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.     

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.     

Gonococcal Antibiotic Resistance in Los Angeles

One hundred and thirty-five gonococcal isolates collected from Los Angeles in 1972 were studied for antibiotic susceptibility to penicillin, ampicillin, carbenicillin, tetracycline, minocycline, doxycycline and spectinomycin. Only 12 percent of the isolates were sensitive to 0.05 μg per ml of penicillin while 35 percent required at least 0.5 μg per ml for inhibition of growth. The results were slightly better with ampicillin and nearly the same with carbenicillin. Nineteen percent of the isolates required at least 1.0 μg per ml of tetracycline for inhibition of growth and the results were similar with either minocycline or doxycycline. Forty-nine percent were sensitive to 2.0 μg per ml spectinomycin, but 37 percent required at least 8.0 μg per ml for inhibition of growth.In this study nine of eleven isolates resistant to 1.0 μg per ml of tetracycline were also resistant to both penicillin and spectinomycin. Six came from endocervical sites of female patients who contributed only 37 percent of the total number of isolates studied.Correlation between the agar dilution and disc diffusion methods was satisfactory with penicillin but not with ether tetracycline or spectinomycin.     

Effects of Aldicarb and Its Sulfoxide and Sulfone on the Biology of Tylenchulus semipenetrans

In laboratory testing, egg hatch of Tylenchulus semipenetrans was stimulated at concentrations of 1 and 10 μg/ml aldicarb solution and inhibited at 50 and 100 μg/ml. Aldicarb was more inhibitory to egg hatch than the aldicarb sulfoxide and the aldicarb sulfone. Inhibition of hatch at the high concentration was associated with delays in the molting processes, lack of larval movement within the egg, and delays in embryonic development. Nematode motility was reduced at 10, 50, and 100 μg/ml of aldicarb and aldicarb sulfoxide solution, and at 50 and 100 μg/ml aldicarb sulfone. Male development was retarded at 10 μg/nrl and almost completely inhibited at 50 and 100 μg/ml of the three chemicals. In greenhouse tests, female development antl reproduction on roots of citrus seedlings were suppressed by aldicarb at rates of 2.6 μg/ml and completely inhibited at 10.6 μg/ml of soil solution during a 50-day experimental period. Under field conditions, there was little systemic movement of aldicarb into roots located outside treated areas. Aldicarb reduced the nematode larvae and the female adult population in the second year after the second treatment. There were no differences in egg hatch and sex ratio of citrus nematodes between treated and nontreated roots.     

In vitro antibacterial activity of kasugamycin

Kasugamycin is an aminoglycosidic antibiotic which was initially reported as being of potential use against Pseudomonas. Our evaluation of this antibiotic does not confirm this expectation. The median minimal inhibitory concentration (MIC) of the Pseudomonas strains tested was 250 μg/ml and the bactericidal level was 500 μg/ml. Kasugamycin was found to be slightly more active in a more basic medium (Mycin Assay broth) in which the median MIC for 11 Pseudomonas strains was 125 μg/ml. Kasugamycin manifests a modest degree of serum binding. Kasugamycin did not have any appreciable effect against a variety of bacteria tested. The only exceptions were several species of gram-negative bacteria, against which more satisfactory antibiotics already exist. Further evaluation of kasugamycin for potential human use as an antipseudomonal agent does not appear warranted.     

Microbial Degradation of a Macrotetrolide Miticide in Soil

Macrotetrolide, a miticide consisting of tetranactin, trinactin, and dinactin, was readily biodegradable and hence did not accumulate in soil. [U-¹⁴C]macrotetrolide was rapidly degraded via its constituent hydroxycarboxylic acids to carbon dioxide and water. In culture media, however, the mixture was hydrolyzed to homononactic and nonactic acids by three strains of Bacillus sp. and two of Micrococcus sp. The latter strains were able to hydrolyze 500 μg of the antibiotic per ml within a few days and to grow in the presence of 4,000 μg of the antibiotic per ml. However, they were unable to assimilate the constituent acids which accumulated in the culture medium.     

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.     

Selective Medium for Recovering Specific Populations of Rhizobia Introduced into Tropical Soils

Experiments were designed to evaluate the usefulness of antifungal agents and streptomycin for recovering low densities of rhizobia inoculated into tropical soils. The results showed that yeast-mannitol agar (pH 6.0) containing 500 μg of streptomycin, 400 μg of cycloheximide and 50 μg of benomyl or chlorothalonil per ml was the best selective medium.     

Energetics of Streptococcal Growth Inhibition by Hydrostatic Pressure

Growth of Streptococcus faecalis in complex media with various fuel sources appeared to be limited by the rate of supply of adenosine-5′ -triphosphate (ATP) at 1 atm and also under 408 atm of hydrostatic pressure. Growth under pressure was energetically inefficient, as indicated by an average cell yield for exponentially growing cultures of only 10.7 g (dry weight) per mol of ATP produced compared with a 1-atm value of 15.6. Use of ATP for pressure-volume work or for turnover of protein, peptidoglycan, or stable ribonucleic acid (RNA) did not appear to be significant causes of growth inefficiency under pressure. In addition, there did not seem to be an increased ATP requirement for ion uptake because cells growing at 408 atm had significantly lower internal K⁺ levels than did those growing at 1 atm. Pressure did stimulate the membrane adenosine triphosphatase (ATPase) or S. faecalis at ATP concentrations greater than 0.5 mM. Intracellular ATP levels were found to vary during the culture cycle from about 2.5 μmol/ml of cytoplasmic water for lag-phase or stationary-phase cells to maxima for exponentially growing cells of about 7.5 μmol/ml at 1 atm and 5.5 μmol/ml at 408 atm. N,N′-dicyclohexylcarbodiimide at a 10 μM concentration improved growth efficiency under pressure, as did Mg²⁺ or Ca²⁺ ions at 50 mM concentration. These agents also enhanced ATP pooling, and it seemed that at least part of the growth inefficiency under pressure was due to increased ATPase activity. In all, it appeared that S. faecalis growing under pressure has somewhat reduced ATP supply but significantly increased demand and that the inhibitory effects of pressure can be interpreted largely in terms of ATP supply and demand.     

Effect of Humic Fractions and Clay on Biodegradation of Phenanthrene by a Pseudomonas fluorescens Strain Isolated from Soil

The mineralization of phenanthrene in pure cultures of a Pseudomonas fluorescens strain, isolated from soil, was measured in the presence of soil humic fractions and montmorillonite. Humic acid and clay, either separately or in combination, shortened the acclimation phase. A higher mineralization rate was measured in treatments with humic acid at 100 μg/ml. Humic acid at 10 μg/ml stimulated the transformation only in the presence of 10 g of clay per liter. We suggest that sorption of phenanthrene to these soil components may result in a higher concentration of substrate in the vicinity of the bacterial cells and therefore may increase its bioavailability.     

Comparative In Vitro Activities of Lysostaphin and Other Antistaphylococcal Antibiotics on Clinical Isolates of Staphylococcus aureus

The in vitro activity of lysostaphin against clinical isolates of Staphylococcus aureus was determined by conventional tube-dilution methods. For comparison, minimal inhibitory concentration (MIC) values were also determined for penicillin G, ampicillin, methicillin, ristocetin, vancomycin, and erythromycin. Phage type and penicillinase and coagulase production were determined for each isolate. The MIC values for lysostaphin ranged from <0.047 to 12.5 μg/ml; 96% of the penicillinase-positive strains were inhibited by 1.56 μg/ml of lysostaphin, whereas 3.12 μg/ml of vancomycin and methicillin were required to attain the same degree of inhibition.     

Effects of the Metalloid Oxyanion Tellurite (TeO32−) on Growth Characteristics of the Phototrophic Bacterium Rhodobacter capsulatus

This work examines the effects of potassium tellurite (K₂TeO₃) on the cell viability of the facultative phototroph Rhodobacter capsulatus. There was a growth mode-dependent response in which cultures anaerobically grown in the light tolerate the presence of up to 250 to 300 μg of tellurite (TeO₃²⁻) per ml, while dark-grown aerobic cells were inhibited at tellurite levels as low as 2 μg/ml. The tellurite sensitivity of aerobic cultures was evident only for growth on minimal salt medium, whereas it was not seen during growth on complex medium. Notably, through the use of flow cytometry, we show that the cell membrane integrity was strongly affected by tellurite during the early growth phase (≤50% viable cells); however, at the end of the growth period and in parallel with massive tellurite intracellular accumulation as elemental Te⁰ crystallites, recovery of cytoplasmic membrane integrity was apparent (≥90% viable cells), which was supported by the development of a significant membrane potential (Δψ = 120 mV). These data are taken as evidence that in anaerobic aquatic habitats, the facultative phototroph R. capsulatus might act as a natural scavenger of the highly soluble and toxic oxyanion tellurite.     

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 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.     

Production of Pyrroloquinoline Quinone by Using Methanol-Utilizing Bacteria

A large number of methanol-utilizing bacteria were screened for extracellular production of pyrroloquinoline quinone (PQQ) by using methanol as the carbon and energy sources. Of the bacteria selected, Hyphomicrobium sp. strain TK 0441 was examined for PQQ production by using a jar fermentor. The amount of PQQ in the broth and the level of methanol dehydrogenase activity in the cells were increased by simply decreasing the amount of Fe added to the medium. On the other hand, extracellularly produced protein which interfered with the purification of PQQ was decreased by simply increasing the amount of Mg added to the medium. A suitable medium that contained 1 μg of Fe per ml, 150 μg of Mg per ml, and trace elements was developed. In this medium, the production of PQQ reached approximately 1 mg/ml and protein formation was low.     

Reversal of the Vancomycin Inhibition of Peptidoglycan Synthesis by Cell Walls

Addition of cell walls to the peptidoglycan synthetase-acceptor system containing vancomycin (50 μg/ml) prevented the inhibition by the antibiotic. In addition, the inhibition of incorporation of [¹⁴C]muramyl-pentapeptide into peptidoglycan in the presence of vancomycin was reversed by the addition of cell walls to the assay mixture at 60 min. Cell walls previously saturated with vancomycin lost their ability to reverse the inhibition by the antibiotic. The inhibition of peptidoglycan synthesis by ristocetin was partially reversed by the addition of cell walls. The initial stage in peptidoglycan synthesis is catalyzed by phospho-N-acetyl(NAc)muramyl-pentapeptide translocase (uridine 5′-phosphate) according to the reaction: UDP-NAc-muramyl-pentapeptide + acceptor acceptor-phospho-NAc-muramyl-pentapeptide + UMP where acceptor is C₅₅-isoprenoid alcohol phosphate. Vancomycin stimulates the transfer of phospho-NAc-muramyl-pentapeptide to the acceptor, and the addition of cell walls to this assay mixture prevented the stimulation of transfer. In addition to the transfer reaction, the enzyme catalyzes the exchange of [³H]uridine monophosphate (UMP) with UDP-NAc-muramyl-pentapeptide. The exchange reaction is effectively inhibited by vancomycin. For example, 60 μg of vancomycin per ml inhibited the rate of exchange by 50%. Addition of cell walls restored the exchange of UMP with the UMP moiety of UDP-NAc-muramyl-pentapeptide. Thus, cell walls appeared to have a higher affinity for vancomycin than did either the peptidoglycan synthetase-acceptor system or phospho-NAc-muramyl-pentapeptide translocase. These results provide support for the proposal made by Best and Durham that the effective binding of vancomycin to the cell wall could result in the inhibition of transfer of membrane-associated peptidoglycan chains to the growing wall.     

Effects of Polychlorinated Biphenyls on Growth and Respiration of Heterotrophic Marine Bacteria

A number of marine bacterial isolates from both near-shore and open-ocean environments were tested for growth inhibition with exposure to low concentrations (1 to 100 μg/liter) of Aroclor 1254, a commercial mixture of polychlorinated biphenyls (PCBs). Of over 17 bacterial cultures tested, growth of only two open-ocean isolates, one a pseudomonad and the other a tetrad-forming coccus, was consistently inhibited by Aroclor at concentrations as low as 10 μg/liter (10 ppb). Growth inhibition was dose dependent over a concentration range of 10 to 100 μg/liter. The effects upon division rates and final cell yields of each bacterial isolate were greatest when PCBs were added to cultures with low cell densities or with lower specific growth rates. The pseudomonad also had reduced carotenoid levels and an altered filamentous morphology with Aroclor present at a concentration of 10 μg/liter, or more. The effects noted were reversible for at least 18 h after initial exposure. Concentrations of Aroclor in excess of those needed to stop growth had no detectable effect upon the respiration rate of cells of either culture. This suggests that the reduced division rates observed were not due to inability of PCB-treated cells to transport or catabolize the carbohydrate or amino acid substrates tested.     

Epidemiology and Prognosis of Coagulase-Negative Staphylococcal Endocarditis: Impact of Vancomycin Minimum Inhibitory Concentration

This study describes coagulase-negative staphylococcal (CoNS) infective endocarditis (IE) epidemiology at our institution, the antibiotic susceptibility profile, and the influence of vancomycin minimum inhibitory concentration (MIC) on patient outcomes. One hundred and three adults with definite IE admitted to an 850-bed tertiary care hospital in Barcelona from 1995-2008 were prospectively included in the cohort. We observed that CoNS IE was an important cause of community-acquired and healthcare-associated IE; one-third of patients involved native valves. Staphylococcus epidermidis was the most frequent species, methicillin-resistant in 52% of patients. CoNS frozen isolates were available in 88 patients. Vancomycin MICs of 2.0 μg/mL were common; almost all cases were found among S. epidermidis isolates and did not increase over time. Eighty-five patients were treated either with cloxacillin or vancomycin: 38 patients (Group 1) were treated with cloxacillin, and 47 received vancomycin; of these 47, 27 had CoNS isolates with a vancomycin MIC <2.0 μg/mL (Group 2), 20 had isolates with a vancomycin MIC ≥2.0 μg/mL (Group 3). One-year mortality was 21%, 48%, and 65% in Groups 1, 2, and 3, respectively (P=0.003). After adjusting for confounders and taking Group 2 as a reference, methicillin-susceptibility was associated with lower 1-year mortality (OR 0.12, 95% CI 0.02-0.55), and vancomycin MIC ≥2.0 μg/mL showed a trend to higher 1-year mortality (OR 3.7, 95% CI 0.9-15.2; P=0.069). Other independent variables associated with 1-year mortality were heart failure (OR 6.2, 95% CI 1.5-25.2) and pacemaker lead IE (OR 0.1, 95%CI 0.02-0.51). In conclusion, methicillin-resistant S.epidermidis was the leading cause of CoNS IE, and patients receiving vancomycin had higher mortality rates than those receiving cloxacillin; mortality was higher among patients having isolates with vancomycin MICs ≥2.0 μg/mL.     

Estimation of the Yield Coefficient of Pseudomonas sp. Strain DP-4 with a Low Substrate (2,4-Dichlorophenol [DCP]) Concentration in a Mineral Medium from Which Uncharacterized Organic Compounds Were Eliminated by a Non-DCP-Degrading Organism

The yield coefficient (YC) of Pseudomonas sp. strain DP-4, a 2,4-dichlorophenol (DCP)-degrading organism, was estimated from the number of CFU produced at the expense of 1 unit amount of DCP at low concentrations. At a low concentration of DCP, the YC can be overestimated in pure culture, because DP-4 assimilated not only DCP but also uncharacterized organic compounds contaminating a mineral salt medium. The concentration of these uncharacterized organic compounds was nutritionally equivalent to 0.7 μg of DCP-C ml⁻¹. A mixed culture with non-DCP-degrading organisms resulted in elimination of ca. 99.9% of the uncharacterized organic compounds, and then DP-4 assimilated only DCP as a substrate. In a mixed culture, DP-4 degraded an initial concentration of 0.1 to 10 μg of C ml of DCP⁻¹ and the number of CFU of DP-4 increased. In the mixed culture, DCP at an initial concentration of 0.07 μg of C ml⁻¹ was degraded. However, the number of CFU of DP-4 did not increase. DCP at an extremely low initial concentration of 0.01 μg of C ml⁻¹ was not degraded in mixed culture even by a high density, 10⁵ CFU ml⁻¹, of DP-4. When glucose was added to this mixed culture to a final concentration of 1 μg of C ml⁻¹, the initial concentration of 0.01 μg of C ml of DCP⁻¹ was degraded. These results suggested that DP-4 required cosubstrates to degrade DCP at an extremely low initial concentration of 0.01 μg of C ml⁻¹. The YCs of DP-4 at the expense of DCP alone decreased discontinuously with the decrease of the initial concentration of DCP, i.e., 1.5, 0.19, or 0 CFU per pg of DCP-C when 0.7 to 10, 0.1 to 0.5, or 0.07 μg of C ml of DCP⁻¹ was degraded, respectively. In this study, we developed a new method to eliminate uncharacterized organic compounds, and we estimated the YC of DP-4 at the expense of DCP as a sole source of carbon.