Effect of dietary fish oil supplements alone or in combination with sunflower and linseed oil on ruminal lipid metabolism and bacterial populations in lactating cows.

Fish oil (FO) alters ruminal biohydrogenation causing trans fatty acid (FA) intermediates to accumulate, but the effects of 18-carbon polyunsaturated FA supply on ruminal long-chain FA metabolism and microbial communities in cattle fed FO are not well established. Four cows fitted with rumen cannula were used in a 4 × 4 Latin square with 21-d experimental periods to evaluate the effects of FO alone or in combination with plant oils high in 18:2n-6 or 18:3n-3 on rumen microbial ecology and flow of FA at the omasum. Treatments comprised a basal grass silage-based diet containing no additional oil (control) or supplements of FO (200 g/d) or FO (200 g/d) plus 500 g/d of sunflower oil (SFO) or linseed oil (LFO). Flow of FA was determined using the omasal sampling technique. The relative abundance of key biohydrogenating bacteria was assessed by quantitative PCR on 16S rRNA genes in omasal digesta. Fish oil-supplemented treatments increased the amounts of trans-18:1, trans-18:2, and 20- to 22-carbon polyunsaturated FA escaping the rumen. Relative to the control, oil supplements had no effect on the amount of 18:0 leaving the rumen, but LFO decreased the flow of 18:0 at the omasum compared with SFO. Both SFO and LFO increased trans-18:1 relative to FO, whereas LFO resulted in the highest trans-18:2 and 20- to 22-carbon FA flow. Supplements of FO plus plant oils shifted biohydrogenation toward trans-10 18:1 formation. Compared with FO alone, the ruminal metabolism of 22:6n-3 in the rumen of lactating cows is more extensive on diets containing higher amounts of 18-carbon polyunsaturated FA. However, the biohydrogenation of 22:5n-3 was less extensive in LFO than SFO, but showed no difference between FO and diets containing plant oils. Ruminal outflow of 20:5n-3 was not altered when plant oils were added to FO. Alterations in the amount of intermediates at the omasum or ruminal biohydrogenation pathways were not accompanied by major changes in analyzed bacterial populations. In conclusion, dietary supplements of FO alone or in combination with plant oils increase the amount of biohydrogenation intermediates containing 1 or more trans double bonds escaping the rumen, which may have implications for host metabolism and the nutritional quality of ruminant foods.

Effectiveness of nitrate addition and increased oil content as methane mitigation strategies for beef cattle fed two contrasting basal diets.

The objectives of this study were to investigate the effects of (1) the addition of nitrate and (2) an increase in dietary oil on methane (CH4) and hydrogen (H2) emissions from 2 breeds (cross-bred Charolais and purebred Luing) of finishing beef cattle receiving 2 contrasting basal diets consisting (grams per kilogram DM) of 500:500 (Mixed) and 80:920 (Concentrate) forage to concentrate ratios. Within each basal diet there were 3 treatments: (i) control treatments (mixed-CTL and concentrate-CTL) contained rapeseed meal as the protein source, which was replaced with either (ii) calcium nitrate (mixed-NIT and concentrate-NIT) supplying 21.5 g nitrate/kg DM, or (iii) rapeseed cake (mixed-RSC and concentrate-RSC) to increase dietary oil from 27 (CTL) to 53 g/kg DM (RSC). Following adaption to diets, CH4 and H2 emissions were measured on 1 occasion from each of the 76 steers over a 13-wk period. Dry matter intakes tended (P = 0.051) to be greater for the concentrate diet than the mixed diet; however, when expressed as grams DMI per kilogram BW, there was no difference between diets (P = 0.41). Dry matter intakes for NIT or RSC did not differ from CTL. Steers fed a concentrate diet produced less CH4 and H2 than those fed a mixed diet (P < 0.001). Molar proportions of acetate (P < 0.001) and butyrate (P < 0.01) were lower and propionate (P < 0.001) and valerate (P < 0.05) higher in the rumen fluid from steers fed the concentrate diet. For the mixed diet, CH4 yield (grams per kilogram DMI) was decreased by 17% when nitrate was added (P < 0.01), while H2 yield increased by 160% (P < 0.001). The addition of RSC to the mixed diet decreased CH4 yield by 7.5% (P = 0.18). However, for the concentrate diet neither addition of nitrate (P = 0.65) nor increasing dietary oil content (P = 0.46) decreased CH4 yield compared to concentrate-CTL. Molar proportions of acetate were higher (P < 0.001) and those of propionate lower (P < 0.01) in rumen fluid from NIT treatments compared to respective CTL treatments. Overall, reductions in CH4 emissions from adding nitrate or increasing the oil content of the mixed diet were similar to those expected from previous reports. However, the lack of an effect of these mitigation strategies when used with high concentrate diets has not been previously reported. This study shows that the effect of CH4 mitigation strategies is basal diet-dependent.

Effect of forage conservation method on ruminal lipid metabolism and microbial ecology in lactating cows fed diets containing a 60:40 forage-to-concentrate ratio.

The effect of forage conservation method on ruminal lipid metabolism and microbial ecology was examined in 2 complementary experiments in cows. Treatments comprised fresh chopped grass, barn-dried hay, or untreated (UTS) or formic acid-treated silage (FAS) prepared from the same grass sward. Preparation of conserved forages coincided with the collection of samples from cows offered fresh grass. In the first experiment, 5 multiparous Finnish Ayrshire cows (229 d in milk) were used to compare the effects of feeding diets based on grass followed by hay during 2 consecutive 14-d periods separated by a 5-d transition during which extensively wilted grass was fed. In the second experiment, 5 multiparous Finnish Ayrshire cows (53 d in milk) were assigned to 1 of 2 blocks and allocated treatments according to a replicated 3×3 Latin square design with 14-d periods to compare the effects of hay, UTS, and FAS. Cows received 7 or 9 kg/d of the same concentrate in experiments 1 and 2, respectively. Conservation of grass by drying, but not ensiling, decreased forage fatty acid content primarily due to losses of 18:2n-6 and 18:3n-3. Compared with grass, feeding hay had no effect on dry matter intake (DMI), rumen pH, or fermentation characteristics, other than increasing ammonia content, but lowered whole-tract organic matter and fiber digestibility (experiment 1). Relative to hay, silage increased DMI, rumen volatile fatty acid (VFA) concentrations, and molar proportions of butyrate, and decreased molar acetate proportions (experiment 2). Compared with UTS, FAS increased DMI, had no effect on rumen ammonia or VFA concentrations, but tended to lower rumen pH and the molar ratio of lipogenic to glucogenic VFA. Conservation method had no substantial effect on ruminal or whole-tract digestibility coefficients. Compared with fresh grass and silages, hay decreased lipolysis and biohydrogenation (BH) of dietary unsaturates in the rumen, resulting in similar flows of 18:2n-6 and 18:3n-3, but lower amounts of trans-11 18:1 and Δ11,13 18:2 at the omasum. The extent of silage fermentation had minimal influence on ruminal lipid metabolism. Treatments were not associated with changes in the relative abundance of specific bacteria known to be capable of BH or rumen protozoal numbers. In conclusion, conservation method altered forage lipids, the extent of lipolysis and BH in the rumen, and the flow of fatty acids at the omasum, in the absence of substantial changes in ruminal Butyrivibrio populations.

Ricinoleic acid inhibits methanogenesis and fatty acid biohydrogenation in ruminal digesta from sheep and in bacterial cultures.

Ricinoleic acid (RA; 12-hydroxy-cis-9-18:1) is the main fatty acid component of castor oil. Although a precursor for CLA synthesis in lactic acid bacteria, RA was found previously not to form CLA in ruminal digesta but to have some inhibitory properties. The present study was undertaken to evaluate the potential of RA to modulate ruminal biohydrogenation and methanogenesis. Ruminal digesta from 4 sheep receiving a mixed hay-concentrate diet was incubated in vitro with 0.167 g/L of linoleic acid (LA; cis-9,cis-12-18:2) or with a combination of LA and RA or LA and castor oil (LA, RA, and castor oil added to a final concentration of 0.167 g/L) in the presence and absence of lipase. The CLA rumenic acid (cis-9,trans-11-18:2) accumulated when either RA or castor oil and lipase was present. Vaccenic acid (VA; trans-11-18:1) also accumulated, and a decrease of the rate of production of stearic acid (SA; 18:0) was observed. When LA was incubated with castor oil in the absence of lipase, no effects on biohydrogenation were observed. Ricinoleic acid at 0.02 g/L did not affect growth of Butyrivibrio fibrisolvens but it inhibited growth of Butyrivibrio proteoclasticus. Butyrivibrio proteoclasticus but not B. fibrisolvens metabolized RA to 12-hydroxystearate. Linoleic acid metabolism by B. proteoclasticus appeared to be unaffected by RA addition whereas rumenic acid accumulation increased (P = 0.015 at 12 h) when RA was added. A 28% decrease (P = 0.004) in methane was obtained in 24 h in vitro incubations of diluted buffered ruminal fluid with added 0.2 g RA/L. There was no effect on the total concentration of VFA after 24 h as a result of RA addition, but the molar proportions of acetate and butyrate were decreased (P = 0.041 and P < 0.001, respectively) whereas that of propionate increased (P < 0.001). It was concluded that, at least in vitro, RA or the combination of castor oil and lipase inhibit biohydrogenation, causing the accumulation of rumenic acid and VA, with potential health benefits for ruminant products. The effect appeared to be mediated via an inhibitory effect on the biohydrogenating activity of B. proteoclasticus. An added environmental benefit could be a concomitant decrease in methane emissions. In vivo studies are now required to confirm the potential of these additives.

Dietary plant bioactives for poultry health and productivity.

1. Plants and their biologically active chemical constituents, sometimes called secondary metabolites or bioactives, present numerous opportunities for the improvement of livestock production by inclusion in the diet. 2. Many such plant derived materials have well established therapeutic values in man; however, their potential as feed additives in animal production, particularly of poultry, remains largely unexploited. 3. There is increasing evidence indicating that they can be efficient in controlling diseases, and plant bioactives may also influence production parameters such as feed efficiency and product quality. 4. It has been reported that they may even replicate some of the effects of antibiotic growth promoters, which were banned from use in Europe from 2006. 5. This review assesses the status of plant bioactives in poultry production and their mode of action on avian physiology, particularly in the digestive tract.

Effect of a blend of essential oil compounds on the colonization of starch-rich substrates by bacteria in the rumen.

AIMS: To investigate the mode of action of a blend of essential oil compounds on the colonization of starch-rich substrates by rumen bacteria. METHODS AND RESULTS: Starch-rich substrates were incubated, in nylon bags, in the rumen of sheep organized in a 4 x 4 latin square design and receiving a 60:40 silage : concentrate diet. The concentrate was either high or low in crude protein, and the diet was supplemented or not with a commercial blend of essential oil compounds (110 mg per day). The total genomic DNA was extracted from the residues in the bags. The total eubacterial DNA was quantified by real-time PCR and the proportion of Ruminobacter amylophilus, Streptococcus bovis and Prevotella bryantii was determined. Neither the supplementation with essential oil compounds nor the amount of crude protein affected the colonization of the substrates by the bacteria quantified. However, colonization was significantly affected by the substrate colonized. CONCLUSIONS: The effect of essential oils on the colonization of starch-rich substrates is not mediated through the selective inhibition of R. amylophilus. SIGNIFICANCE AND IMPACT OF THE STUDY: This study enhances our understanding of the colonization of starch-rich substrates, as well as of the mode of action of the essential oils as rumen manipulating agents.

Results of a screening programme to identify plants or plant extracts that inhibit ruminal protein degradation.

One aim of the EC Framework V project, 'Rumen-up' (QLK5-CT-2001-00 992), was to find plants or plant extracts that would inhibit the nutritionally wasteful degradation of protein in the rumen. A total of 500 samples were screened in vitro using 14C-labelled casein in a 30-min incubation with ruminal digesta. Eight were selected for further investigation using a batch fermentation system and soya protein and bovine serum albumin as proteolysis substrates; proteolysis was monitored over 12 h by the disappearance of soluble protein and the production of branched SCFA and NH3. Freeze-dried, ground foliage of Peltiphyllum peltatum, Helianthemum canum, Arbutus unedo, Arctostaphylos uva-ursi and Knautia arvensis inhibited proteolysis (P < 0.05), while Daucus carota, Clematis vitalba and Erica arborea had little effect. Inhibition by the first four samples appeared to be caused by the formation of insoluble tannin-protein complexes. The samples were rich in phenolics and inhibition was reversed by polyethyleneglycol. In contrast, K. arvensis contained low concentrations of phenolics and no tannins, had no effect in the 30-min assay, yet inhibited the degradation rate of soluble protein (by 14 %, P < 0.0001) and the production of branched SCFA (by 17 %, P < 0.05) without precipitating protein in the 12-h batch fermentation. The effects showed some resemblance to those obtained in parallel incubations containing 3 mum-monensin, suggesting that K. arvensis may be a plant-derived feed additive that can suppress growth and activity of key proteolytic ruminal micro-organisms in a manner similar to that already well known for monensin.

Influence of fish oil on ruminal biohydrogenation of C18 unsaturated fatty acids.

Dietary cis-9, trans-11-conjugated linoleic acid (CLA) is generally thought to be beneficial for human health. Fish oil added to ruminant diets increases the CLA concentration of milk and meat, an increase thought to arise from alterations in ruminal biohydrogenation of unsaturated fatty acids. To investigate the mechanism for this effect, in vitro incubations were carried out with ruminal digesta and the main biohydrogenating ruminal bacterium, Butyrivibrio fibrisolvens. Linoleic acid (LA) or alpha-linolenic acid (LNA) was incubated (1.67 g/l) with strained ruminal digesta from sheep receiving a 50:50 grass hay-concentrate ration. Adding fish oil (up to 4.17 g/l) tended to decrease the initial rate of LA (P=0.025) and LNA (P=0.137) disappearance, decreased (P<0.05) the transient accumulation of conjugated isomers of both fatty acids, and increased (P<0.05) the accumulation of trans-11-18:1. Concentrations of EPA (20:5n-3) or DHA (22:6n-3), the major fatty acids in fish oil, were low (100 mg/l or less) after incubation of fish oil with ruminal digesta. Addition of EPA or DHA (50 mg/l) to pure cultures inhibited the growth and isomerase activity of B. fibrisolvens, while fish oil had no effect. In contrast, similar concentrations of EPA and DHA had no effect on biohydrogenation of LA by mixed digesta, while the addition of LA prevented metabolism of EPA and DHA. Neither EPA nor DHA was metabolised by B. fibrisolvens in pure culture. Thus, fish oil inhibits ruminal biohydrogenation by a mechanism which can be interpreted partly, but not entirely, in terms of its effects on B. fibrisolvens.

Influence of supplementary fibrolytic enzymes on the fermentation of corn and grass silages by mixed ruminal microorganisms in vitro.

This study was done to determine the effectiveness of supplementary enzymes at increasing the fiber digestion by ruminal microorganisms and to assess whether enzyme activity limits the rate of fiber digestion in ruminal digesta. In vitro comparisons of enzyme activities in two feed enzyme preparations (A and B) with enzyme activities extracted from ruminal fluid indicated that the addition of fibrolytic enzymes at the application rates recommended by the manufacturers would not be expected to increase significantly glycanase and polysaccharidase activities in ruminal fluid. Preparations A and B both increased (P < 0.001) the rate of gas production from freeze-dried corn and grass silages in in vitro incubations with ruminal fluid, but only at concentrations much higher than recommended application rates. Autoclaved controls had little or no effect. Ultrafiltration of enzyme B indicated that most stimulation was due to components >100 kDa, which is consistent with the cause of the stimulation being enzyme activity. Fibrolytic enzymes from other sources were also able to stimulate gas production: increased rates of gas production were observed in seven out of eight combinations of "cellulase" and corn or grass silage (P < 0.05). The comparison of glycanase and polysaccharidase activities with gas-stimulatory activity in the different enzyme preparations indicated that the highest correlation was between increased gas production and enzyme activity against microgranular cellulose (P < 0.05). In a wider range of fibrolytic enzyme preparations, those with endo-(beta-1,4)- or exo-(beta-1,4)-xylanase activity equal to that of preparation A did not produce similar increased rates of fermentation of corn silage when glucanase activity was low (P > 0.05). In contrast, preparations with glucanase activity similar to enzyme A gave at least as great (P < 0.05) an improvement in gas production than enzyme A, irrespective of xylanase activity. It was concluded that enzyme activity, probably a type of endo-(beta-1,4)-glucanase activity, limits the rate of fermentation of corn and grass silage in the rumen. Enzyme supplements of the type used in these experiments are unlikely to possess sufficient activity to overcome this limitation by direct application to ruminal digesta, implying that treatment of the ration prefeeding will be key to harnessing the potential of exogenous fibrolytic enzymes in ruminant nutrition.

Abscesses due to mycobacterium abscessus linked to injection of unapproved alternative medication.

An unlicensed injectable medicine sold as adrenal cortex extract (ACE*) and distributed in the alternative medicine community led to the largest outbreak of Mycobacterium abscessus infections reported in the United States. Records from the implicated distributor from January 1, 1995, to August 18, 1996, were used to identify purchasers; purchasers and public health alerts were used to identify patients. Purchasers and patients were interviewed, and available medical records were reviewed. Vials of ACE* were tested for mycobacterial contamination, and the product was recalled by the U.S. Food and Drug Administration. ACE* had been distributed to 148 purchasers in 30 states; 87 persons with postinjection abscesses attributable to the product were identified. Patient and vial cultures contained M. abscessus identical by enzymatic and molecular typing methods. Unusual infectious agents and alternative health practices should be considered in the diagnosis of infections that do not respond to routine treatment.

Influence of sodium fumarate addition on rumen fermentation in vitro.

The influence of sodium fumarate on rumen fermentation was investigated in vitro using batch and semi-continuous cultures of mixed rumen micro-organisms taken from three sheep receiving a basal diet of hay, barley, molasses, fish meal and a mineral-vitamin supplement (500, 299.5, 100, 91 and 9.5 g/kg DM respectively). Batch cultures consisted of 10 ml strained rumen fluid in 40 ml anaerobic buffer containing 200 mg of the same feed given to the sheep. Sodium fumarate was added to achieve a final concentration of 0, 5 or 10 mmol/l, as a result of the addition of 0, 250 or 500 mumol, equivalent to 0, 200 and 400 g/kg feed. CH4 production at 24 h (360 mumol in the control cultures) fell (P < 0.05) by 18 and 22 mumol respectively (SED 7.5). Total gas production was increased by the addition of fumarate without significant accumulation of H2. Substantial increases in acetate production (92 and 194 mumol; SED 26.7, P < 0.01) were accompanied by increases in propionate formation (212 and 396 mumol; SED 13.0, P < 0.001). Longer-term effects of fumarate supplementation on ruminal fermentation and CH4 production were investigated using the rumen simulation technique (Rusitec). Eight vessels were given 20 g basal diet/d, and half of them received a supplement of fumarate (disodium salt) over a period of 19 d. The response to the daily addition of 6.25 mmol sodium fumarate was a decrease in CH4 production of 1.2 mmol (SED 0.39, P < 0.05), equivalent to the consumption of 4.8 mmol H2, and an increase in propionate production of 4.9 mmol (from 10.4 to 15.3 (SED 1.05) mmol/d, P < 0.01). The inhibition of CH4 production did not decline during the period of time that fumarate was added to the vessels. Thus, the decrease in CH4 corresponded well to the fraction of the fumarate that was converted to propionate. Fumarate had no significant (P > 0.05) effect on total bacterial numbers or on the number of methanogenic archaea, but numbers of cellulolytic bacteria were increased (8.8 v. 23.9 (SED 2.49) x 10(5) per ml, P < 0.01). Fumarate also increased DM digestibility of the basal diet after 48 h incubation (0.476 v. 0.508 (SED 0.0123), P < 0.05). Thus, it was concluded that sodium fumarate may be a useful dietary additive for ruminants, because it diverts some H2 from CH4 production and because it is able to stimulate proliferation of cellulolytic bacteria and digestion of fibre.

Influence of peptides and amino acids on fermentation rate and de novo synthesis of amino acids by mixed micro-organisms from the sheep rumen.

The influence of different N sources on fermentation rate and de novo amino acid synthesis by rumen micro-organisms was investigated in vitro using rumen fluid taken from four sheep receiving a mixed diet comprising (g/kg DM): grass hay 500, barley 299.5, molasses 100, fish meal 91, minerals and vitamins 9.5. Pancreatic casein hydrolysate (P; comprising mainly peptides with some free amino acids; 10 g/l), free amino acids (AA; casein acid hydrolysate + added cysteine and tryptophan; 10 g/l), or a mixture of L-proline, glycine, L-valine and L-threonine (M; 0.83 g/l each) were added to diluted (1:3, v/v), strained rumen fluid along with 15NH4Cl (A; 1.33 g/l) and 6.7 g/l of a mixture of starch, cellobiose and xylose (1:1:1, by weight). P and AA, but not M, stimulated net gas production after 4 and 8 h incubation (P < 0.05) in comparison with A alone. P increased microbial-protein synthesis (P < 0.05) compared with the other treatments. All of the microbial-N formed after 10 h was synthesized de novo from 15NH3 in treatment A, and the addition of pre-formed amino acids decreased the proportion to 0.37, 0.55, and 0.86 for P, AA, and M respectively. De novo synthesis of amino acids (0.29, 0.42 and 0.69 respectively) was lower than cell-N. Enrichment of alanine, glutamate and aspartate was slightly higher than that of other amino acids, while enrichment in proline was much lower, such that 0.83-0.95 of all proline incorporated into particulate matter was derived from pre-formed proline. Glycine, methionine, lysine, valine and threonine tended to be less enriched than other amino acids. The form in which the amino acids were supplied, as P or AA, had little influence on the pattern of de novo synthesis. When the concentration of peptides was decreased, the proportion of microbial-N formed from NH3 increased, so that at an initial concentration of 1 g peptides/l, similar to the highest reported ruminal peptide concentrations, 0.68 of cell-N was formed from NH3. Decreasing the NH3 concentration at 1.0 g peptides/l caused proportionate decreases in the fraction of cell-N derived from NH3, from 0.81 at 0.53 g NH3-N/l to 0.40 at 0.19 g NH3-N/l. It was concluded that different individual amino acids are synthesized de novo to different extents by mixed rumen micro-organisms when pre-formed amino acids are present, and that the source of N used for synthesis of cell-N and amino acids depends on the respective concentrations of the different N sources available; however, supplementing only with amino acids whose synthesis is lowest when pre-formed amino acids are present does not stimulate fermentation or microbial growth.

Report on an outbreak of postinjection abscesses due to Mycobacterium abscessus, including management with surgery and clarithromycin therapy and comparison of strains by random amplified polymorphic DNA polymerase chain reaction.

An outbreak of postinjection abscesses occurred in Barranquilla, Colombia, and was associated with local injections of lidocaine given in a single physician's office. Over a 5-month period, 350 (18%) of approximately 2,000 injected patients developed localized cutaneous abscesses or cellulitis; of 210 abscess specimens that were cultured, 205 were positive for rapidly growing mycobacteria, subsequently identified as Mycobacterium abscessus. The source of the outbreak was not identified. M. abscessus could not be characterized by pulsed-field gel electrophoresis, but all isolates were identical in terms of drug and heavy metal resistance patterns and random amplified polymorphic DNA PCR profiles. We believe this is the first report of the use of this latter technique for investigation of an outbreak due to M. abscessus. Therapy with a combination of surgical excision and 3-6 months' administration of clarithromycin was successful for 95% of 148 patients treated in this manner; in contrast, therapy was successful for less than one-third of patients treated with surgery alone or clarithromycin alone. This is the largest of the nine known outbreaks of postinjection abscesses that have occurred due to rapidly growing mycobacteria and is the first in which an effective method of therapy was demonstrated.

Different strains of Saccharomyces cerevisiae differ in their effects on ruminal bacterial numbers in vitro and in sheep.

A ruminal simulation device (Rusitec) was used to compare the effects of Saccharomyces cerevisiae strains NCYC 240, NCYC 694, NCYC 1026, NCYC 1088, and Yea-Sacc (a commercial product containing S. cerevisiae) on ruminal fermentation. S. cerevisiae NCYC 240, NCYC 1088, NCYC 1026, and NCYC 694 were grown on malt extract at 30 degrees C in aerated fed-batch culture and harvested along with spent growth medium by freeze-drying. Each vessel received daily 20 g of a basal diet consisting of hay, barley, molasses, fishmeal, and a minerals/vitamins mixture at 500, 299.5, 100, 91, and 9.5 g/kg of DM, respectively. Yeast preparations (500 mg/d) were added along with the feed. S. cerevisiae NCYC 240, NCYC 1026, and Yea-Sacc stimulated total and cellulolytic bacterial numbers, whereas S. cerevisiae NCYC 694 and NCYC 1088 had no effect on the numbers of bacteria. The effects of S. cerevisiae NCYC 240, NCYC 1026, and Yea-Sacc on ruminal fermentation were further investigated in vivo using ruminally cannulated sheep fed 1.5 kg/d of the diet used in Rusitec, supplemented with 2 g/d of yeast culture. All treatments tended to stimulate total and cellulolytic bacterial numbers. However, the stimulation was only statistically significant for S. cerevisiae NCYC 1026 with total bacterial numbers and S. cerevisiae NCYC 240 with cellulolytic bacteria (P < .05). Increased bacterial numbers were associated with an increase in the rate of straw degradation in the rumen and a nonsignificant (P > .05) increase in the excretion of purine derivatives in the urine, measured as an index of microbial nitrogen leaving the rumen.(ABSTRACT TRUNCATED AT 250 WORDS)

Rapid development of resistance to clarithromycin following monotherapy for disseminated Mycobacterium chelonae infection in a heart transplant patient.

Mycobacterium chelonae (formerly known as M. chelonae subspecies chelonae) is a rapidly growing mycobacterium that can cause disseminated infections, especially in immunocompromised hosts. The bacterium is typically resistant to antimicrobial agents; less than 20% of M. chelonae isolates are susceptible to trimethoprim-sulfamethoxazole, doxycycline, erythromycin, or ciprofloxacin. Findings in a recent study suggested that clarithromycin may be the drug of choice for the treatment of cutaneous (disseminated) disease due to M. chelonae. We describe a 60-year-old heart transplant patient with disseminated M. chelonae infection for whom monotherapy with clarithromycin failed because of the rapid development of resistance to the drug.

Influence of Yucca shidigera extract on ruminal ammonia concentrations and ruminal microorganisms.

An extract of the desert plant Yucca shidigera was assessed for its possible benefit in ruminal fermentation. The extract bound ammonia in aqueous solution when concentrations of ammonia were low (up to 0.4 mM) and when the extract was added at a high concentration to the sample (20%, vol/vol). The apparent ammonia-binding capability was retained after autoclaving and was decreased slightly following dialysis. Acid-precipitated extract was inactive. No evidence of substantial ammonia binding was found at higher ammonia concentrations (up to 30 mM). When Y. shidigera extract (1%, vol/vol) was added to strained rumen fluid in vitro, a small (6%) but significant (P < 0.05) decrease in ammonia concentration occurred, apparently because of decreased proteolysis. Inclusion of Y. shidigera extract (1%, vol/vol) in the growth medium of the rumen bacterium Streptococcus bovis ES1 extended its lag phase, while growth of Butyrivibrio fibrisolvens SH13 was abolished. The growth of Prevotella (Bacteroides) ruminicola B(1)4 was stimulated, and that of Selenomonas ruminantium Z108 was unaffected. Protozoal activity, as measured by the breakdown of 14C-leucine-labelled S. ruminantium in rumen fluid incubated in vitro, was abolished by the addition of 1% extract. The antimicrobial activities were unaffected by precipitating tannins with polyvinylpyrrolidone, but a butanol extract, containing the saponin fraction, retained its antibacterial and antiprotozoal effects. Saponins from other sources were less effective against protozoa than Y. shidigera saponins. Y. shidigera extract, therefore, appears unlikely to influence ammonia concentration in the rumen directly, but its saponins have antimicrobial properties, particularly in suppressing ciliate protozoa, which may prove beneficial to ruminal fermentation and may lead indirectly to lower ruminal ammonia concentrations.

Rifampin-resistant Mycobacterium kansasii.

We identified 36 rifampin-resistant Mycobacterium kansasii isolates, including 17 (4%) of 464 isolates recovered in Texas between 1989 and 1992. Of 29 patients infected with rifampin-resistant M. kansasii whose history of medication was known, 90% had previously received rifampin, and 58% of these patients had been treated with one or two effective drugs. Thirty-two percent of rifampin-resistant isolates recovered since 1989 were from patients who were seropositive for human immunodeficiency virus (HIV) infection. Twenty courses of therapy with a four-drug regimen determined on the basis of in vitro susceptibilities were administered to 16 patients from whom rifampin-resistant isolates were recovered; the therapy did not include surgery. Sputum cultures converted to negative as the result of 90% of treatments (time to conversion: mean, 11 weeks; range, 4-20 weeks). Bacteriologic relapses occurred in four of five patients who withdrew from therapy after being culture negative for < or = 6 months of therapy and in one of 12 patients who were culture negative for at least 12 months of therapy (mean, 16.3 months). This study suggests that the prognosis for cure of infection due to rifampin-resistant M. kansasii with chemotherapy alone is excellent, although the number of cases appears to be increasing, in part because of the HIV disease epidemic.

Initial clarithromycin monotherapy for Mycobacterium avium-intracellulare complex lung disease.

Sputum conversion rates in Mycobacterium avium-intracellulare (MAI) complex lung disease have ranged from only 50 to 80% despite the use of three to five antituberculosis agents. We initiated a prospective, open, noncomparative trial of initial clarithromycin monotherapy at 500 mg twice a day for 4 months in HIV-negative patients with MAI lung disease. The primary study end point was microbiologic improvement. Of 30 patients enrolled, 20 completed therapy. This latter group was predominantly male (60%), smokers (70%), older than 45 yr of age (90%), infected with Mycobacterium intracellulare (70%) and with bilateral disease (85%). Of 19 patients with pretreatment minimum inhibitory concentrations (MIC) for clarithromycin < 16 micrograms/ml, 58% became sputum-negative, and 21% showed significant reductions in sputum positivity. Heavily positive sputum cultures (> 200 colonies) were reduced from 30 to 47 samples pretherapy (64%) to three of 54 (6%) post-therapy (p < 0.0001); 18 of 19 patients (95%) showed an improvement in sputum cultures, chest radiographs, or both. Only two patients (7%) discontinued the drug because of adverse events. Only three (16%) of 19 isolates developed clarithromycin resistance (MIC > 32 micrograms/ml). Clarithromycin-susceptible and -resistant MAI isolates from the same patient had identical DNA large-restriction fragment patterns. Clarithromycin is the first single agent to be shown efficacious in the treatment of MAI lung disease.

Clinical trial of clarithromycin for cutaneous (disseminated) infection due to Mycobacterium chelonae.

OBJECTIVE: To determine if clarithromycin monotherapy is safe and effective in treating cutaneous disease (especially disseminated disease) due to Mycobacterium chelonae (formerly M. chelonae subspecies chelonae). DESIGN: An open, noncomparative trial of clarithromycin as single-drug therapy. SETTING: Nationwide referrals. PATIENTS: Culture-positive patients whose M. chelonae came from a cutaneous source and whose isolate was submitted to a single referral laboratory for susceptibility testing. INTERVENTION: Clarithromycin, 500 mg twice a day by mouth for 6 months. No attempt was made to alter use of immunosuppressive drugs. MAIN OUTCOME MEASURES: Acid-fast bacilli smears and cultures of skin lesions during and after treatment, with monitoring of clinical response, side effects, and development of new lesions. RESULTS: Fourteen patients (10 with disseminated disease) were enrolled in the study and completed at least 3 months of therapy. Underlying diseases included rheumatoid arthritis, other autoimmune disorders, and organ transplantation. All were taking corticosteroids (93%) or cyclophosphamide (7%). All patients had an excellent response to therapy, with only mild side effects from the drug. Two patients died of other diseases after improving clinically but while still taking medication. One noncompliant patient who prematurely discontinued therapy after 3.5 months relapsed 1 month later with an isolate resistant to clarithromycin. The remaining 11 patients have all completed therapy given for a mean of 6.8 months (range, 4.5 to 9 months). Therapy has been discontinued for 9 of the 11 patients for at least 6 months (mean, 7.1 months; range, 6 to 12 months), with no evidence of relapse. No remaining patient had positive acid-fast bacilli smears or cultures of skin lesions after 1 month of therapy. CONCLUSIONS: Clarithromycin may be the drug of choice for cutaneous (disseminated) disease due to M. chelonae, although more patients with long-term clinical follow-up need to be studied.

Acquired resistance of Nocardia brasiliensis to clavulanic acid related to a change in beta-lactamase following therapy with amoxicillin-clavulanic acid.

Previous studies have demonstrated that Nocardia brasiliensis is susceptible to amoxicillin-clavulanic acid and that its beta-lactamases are inhibited in vitro by clavulanic acid. A cardiac transplant patient with disseminated infection caused by N. brasiliensis was treated with this drug combination with good response, but relapsed while still on therapy. The relapse isolate was found to be identical to the initial isolate by using genomic DNA restriction fragment patterns obtained by pulsed field gel electrophoresis, but it was resistant to amoxicillin-clavulanic acid. On isoelectric focusing, the beta-lactamase from the relapse isolate exhibited a shift in the isoelectric point (pI) of its major band from 5.10 to 5.04 compared with the enzyme from the pretreatment isolate. As determined by using values of the amount of beta-lactamase inhibitor necessary to give 50 +/- 5% inhibition of beta-lactamase-mediated hydrolysis of 50 microM nitrocefin, the beta-lactamase of the relapse isolate was also 200-fold more resistant than the enzyme from the pretreatment isolate to clavulanic acid and was more resistant to sulbactam, tazobactam, cloxacillin, and imipenem. The beta-lactamase of the relapse isolate exhibited a 10-fold decrease in hydrolytic activity for cephaloridine and other hydrolyzable cephalosporins compared with that for nitrocefin. Acquired resistance to amoxicillin-clavulanic acid in this isolate of N. brasiliensis appears to have resulted from a mutational change affecting the inhibitor and active site(s) in the beta-lactamase.