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.     

Effect of Cadmium on Fungi and on Interactions Between Fungi and Bacteria in Soil: Influence of Clay Minerals and pH

Fungi (Rhizopus stolonifer, Trichoderma viride, Fusarium oxysporum f. sp. conglutinans, Cunninghamella echinulata, and several species of Aspergillus and Penicillium) tolerated higher concentrations of cadmium (Cd) when grown in soil than when grown on laboratory media, indicating that soil mitigated the toxic effects of Cd. In soil amended with clay minerals, montmorillonite provided partial or total protection against fungistatic effects of Cd, whereas additions of kaolinite provided little or no protection. Growth rates of Aspergillus niger were inhibited to a greater extent by 100 or 250 μg of Cd per g in soil adjusted to pH 7.2 than in the same soil at its natural pH of 5.1. However, there were no differences in the growth rates of Aspergillus fischeri with 100 or 250 μg of Cd per g in the same soil, whether at pH 5.1 or adjusted to pH 7.2. Growth of A. niger and A. fischeri in a soil contaminated with a low concentration of Cd (i.e., 28 μg/g), obtained from a site near a Japanese smelter, did not differ significantly from growth in a soil collected some distance away and containing 4 μg of Cd per g. Growth of A. niger in sterile soil amended with 100 μg of Cd per g and inoculated with Bacillus cereus or Agrobacterium tumefaciens was reduced to a greater extent than in the same soil containing 100 μg of Cd per g but no bacteria. The inhibitory effects of Agrobacterium radiobacter to A. niger were slightly reduced in the presence of 100 μg of Cd per g, whereas the inhibitory effects of Serratia marcescens were enhanced.     

In Vitro Sensitivity of Torulopsis glabrata to Amphotericin B, 5-Fluorocytosine, and Clotrimazole (Bay 5097)

Thirty-five strains of Torulopsis glabrata were tested by a tube dilution method for their susceptibility to amphotericin B, 5-fluorocytosine, and clotrimazole (Bay 5097). Amphotericin B was the most active in vitro, inhibiting all strains at a concentration of 1 μg/ml and killing all strains at 2 μg/ml. 5-Fluorocytosine inhibited over 80% of strains at 0.24 μg/ml, but three strains required ≥7.8 μg/ml for killing. A concentration of 2 μg of clotrimazole per ml inhibited less than 50% of strains, and 8 μg/ml killed only 10% of strains. Most strains of T. glabrata were killed by therapeutically achievable concentrations of amphotericin B and 5-fluorocytosine, but not clotrimazole.     

In Vitro Antimicrobial Activity and Human Pharmacology of Cephalexin, a New Orally Absorbed Cephalosporin C Antibiotic

Concentrations of cephalexin (an orally absorbed derivative of cephalosporin C) in serum and urine were determined in normal volunteers and patients. The in vitro antibacterial activity was also studied. All strains of group A β-hemolytic streptococci and Diplococcus pneumoniae were inhibited by 3.1 μg/ml. Of the Staphylococcus aureus strains, 88% were inhibited by 6.3 μg/ml, and 12.5 μg/ml was inhibitory for all S. aureus, 80% of Escherichia coli, 72% of Klebsiella-Aerobacter, and 56% of Proteus mirabilis strains. About 90 to 96% of E. coli, Klebsiella Aerobacter, and P. mirabilis strains were inhibited by 25 μg of cephalexin per ml. Pseudomonas and indole-positive Proteus strains proved to be quite resistant to cephalexin. Cephalexin was well absorbed after oral administration. A peak serum concentration of cephalexin of at least 5 μg/ml was achieved in each volunteer with 250 and 500-mg doses. A mean peak serum concentration of 7.7 μg/ml was achieved with 250-mg doses; 12.3μg/ml was achieved with 500-mg doses of antibiotic. Food did not interfere with absorption. Probenecid enhanced both the peak serum concentration and the duration of antibiotic activity in the serum. Over 90% of the administered dose was excreted in the urine within 6 hr. The mean peak serum concentration of cephalexin after an oral dose of 500 mg was adequate to inhibit all group A streptococci, D. pneumoniae, and S. aureus, 85% of E. coli, and about 40 to 75% of Klebsiella-Aerobacter and P. mirabilis strains. Levels of cephalexin in urine were adequate to inhibit over 90% of E. coli, and P. mirabilis and 80 to 96% of Klebsiella-Aerobacter strains.     

Effect of Gramicidin on Methanogenesis by Various Methanogenic Bacteria

Methanogenesis by Methanobacterium thermoautotrophicum strains was extremely sensitive to gramicidin, total inhibition being observed at 0.2 μg/ml. In contrast, methane synthesis by Methanococcus voltae, Methanogenium marisnigri, Methanosarcina mazei, and Methanospirillum hungatei were resistant to the highest concentrations of gramicidin tested (40 μg/ml), although spheroplasts of Methanospirillum hungatei were extremely sensitive. Other species tested showed intermediate sensitivity to gramicidin, methanogenesis inhibition occurring at 4 to 20 μg/ml.     

In Vitro Studies of a New Semisynthetic Penicillin, 6-(D-α-Sulfoaminophenylacetamido)-Penicillanic Acid

The activity of 6-(D-α-sulfoaminophenylacetamido)-penicillanic acid was determined against 357 clinical isolates of gram-negative bacilli by use of the tube-dilution technique. The majority of the isolates of Pseudomonas species were inhibited by 200 μg/ml or less of this antibiotic. Most of the isolates of Escherichia coli had a minimal inhibitory concentration of 50 μg/ml or less. Seventy-three per cent of the isolates of P. mirabilis, 40% of the isolates of P. morganii, and 45% of the isolates of Enterobacter species were inhibited by 12.5 μg/ml or less, whereas most of the isolates of Klebsiella species and Serratia species were resistant. The activity of this semisynthetic penicillin was affected by the size of the inoculum. The drug was bactericidal against all isolates of E. coli and Proteus species that were sensitive to it, but it was bactericidal against only 32% of the sensitive isolates of Pseudomonas species.     

Effects of Varying Concentrations of Novobiocin Incorporated into Two Salmonella Plating Media on the Recovery of Four Enterobacteriaceae

Hydrogen sulfide-producing strains of salmonellae, Escherichia coli, Citrobacter freundii, and Proteus mirabilis were isolated from fresh pork sausage. All the strains produced black-centered colonies on Hektoen enteric agar (HE). On xylose lysine deoxycholate agar (XLD), C. freundii produced yellow colonies, and the strains of the other three genera formed black-centered colonies. The selectivity of HE and XLD for salmonellae was improved by the addition of novobiocin to both media. With increasing concentrations of novobiocin, the degree of growth inhibition for the four genera was less on HE than on XLD. Novobiocin concentrations of 80 μg/ml in HE and 5 μg/ml in XLD did not affect the growth or colonial morphology of salmonellae. When 80 μg of novobiocin per ml was incorporated into HE, P. mirabilis strains were not recovered, 40% of C. freundii strains failed to form black-centered colonies, and growth of E. coli strains was not affected but colonies were altered without eliminating the black centers. When novobiocin at 5 μg/ml was incorporated into XLD, colonies of P. mirabilis strains were not recovered, C. freundii formed yellow colonies, and the colonies of the H₂S-producing E. coli strains were unaffected.     

In Vitro Susceptibility of Atypical Mycobacteria To Rifampin

Atypical mycobacteria (209 strains) were examined for susceptibility to rifampin by the proportion method by using Middlebrook 7H-10 agar. All strains of Mycobacterium kansasii and tap-water scotochromogens were inhibited by 0.25 to 1 μg of the drug per ml. Seventy-six per cent of M. scrofulaceum and 61% of M. intracellulare strains were susceptible to 4 μg/ml or less; 5% of the former and 8% of the latter were resistant to 16 μg/ml. All strains of M. gastri and M. triviale and most strains of M. terrae were sensitive to 1 to 4 μg/ml. Two strains of M. borstelense were both inhibited by 8 μg/ml. Nearly all strains of M. fortuitum were resistant to the drug. The results of this study suggest that rifampin may be a valuable agent for the treatment of many atypical mycobacterial infections.     

Growth of Potato and Control of Pratylenchus penetrans with Oxamyl-treated Seed Pieces in Greenhouse Studies

Oxamyl was applied to both uncut and cut potato tubers in aqueous solutions of 1,000 to 32,000 μg/ml. Emergence in greenhouse pots was delayed for a day or more after soaking cut tuber pieces in 32,000 μg/ml. After 10 weeks plant growth was greater, relative to the control, when Pratylenchus penetrans-infested soil was planted with cut tubers soaked for 20 minutes in 32,000 μg/ml. Soaking for 40 minutes did not increase nematode control nor affect plant growth. Oxamyl applied to tubers at 1,000 μg/ml reduced the numbers of P. penetrans in the soil by 20% and in the roots by 35%; at 32,000 μg/ml, the numbers of P. penetrans in the soil were reduced by 73-86% and in the roots by 86-97%. The numbers of P. penetrans did not increase in the roots of plants developed from cut tubers soaked in 32,000 μg/ml over a period of 10 weeks, but numbers of lesion nematodes had begun to increase in the soil.     

A combination of sbmA and tolC mutations in Escherichia coli K-12 Tn10-carrying strains results in hypersusceptibility to tetracycline

Previously, we demonstrated that Escherichia coli tolC mutations reduce the high-level resistance to tetracycline afforded by the transposon Tn10-encoded TetA pump from resistance at 200 μg/ml to resistance at 40 μg/ml. In this study, we found that the addition of an sbmA mutation to a tolC::Tn10 mutant exacerbates this phenotype: the double mutant did not form colonies, even in the presence of tetracycline at a concentration as low as 5 μg/ml. Inactivation of sbmA alone partially inhibited high-level tetracycline resistance, from resistance at 200 μg/ml to resistance at 120 μg/ml. There thus appears to be an additive effect of the mutations, resulting in almost complete suppression of the phenotypic expression of Tn10 tetracycline resistance.     

Evaluation of Chemicals for Restricting Colony Spreading by a Xerophilic Mold, Eurotium amstelodami, on Dichloran-18% Glycerol Agar

Twenty chemicals were screened for their effectiveness in restricting colony spreading by four strains of a xerophilic mold, Eurotium amstelodami, on dichloran-18% glycerol agar. Triton X-100, Triton X-301, Tergitol NP-7, and Tergitol 15-S-3 (each at 200 μg/ml) and 1,000 μg of sodium deoxycholate, 1 μg of iprodione, 0.1 μg of propiconazole, and 0.01 μg of Maxim per ml were judged to be most effective for restricting the rate of colony spreading.     

Supplemental Substrate Enhancement of 2,4-Dinitrophenol Mineralization by a Bacterial Consortium

A Janthinobacterium sp. and an actinomycete, both capable of mineralizing 2,4-dinitrophenol (DNP), were used to construct a consortium to mineralize DNP in nonaxenic bench-scale sequencing batch reactors (SBRs). Average K_m values for DNP mineralization by pure cultures of the Janthinobacterium sp. and the actinomycete were 0.01 and 0.13 μg/ml, respectively, and the average maximum specific growth rate (μ_max) values for them were 0.06 and 0.23/h, respectively. In the presence of NH₄Cl, nitrite accumulation in pure culture experiments and in the SBRs was stoichiometric to initial DNP concentration and the addition of nitrogen enhanced DNP mineralization in the SBRs. Mineralization of 10 μg of DNP per ml was further enhanced in SBRs by the addition of glucose at concentrations of 100 and 500 μg/ml but not at 10 μg/ml. Possible mechanisms for this enhanced DNP mineralization in SBRs were suggested by kinetic analyses and biomass measurements. Average μ_max values for DNP mineralization in the presence of 0, 10, 100, and 500 μg of glucose per ml were 0.33, 0.13, 0.42, and 0.59/h, respectively. In addition, there was greater standing biomass in reactors amended with glucose. At steady-state operation, all SBRs contained heterogeneous microbial communities but only one organism, an actinomycete, that was capable of mineralizing DNP. This research demonstrates the usefulness of supplemental substrates for enhancing the degradation of toxic chemicals in bioreactors that contain heterogeneous microbial communities.     

In Vitro Studies of α-Carboxyl-3-Thienylmethyl Penicillin,a New Semisynthetic Penicillin

The activity of a new semisynthetic penicillin, α-carboxyl-3-thienylmethyl penicillin (BRL-2288) was determined against 535 clinical isolates of gram-negative bacilli, by using the tube dilution technique. Nearly 80% of isolates of Proteus spp. were inhibited by 3.12 μg or less of this antibiotic per ml. BRL-2288 was as active as ampicillin against Escherichia coli. It was slightly more active than carbenicillin or 6-(d-α-sulfoaminophenylacetamido)-penicillanic acid against Pseudomonas sp., with over half of the isolates being inhibited by 50 μg or less of BRL-2288 per ml. Isolates of Klebsiella sp. were routinely resistant to this antibiotic. The drug was bactericidal against most sensitive organisms. BRL-2288 was less active against large inocula. A strain of Pseudomonas sp. which developed resistance to carbenicillin also developed resistance to BRL-2288 simultaneously.     

Mesoionic compounds with antifungal activity against Fusarium verticillioides

Background

Fungi contaminate the food of humans and animals, are a risk to health, and can cause financial losses. In this work, the antifungal activities of 16 mesoionic compounds (MI 1–16) were evaluated against mycotoxigenic fungi, including Aspergillus spp., Fusarium verticillioides and Penicillium citrinum. Furthermore, the decreased ergosterol in the total lipid content of Fusarium verticillioides was investigated.

Results

F. verticillioides was the most sensitive fungus to the mesoionic compounds. Among the evaluated compounds, MI-11 and MI-16 presented higher antifungal effects against F. verticillioides, with MIC values of 7.8 μg/ml, and MI-2 and MI-3 followed, with MICs of 15.6 μg/ml. The most active compounds were those with heterocyclic ring phenyl groups substituted by electron donor moieties (MI-11 and MI-16). Among some compounds with higher activity (MI-2, MI-11 and MI-16), decreased ergosterol content in the total lipid fraction of F. verticillioides was demonstrated. MI-2 reduced the ergosterol content approximately 40% and 80% at concentrations of 7.8 μg/ml and 15.6 μg/ml, respectively, and MI-11 and MI-16 decreased the content by 30% and 50%, respectively, when at a concentration of 7.8 μg/ml.

Conclusion

These findings indicate that mesoionic compounds have significant antifungal activity against F. verticillioides.     

Utilization of Low Concentrations of Starch by a Flavobacterium Species Isolated from Tap Water

Experiments in well-cleaned glass flasks revealed that addition of starch in concentrations of 10 and 25 μg of substrate C per liter to the filtrate of slow sand filters stimulated the development of a yellow-pigmented bacterium which was identified as a Flavobacterium species. The isolate was able to multiply in tap water without substrates added, but addition of starch and glucose in amounts as low as 1 μg of substrate C per liter clearly enhanced growth. The substrate affinities of the Flavobacterium for these compounds were 3.9 μg of starch C and 3.3 μg of glucose C per liter. The results of this study indicate that microorganisms which rapidly utilize starch at a level of a few micrograms per liter commonly occur in water.     

Correspondence of High Levels of Beta-Exotoxin I and the Presence of cry1B in Bacillus thuringiensis

Examination of 640 natural isolates of Bacillus thuringiensis showed that the 58 strains (9%) whose supernatants were toxic to Anthonomus grandis (Coleoptera: Curculionidae) produced between 10 and 175 μg of β-exotoxin I per ml. We also found that 55 (46%) of a sample of 118 strains whose culture supernatants were not toxic to A. grandis nevertheless produced between 2 and 5 μg/ml. However, these amounts of β-exotoxin I were below the threshold for detectable toxicity against this insect species. Secretion of large amounts of β-exotoxin I was strongly associated with the presence of cry1B and vip2 genes in the 640 natural B. thuringiensis isolates studied. We concluded that strains carrying cry1B and vip2 genes also possess, on the same plasmid, genetic determinants necessary to promote high levels of production of β-exotoxin I.     

Mode of Action of Lomofungin

Lomofungin inhibited the growth of some yeasts and mycelial fungi at concentrations between 5 and 10 μg/ml. At such concentrations, there was no decrease in endogenous and exogenous oxygen consumption, and even 50 μg of antibiotic per ml caused only slight decreases. The permeation of the cell membrane was changed so that leakage of ninhydrin-positive substances was reduced, and the uptake of ¹⁴C-labeled glucose, amino acids, uracil, and thymidine was decreased at concentrations as low as 4 μg/ml. Protein synthesis in whole cells of Saccharomyces cerevisiae was reduced 35% at 10 μg/ml. However, the antibiotic did not reduce the incorporation of phenylalanine-U-¹⁴C into polypeptides with cell-free systems of Rhizoctonia solani and S. cerevisiae. The synthesis of ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) was inhibited even at concentrations of lomofungin of 4 μg/ml. Since RNA synthesis was inhibited at lower concentrations and earlier than DNA synthesis, the primary site of action of the antibiotic appears to be the synthesis of RNA.     

Mode of Action of Lomofungin

Lomofungin inhibited the growth of some yeasts and mycelial fungi at concentrations between 5 and 10 μg/ml. At such concentrations, there was no decrease in endogenous and exogenous oxygen consumption, and even 50 μg of antibiotic per ml caused only slight decreases. The permeation of the cell membrane was changed so that leakage of ninhydrin-positive substances was reduced, and the uptake of ¹⁴C-labeled glucose, amino acids, uracil, and thymidine was decreased at concentrations as low as 4 μg/ml. Protein synthesis in whole cells of Saccharomyces cerevisiae was reduced 35% at 10 μg/ml. However, the antibiotic did not reduce the incorporation of phenylalanine-U-¹⁴C into polypeptides with cell-free systems of Rhizoctonia solani and S. cerevisiae. The synthesis of ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) was inhibited even at concentrations of lomofungin of 4 μg/ml. Since RNA synthesis was inhibited at lower concentrations and earlier than DNA synthesis, the primary site of action of the antibiotic appears to be the synthesis of RNA.     

Accumulation of Trehalose and Sucrose in Cyanobacteria Exposed to Matric Water Stress

The drought-resistant cyanobacteria Phormidium autumnale, strain LPP₄, and a Chroococcidiopsis sp. accumulated trehalose, sucrose, and both trehalose and sucrose, respectively, in response to matric water stress. Accumulated sugar concentrations reached values of up to 6.2 μg of trehalose per μg of chlorophyll in P. autumnale, 6.9 μg of sucrose per μg of chlorophyll in LPP₄, and 4.1 μg of sucrose and 3.2 μg of trehalose per μg of chlorophyll in the Chroococcidiopsis sp. The same sugars were accumulated by these cyanobacteria in similar concentrations under osmotic water stress. Cyanobacteria that did not show drought resistance (Plectonema boryanum and Synechococcus strain PCC 7942) did not accumulate significant amounts of sugars when matric water stress was applied.     

Ingestion of Salmonella enterica Serotype Poona by a Free-Living Nematode, Caenorhabditis elegans, and Protection against Inactivation by Produce Sanitizers

Free-living nematodes are known to ingest food-borne pathogens and may serve as vectors to contaminate preharvest fruits and vegetables. Caenorhabditis elegans was selected as a model to study the effectiveness of sanitizers in killing Salmonella enterica serotype Poona ingested by free-living nematodes. Aqueous suspensions of adult worms that had fed on S. enterica serotype Poona were treated with produce sanitizers. Treatment with 20 μg of free chlorine/ml significantly (α = 0.05) reduced the population of S. enterica serotype Poona compared to results for treating worms with water (control). However, there was no significant difference in the number of S. enterica serotype Poona cells surviving treatments with 20 to 500 μg of chlorine/ml, suggesting that reductions caused by treatment with 20 μg of chlorine/ml resulted from inactivation of S. enterica serotype Poona on the surface of C. elegans but not cells protected by the worm cuticle after ingestion. Treatment with Sanova (850 or 1,200 μg/ml), an acidified sodium chlorite sanitizer, caused reductions of 5.74 and 6.34 log₁₀ CFU/worm, respectively, compared to reductions from treating worms with water. Treatment with 20 or 40 μg of Tsunami 200/ml, a peroxyacetic acid-based sanitizer, resulted in reductions of 4.83 and 5.34 log₁₀ CFU/worm, respectively, compared to numbers detected on or in worms treated with water. Among the organic acids evaluated at a concentration of 2%, acetic acid was the least effective in killing S. enterica serotype Poona and lactic acid was the most effective. Treatment with up to 500 μg of chlorine/ml, 1% hydrogen peroxide, 2,550 μg of Sanova/ml, 40 μg of Tsunami 200/ml, or 2% acetic, citric, or lactic acid had no effect on the viability or reproductive behavior of C. elegans. Treatments were also applied to cantaloupe rind and lettuce inoculated with S. enterica serotype Poona or C. elegans that had ingested S. enterica serotype Poona. Protection of ingested S. enterica serotype Poona against sanitizers applied to cantaloupe was not evident; however, ingestion afforded protection of the pathogen on lettuce. These results indicate that S. enterica serotype Poona ingested by C. elegans may be protected against treatment with chlorine and other sanitizers, although the basis for this protection remains unclear.