) factor.

Butyrate and isobutyrate (after isomerization to n-butyrate) are specific precursors for the biosynthesis of monensin A in Streptomyces cinnamonensis. High concentrations of both butyrate and isobutyrate (greater than 20 and 10 mM, respectively) were toxic to S. cinnamonensis plated on solid medium. Spontaneous mutants resistant to these substances were isolated. These new strains produced monensins at even higher concentrations of butyrate or isobutyrate, with an increased yield of monensin A. S. cinnamonensis produced an anti-isobutyrate (AIB) factor, which was originally found to be excreted by some isobutyrate-resistant stains growing on solid medium containing isobutyrate. On plates, the AIB factor efficiently counteracted toxic concentrations not only of isobutyrate, but also of acetate, propionate, butyrate, 2-methylbutyrate, valerate and isovalerate against S. cinnamonensis as well as other Streptomyces species. Although the AIB factor enabled normal growth, sporulation and monensin production on plates, it did not have positive effects on submerged cultures of S. cinnamonensis with isobutyrate. The partial purification of the AIB factor was achieved. The role of the AIB factor during spore germination on solid medium containing isobutyrate or its homologues is discussed.     

Effect of Monensin and Lasalocid-Sodium on the Growth of Methanogenic and Rumen Saccharolytic Bacteria

It is thought that monensin increases the efficiency of feed utilization by cattle by altering the rumen fermentation. We studied the effect of monensin and the related ionophore antibiotic lasalocid-sodium (Hoffman-LaRoche) on the growth of methanogenic and rumen saccharolytic bacteria in a complex medium containing rumen fluid. Ruminococcus albus, Ruminococcus flavefaciens, and Butyrivibrio fibrisolvens were inhibited by 2.5 μg of monensin or lasalocid per ml. Growth of Bacteroides succinogenes and Bacteroides ruminicola was delayed by 2.5 μg of monensin or lasalocid per ml. Populations of B. succinogenes and B. ruminicola that were resistant to 20 μg of either drug per ml were rapidly selected by growth in the presence of each drug at 5.0 μg/ml. Selenomonas ruminantium was insensitive to 40 μg of monensin or lasalocid per ml. Either antibiotic (10 μg/ml) inhibited Methanobacterium MOH, Methanobacterium formicicum, and Methanosarcina barkeri MS. Methanobacterium ruminantium PS was insensitive to 40 μg of monensin or 20 μg of lasalocid per ml. The methanogenic strain 442 was insensitive to 40 μg of monensin but sensitive to 10 μg of lasalocid per ml. The results suggest that monensin or lasalocid acts in the rumen by selecting for succinate-forming Bacteroides and for S. ruminantium, a propionate producer that decarboxylates succinate to propionate. The selection could lead to an increase in rumen propionate formation. Selection against H₂ and formate producers, e.g. R. albus, R. flavefaciens, and B. fibrisolvens, could lead to a depression of methane production in the rumen.     

Performance and plasma metabolites of dairy calves fed starter containing sodium butyrate, calcium propionate or sodium monensin

This study was conducted to examine the influence of supplementation of sodium butyrate, sodium monensin or calcium propionate in a starter diet on the performance and selected plasma metabolites (plasma glucose, non-esterified fatty acids and β-hydroxybutyrate) of Holstein calves during pre- and post-weaning periods. Twenty-four newborn Holstein calves were housed in individual hutches until 10 weeks of life, receiving water free choice, and fed four liters of milk daily. Calves were blocked according to weight and date of birth, and allocated to one of the following treatments, according to the additive in the starter: (i) sodium butyrate (150 g/kg); (ii) sodium monensin (30 mg/kg); and (iii) calcium propionate (150 g/kg). During 10 weeks, calves received starter ad libitum, while coast cross hay (Cynodon dactylon (L.) pers.) was offered after weaning, which occurred at the 8th week of age. Weekly, calves were weighted and evaluated for body measurements. Blood samples were taken weekly after the fourth week of age, 2 hours after the morning feeding, for determination of plasma metabolites. No differences were observed among treatments for starter or hay intake, BW and daily gain of the animals. Mean concentrations of selected plasma metabolites were similar in calves fed a starter supplemented with sodium butyrate, sodium monensin and calcium propionate. There was significant reduction in the concentrations of plasma glucose as calves aged. The inclusion of sodium butyrate, calcium propionate or sodium monensin as additives in starter feeds resulted in equal animal performance, before and after weaning, suggesting that sodium monensin may be replaced by organic acid salts.     

Effects of type of ionophore and carrier on in vitro ruminal dry matter disappearance,gas production,and fermentation end products of a concentrate substrate

Effects of ionophore type and carrier on in vitro ruminal digestion and fermentation patterns of a concentrate substrate were evaluated at various incubation times. Treatments were: control (no ionophore); lasalocid sodium commercial premix (Bov); lasalocid sodium mycelium cake (LasBio); laidlomycin sodium salt (LaidNa); laidlomycin propionate commercial premix (LaidPro); monensin sodium salt (Mon); and monensin sodium commercial premix (Rum). The Bov, LasBio, Mon, and Rum treatments supplied 4 μg of ionophore/mL of culture volume, whereas the LaidNa and LaidPro treatments supplied 1.33 μg of ionophore/mL. Total gas and methane production did not differ among treatments at any of the incubation times (P>0.09). Similarly, in vitro dry matter disappearance (IVDMD) was not affected by treatment (P>0.28) at 6, 18, and 24 h of incubation; however, IVDMD (P=0.03) was greater for ionophores than for the control at 12 h of incubation. Molar proportions of acetate (P<0.01), acetate:propionate (P<0.01), and total volatile fatty acid (VFA) concentrations (P<0.01) were decreased and propionate was increased (P<0.001) for the average of all ionophore-containing substrates compared with the control. Total VFA were decreased by Bov, LaidNa, and Rum, contrasted with their specific counterparts (LasBio, LaidPro, and Mon, respectively; P<0.05). Differences were detected among ionophore types for acetate (lasalocid vs. laidlomycin; P<0.05), propionate (lasalocid vs. monensin; P<0.05), and butyrate (monensin vs. lasalocid or laidlomycin; P<0.05). Capture of metabolic hydrogen in end products of fermentation was greater for ionophore-containing treatments (P<0.01) than for the control. These data suggest limited unique effects of ionophore type or carrier on IVDMD, total gas production, and methane; however, VFA proportions varied among ionophore types and carriers, which deserves further study.     

Monensin-resistant bacteria in the rumens of calves on monensin-containing and unmedicated diets.

Total and monensin-resistant anaerobic bacterial populations and volatile fatty acid concentrations were examined in the rumens of steers fed monensin-containing (33 mg/kg) and unmedicated diets. Total anaerobic counts on a habitat-simulating medium ranged from 7.1 X 10(8) to 7.1 X 10(9) CFU/g of rumen ingesta and were not significantly different in animals fed the two diets. The mean percentage of the anaerobic population resistant to monensin (10 micrograms/ml) was significantly greater in animals receiving the monensin-supplemented diet for 33 days than in those receiving the unmedicated diet (63.6 and 32.8%, respectively). Treatment group differences in monensin resistance tended to develop later than characteristic differences in acetate/propionate ratios. Relative proportions of resistant organisms in monensin-fed animals remained significantly greater for at least 18 days after monensin was deleted from the ration, whereas acetate/propionate ratios increased to values comparable to those in the control within 10 days. These data suggest that monensin-resistant bacteria may be present in greater numbers in the rumens of animals fed monensin-supplemented diets. However, greater proportions of monensin-resistant organisms were not necessarily associated with altered fermentation patterns.     

Insertional Inactivation of Methylmalonyl Coenzyme A (CoA) Mutase and Isobutyryl-CoA Mutase Genes in Streptomyces cinnamonensis: Influence on Polyketide Antibiotic Biosynthesis

The coenzyme B(12)-dependent isobutyryl coenzyme A (CoA) mutase (ICM) and methylmalonyl-CoA mutase (MCM) catalyze the isomerization of n-butyryl-CoA to isobutyryl-CoA and of methylmalonyl-CoA to succinyl-CoA, respectively. The influence that both mutases have on the conversion of n- and isobutyryl-CoA to methylmalonyl-CoA and the use of the latter in polyketide biosynthesis have been investigated with the polyether antibiotic (monensin) producer Streptomyces cinnamonensis. Mutants prepared by inserting a hygromycin resistance gene (hygB) into either icmA or mutB, encoding the large subunits of ICM and MCM, respectively, have been characterized. The icmA::hygB mutant was unable to grow on valine or isobutyrate as the sole carbon source but grew normally on butyrate, indicating a key role for ICM in valine and isobutyrate metabolism in minimal medium. The mutB::hygB mutant was unable to grow on propionate and grew only weakly on butyrate and isobutyrate as sole carbon sources. (13)C-labeling experiments show that in both mutants butyrate and acetoacetate may be incorporated into the propionate units in monensin A without cleavage to acetate units. Hence, n-butyryl-CoA may be converted into methylmalonyl-CoA through a carbon skeleton rearrangement for which neither ICM nor MCM alone is essential.     

Some growth and metabolic characteristics of monensin-sensitive and monensin-resistant strains of Prevotella (Bacteroides) ruminicola.

New strains with enhanced resistance to monensin were developed from Prevotella (Bacteroides) ruminicola subsp. ruminicola 23 and P. ruminicola subsp. brevis GA33 by stepwise exposure to increasing concentrations of monensin. The resulting resistant strains (23MR2 and GA33MR) could initiate growth in concentrations of monensin which were 4 to 40 times greater than those which inhibited the parental strains. Resistant strains also showed enhanced resistance to nigericin and combinations of monensin and nigericin but retained sensitivity to lasalocid. Glucose utilization in cultures of the monensin-sensitive strains (23 and GA33) and one monensin-resistant strain (23MR2) was retarded but not completely inhibited when logarithmic cultures were challenged with monensin (10 mg/liter). Monensin challenge of cultures of the two monensin-sensitive strains (23 and GA33) was characterized by 78 and 51% decreases in protein yield (milligrams of protein per mole of glucose utilized), respectively. Protein yields in cultures of resistant strain 23MR2 were decreased by only 21% following monensin challenge. Cell yields and rates of glucose utilization by resistant strains GA33MR were not decreased by challenge with 10 mg of monensin per liter. Resistant strains produced greater relative proportions of propionate and less acetate than the corresponding sensitive strains. The relative amounts of succinate produced were greater in cultures of strains 23, GA33, and 23MR2 following monensin challenge. However, only minor changes in end product formation were associate with monensin challenge of resistant strain GA33MR. These results suggest that monensin has significant effects on both the growth characteristics and metabolic activities of these predominant, gram-negative ruminal bacteria.     

Some growth and metabolic characteristics of monensin-sensitive and monensin-resistant strains of Prevotella (Bacteroides) ruminicola.

New strains with enhanced resistance to monensin were developed from Prevotella (Bacteroides) ruminicola subsp. ruminicola 23 and P. ruminicola subsp. brevis GA33 by stepwise exposure to increasing concentrations of monensin. The resulting resistant strains (23MR2 and GA33MR) could initiate growth in concentrations of monensin which were 4 to 40 times greater than those which inhibited the parental strains. Resistant strains also showed enhanced resistance to nigericin and combinations of monensin and nigericin but retained sensitivity to lasalocid. Glucose utilization in cultures of the monensin-sensitive strains (23 and GA33) and one monensin-resistant strain (23MR2) was retarded but not completely inhibited when logarithmic cultures were challenged with monensin (10 mg/liter). Monensin challenge of cultures of the two monensin-sensitive strains (23 and GA33) was characterized by 78 and 51% decreases in protein yield (milligrams of protein per mole of glucose utilized), respectively. Protein yields in cultures of resistant strain 23MR2 were decreased by only 21% following monensin challenge. Cell yields and rates of glucose utilization by resistant strains GA33MR were not decreased by challenge with 10 mg of monensin per liter. Resistant strains produced greater relative proportions of propionate and less acetate than the corresponding sensitive strains. The relative amounts of succinate produced were greater in cultures of strains 23, GA33, and 23MR2 following monensin challenge. However, only minor changes in end product formation were associate with monensin challenge of resistant strain GA33MR. These results suggest that monensin has significant effects on both the growth characteristics and metabolic activities of these predominant, gram-negative ruminal bacteria.     

Use of potassium depletion to assess adaptation of ruminal bacteria to ionophores.

When mixed ruminal bacteria from cattle fed timothy hay were suspended in a medium containing a low concentration of potassium, monensin and lasalocid catalyzed a rapid depletion of potassium from cells. The ionophore-mediated potassium depletion was concentration dependent, and it was possible to describe the relationship with saturation constants. Mixed ruminal bacteria never lost more than 50% of their potassium (Kmax = 46%), and the concentrations of monensin and lasalocid needed to cause half-maximal potassium depletion (Kd) were 178 and 141 nM, respectively. When cattle were fed 350 mg of monensin per day, the ratio of ruminal acetate to propionate decreased from 4.2 to 2.9, and the Kd of monensin was eightfold greater than the value for mixed ruminal bacteria from control animals. Monensin supplementation also caused a twofold increase in the Kd of lasalocid. Lasalocid supplementation (350 mg per day) had no effect on the ruminal acetate-to-propionate ratio, but it caused a twofold increase in the Kd values of monensin and lasalocid. Increases in Kd occurred almost immediately after ionophore was added to the ration, and the Kd values returned to their prefeeding values within 14 days of withdrawal. Ionophore supplementation had no effect on the Kmax values, and approximately 50% of the population was always highly ionophore resistant. Because the Kd values of even adapted ruminal bacteria were low (< 1.5 microM), it appears that a large proportion of the ruminal ionophore is bound nonselectively to feed particles or ionophore-resistant bacteria.     

MeaA, a putative coenzyme B12-dependent mutase, provides methylmalonyl coenzyme A for monensin biosynthesis in Streptomyces cinnamonensis

The ratio of the major monensin analogs produced by Streptomyces cinnamonensis is dependent upon the relative levels of the biosynthetic precursors methylmalonyl-coenzyme A (CoA) (monensin A and monensin B) and ethylmalonyl-CoA (monensin A). The meaA gene of this organism was cloned and sequenced and was shown to encode a putative 74-kDa protein with significant amino acid sequence identity to methylmalonyl-CoA mutase (MCM) (40%) and isobutyryl-CoA mutase (ICM) large subunit (36%) and small subunit (52%) from the same organism. The predicted C terminus of MeaA contains structural features highly conserved in all coenzyme B12-dependent mutases. Plasmid-based expression of meaA from the ermE* promoter in the S. cinnamonensis C730.1 strain resulted in a decreased ratio of monensin A to monensin B, from 1:1 to 1:3. Conversely, this ratio increased to 4:1 in a meaA mutant, S. cinnamonensis WM2 (generated from the C730.1 strain by insertional inactivation of meaA by using the erythromycin resistance gene). In both of these experiments, the overall monensin titers were not significantly affected. Monensin titers, however, did decrease over 90% in an S. cinnamonensis WD2 strain (an icm meaA mutant). Monensin titers in the WD2 strain were restored to at least wild-type levels by plasmid-based expression of the meaA gene or the Amycolatopsis mediterranei mutAB genes (encoding MCM). In contrast, growth of the WD2 strain in the presence of 0.8 M valine led only to a partial restoration (<25%) of monensin titers. These results demonstrate that the meaA gene product is significantly involved in methylmalonyl-CoA production in S. cinnamonensis and that under the tested conditions the presence of both MeaA and ICM is crucial for monensin production in the WD2 strain. These results also indicate that valine degradation, implicated in providing methylmalonyl-CoA precursors for many polyketide biosynthetic processes, does not do so to a significant degree for monensin biosynthesis in the WD2 mutant.     

Investigations on the role of Golgi-mediated, ligand-receptor processing in the activation of granulocytes by chemoattractants: differential effects of monensin

Human granulocytes were exposed to different concentrations of the ionophore monensin for 20 min at 37 degrees C. Subsequent exposure to 50 nM of the chemoattractant fMet-Leu-[3H]Phe for up to 30 min at 37 degrees C resulted in a receptor-mediated uptake that was inhibited 80% at a monensin concentration of 30 microM. 50% inhibition was observed at 1-10 microM monensin with no significant change in fMet-Leu-Phe dose dependency. Subcellular fractionation of cells treated with monensin, indicated that the low density UDP-galactosyltransferase activity associated with internalized receptor-fMet-Leu-Phe complexes in untreated cells was absent. The high density galactosyltransferase activity cosedimenting with specific granule markers, however, was unaffected. Monensin also inhibited chemotaxis toward fMet-Leu-Phe as measured by migration of granulocytes through millipore filters and fMet-Leu-Phe induction of polarized morphology. Incubation of cell suspensions with up to 30 microM monensin, both before and during measurement of fMet-Leu-Phe stimulated superoxide production, did not affect the magnitude, kinetics, or transiency of the radical generation. Monensin did, however, shift the dose dependency of superoxide production of fMet-Leu-Phe to higher concentrations. These differential effects of monensin suggest that endocytosis of complexes of the chemoattractant and receptor is not involved in the activation or termination of the fMet-Leu-Phe stimulated superoxide production. They also are consistent with a role for receptor modulation and processing in the chemotactic response.     

Inhibition of growth,lipid, and sterol biosynthesis by monensin in fungi

The effects of monensin on the growth, morphology, and lipid metabolism of four fungi were determined. The growth of Hypomyces chlorinus, Neurospora crassa, Achlya bisexualis, and Taphrina deformans was suppressed by approximately 50% at 1, 2 to 5, 10, and 20 μg/ml monensin, respectively. Total lipid production, and more specifically the sterols, was reduced in each species by monensin. The hyphal tips of H. chlorinus cultured in the presence of monensin became swollen, whereas no morphological responses were observed in A. bisexualis. The results reported here suggest that growth suppression by monensin is possibly due to the interruption of endomembrane function, perhaps Golgi or their equivalents, which may result from the inhibition of sterol biosynthesis.