Efficacy of Novel Antimicrobials Against Clinical Isolates of Opportunistic Amebas

ABSTRACT We examined the effects of the macrolide antimicrobial agent azithromycin and phenothiazine compounds against clinical isolates of Acanthamoeba spp. and Balamuthia mandrillaris , opportunistic pathogens of human beings and other animals. Acanthamoeba growth was inhibited in vitro at 1,5, and 10 μg/ml of azithromycin, but not the macrolides, erythromycin, and clarithromycin. In experiments attempting to simulate in vivo conditions, azithromycin protected monolayers of rat glioma cells from destruction by Acanthamoeba at a concentration of 0.1 μg/ml, and delayed destruction at concentrations of 0.001 and 0.01 μg/ml. We concluded that the minimal inhibitory concentration of azithromycin was 0.1 μg/ml. Our results, however, suggested that the drug was amebastatic but not amebicidal, since ameba growth eventually resumed after drug removal. The phenothiazines (chlorpromazine, chlorprothixene, and triflupromazine) inhibited Acanthamoeba growth by 70-90% at 5 and 10 μg/ml, but some of these compounds were toxic for rat glioma cells at 10 μg/ml. Azithromycin was not very effective against B. mandrillaris in an in vitro setting, but was amebastatic in tissue culture monolayers at concentrations of 0.1 μg/ml and higher. Balamuthia amebas showed in vitro sensitivity to phenothiazines. Ameba growth was inhibited 30-45% at 5 μg/ml in vitro, but completely at 5 μg/ml in the rat glioma model. In spite of their potential as antiamebic drugs in Balamuthia infections, toxicity of phenothiazines limits their use in clinical settings.     

The Semi Large-scale Production, Extraction, Purification and Properties of an Antibiotic Produced by Bacillus licheniformis Strain 2725

S ummary : Production, extraction, purification and properties of an antibiotic produced by Bacillus liclieniformis , strain 2725 are described. Extraction efficiency was c. 50% with 8–10 g of product/350 1 batch culture, the product having an activity of 2000 units/mg (0·5 μg/ml) against Staphylococcus aureus Paler. The antibiotic was a peptide, was active against Gram positive and negative bacteria; it was stable over a wide pH range, its bactericidal activity was enhanced by serum, but it had a high toxicity and was haemolytic.     

Rapid methods for differentiating Gram-positive from Gram-negative aerobic and facultative anaerobic bacteria

M anafi , M. & K neifel , W. 1990. Rapid methods for differentiating Gram-positive from Gram-negative aerobic and facultative anaerobic bacteria. Journal of Applied Bacteriology 69 , 822–827.
Different tests based on lysis by KOH and on reaction with fluorogenic and chromogenic substrates, L-alanine-4-nitroanilide (LANA); L-alanine-4-methoxy-β-naphthylamide (MNA); 4-alanine-2-amidoacridone (AAA); L-alanine-7-amido-4-methylcoumarin (AAMC); 8-anilino-l-naphthalene-sulphonic acid (ANS) were compared for their suitability to distinguish Gram-positive from Gram-negative bacteria. A concentration of 100 μg/ml was chosen for incorporating LANA, AAA, AAMC and ANS into the growth medium, based on sensitivity tests. MNA did not show any detectable reaction over a concentration range from 50 to 200 μg/ml, and led to inhibition of all bacteria at 200 μ/ml. In the examination of a total of 146 bacterial strains, including Yersinia enterocoiitica, Bacillus cereus , and B. subtilis the KOH test was not comparable with the Gram staining. A good correlation with Gram staining was found between LANA, AAA and AAMC added to plate count agar on one hand, and LANA and AAMC impregnated paper strips on the other hand, thereby utilizing the aminopeptidase activity. Agar containing ANS showed detectable fluorescence with all Gram-negative strains, but with Staphylococcus aureus and Staph. epidermidis a weak reaction was also observed. AAMC was selected for a rapid paper strip test With this substrate a pronounced blue fluorescence was obtained with Gram-negative colonies.     

Anti-bacterial activity of synthetic N-heterocyclic oxidizing compounds

Synthetic chlorochromate derivatives of pyridine and quinoline were active in vitro against type cultures of Escherichia coli (ATCC 128), Staphylococcus aureus (ATCC 14775), Pseudomonas aeruginosa (ATCC 10145) and Bacillus subtilis (NCTC 8236). The minimum inhibitory concentrations (MIC) were 125–250 μg ml⁻¹ and 250–500 μg ml⁻¹ for pyridinium chlorochromate and quinolinium chlorochromate, respectively. An established derivative of quinoline (Perfloxacin) had an MIC of 125–250 μg ml⁻¹. The extinction time for 10⁵ cfu in broth was 90 min for pyridinium chlorochromate and 120 min for quinolinium chlorochromate, except for B. subtilis which survived up to about 180 min and 360 min. A combination of the two compounds produced an antagonistic effect. The 50% lethal dose (LD₅₀ toxicity) in mice was estimated at 76 μg g⁻¹ and 33 μg g⁻¹ body weight for the quinolinium and pyridinium chlorochromates. The compounds also exhibited some potential for suppressing a simulated staphylococcal infection in mice at the dosage levels of ca 22 μg g⁻¹ for pyridinium chlorochromate and 45 μg g⁻¹ for quinolinium chlorochromate.     

MDL 72145, an Enzyme-Activated Irreversible Inhibitor with Selectivity for Monoamine Oxidase Type B

Abstract: MDL 72145, ( E )-2-(3',4'-dimethoxyphenyl)-3-fluoroallylamine hydrochloride, was designed and synthesised as a potential enzyme-activated irreversible inhibitor of monoamine oxidase (MAO). In vitro , the compound displayed time-dependent pseudo-first-order irreversible inhibitory characteristics with high selectivity for the B form of rat brain mitochondrial MAO At 10°C the K_t and T₅₀ values for the B enzyme were 40 μ M and 1.7 min, respectively, while these same kinetic constants for the A enzyme were 131 μ M and 14.5 mm, respectively. Selective protection against inactivation of the two forms of MAO by MDL 72145 was obtained by preincu-bating the enzyme with suitable concentrations of the selective A and B substrates, 5-hydroxytryptamine and benzylamine.     

In Vitro Antibacterial Activity of 4-Phenyl-1-(2-phenyl-allyl)pyridinium bromide: A Novel Class of Pyridinium Based Antibacterial Compounds

The continuing increase in the incidence of multi drug resistant pathogenic bacteria and shortage of new antimicrobial agents are the prime driver in efforts to identify the novel antimicrobial classes. In vitro antibacterial activity of 4-phenyl-1-(2-phenylallyl) pyridinium bromide was tested against Gram positive Staphylococcus aureus, Streptococcus species, Bacillus subtilis, and Gram negative Klebsiella aerogenes and Escherichia coli using disk diffusion method. Among them S. aureus showed strong antibacterial activity (21.99 ± 0.03 mm) while E. coli showed very little activity (8.97 ± 0.06 mm) towards the compound. The MIC of 4-phenyl-1-(2-phenyl-allyl)-pyridinium bromide for 90% S. aureus was ≤20 μg/ml and was compared with phenoxymethylpenicillin, cloxacillin, erythromycin and vancomycin. When 4-phenyl-1-(2-phenyl-allyl)pyridinium bromide showed MIC at ≤20 μg/ml, all others showed MIC at ≤100 μg/ml. Strong antibacterial activity of 4-phenyl-1-(2-phenyl-allyl)pyridinium bromide against S. aureus indicates that there is a possibility to use it as an effective antibacterial agent.     

Yersinia intermedia: A new species of enterobacteriaceae composed of rhamnose-positive,melibiose-positive,raffinose-positive strains (formerly calledYersinia enterocolitica orYersinia enterocolitica-like)

The drugs griseofulvin (10 μg/ml), nalidixic acid (0.05 μg/ml), quinine dihydrochloride (50 μg/ml), quinine ethylcarbonate (50 μg/ml), quinine urea hydrochloride (50 μg/ml), quinine lactate (50 μg/ml), and pamaquine (50 μg/ml) were chosen for laboratory studies. The minimal inhibitory concentration of the drug was used for determining the range of drug concentration needed to produce “mutational synergism” with ultraviolet radiation. Forward mutation from streptomycin sensitivity to resistance was used as a marker for mutagenicity. No stimulatory or inhibitory effects were noted on viable counts and mutation frequency, when the drugs were added (20–60 μg/ml) to the growth medium of unirradiatedEscherichia coli HCR⁺, HCR⁻, and irradiated HCR⁻ strains. These drugs increased mutation frequency and lethality of irradiated HCR⁺ bacteria. Incorporation of adenine (6 μm) into the minimal expression medium reverses the mutagenic effect of chloroquine. Chloroquine (50 μg/ml) did not interfere with the photoactivation of irradiated HCR⁺ cells. Our findings suggest that these chemicals selectively interfere with excision-repair.     

Susceptibility to Phosphomycin as a Differential Character for Gram Negative Anaerobic Bacilli

On the basis of their sensitivity to phosphomycin, various species of anaerobic Gram negative bacilli examined fell into one of two groups. All strains of Bacteroides were resistant to 500 umg/ml whereas strains of Fusobacterium were uniformly sensitive to 62.5 μg/ml. Disc sensitivity testing to phosphomycin at concentrations of 200–500 μg/ml provides a reliable and rapid means of distinguishing fusobacteria from bacteroides.     

The Semi Large-scale Production, Extraction, Purification and Properties of an Antibiotic Produced by Pseudomonas fluorescens Strain 175

S ummary : Production, extraction, purification and properties of an antibiotic produced by Pseudomonas fluorescens , strain 175, are described. Extraction efficiency was c. 57% with 3–5 g of product/350 1 batch culture, the product having an activity of 7900 units/mg (0· 12 μg/ml) against Staphylococcus aureus Paler. The antibiotic had the properties, of a weak acid and was active against both Gram positive and Gram negative bacteria; it was stable over a wide pH range, was bactericidal, inactivated by serum, had low toxicity and was haemolytic at relatively high concentrations.     

Cytokinesis of Candida albicans by Diethylstilbestrol

In vitro effects of the synthetic oestrogenic hormone diethylstilbestrol (DS) and diethylstilbestrol dipropionate (DSP) on Candida albicans have been assessed. At a concentration of 5–20 μg/ml. these compounds suppressed the growth of C. albicans even though the multiplication of the organism was not influenced immediately. When C. albicans was grown for approximately 4 h in tryptic soy broth, its multiplication was rapidly retarded by these two substances. Since C. albicans must grow on a suitable culture medium in order to absorb DS and DSP, it was not surprising that respiration, the uptake, and incorporation of nutrients by C. albicans was not influenced when the cells were 'resting'. Such plasma steroids as androsterone (0·5 μg/ml), 5α-androstan-3 β-diol (0·25 μg/ml), dehydroisoandrosterone (2 μg/ml), epiandrosterone (0·1 μg/ml), oestrone (0·1 μg/ml), progesterone (0·4 μg/ml), cortisol (0·2 μg/ml), cholesterol (10 μg/ml) in combination with DSP did not antagonize the retardive action of DSP for C. albicans .     

Inhibition of fungal growth and infection in maize grains by spice oils

Essential oils of 12 spices were tested for inhibitory activity against fungal infection and mycelial growth in post-harvest maize grains during storage. It was observed that the oils of cassia, clove (30 μl/g grain and above), star-anise (40 μl/g grain and above, geranium (30 μl/g grain and above) and basil (50 μl/g grain) inhibit the in viuo mycelial growth of established seed-borne infections of Aspergillus flavus, Curvularia pallescens and Chaetomium indicum as well as preventing infection following inoculation with A. flavus, A. glaucus, A. niger and A. sydowi . These oils also preserved the grain from natural A. flavus infection during the experimental period. Nutmeg, ginger and cumin oil (all at 50 μ1/g grain) could check mould growth and grain infection for only a brief period.     

Transformation of the entomopathogenic fungus Metarhizium anisopliae to benomyl resistance

Abstract Protoplasts of the entomopathogenic fungus Metarhizium anisopliae were transformed to benomyl resistance using cosmid pSV50 which harbours a β-tubulin gene cloned from a Neurospora crassa benomyl-resistant mutant. Transformant colonies, which appeared at a frequency of 4 per 50 μg DNA, grew and sporulated on 10 μg/ml benomyl, whereas the wild type was inhibited by 3 μg/ml. Southern blot hybridization of DNA from transformants showed that, in each case, tandem repeats of the cosmid had integrated at several chromosomal loci. The transformants were mitotically stable when subcultured on non-selective agar and retained the ability to infect and kill larvae of Manduca sexta . Two transformants were less virulent than the wild type and one of them showed slower in vitro spore germination. The benomyl-resistant phenotype persisted in reisolates from insect cadavers.     

β-Lactamase production and in vitro antimicrobial susceptibility of Porphyromonas gingivalis

Abstract β-Lactamase production by 98 Porphyromonas strains was investigated by the nitrocefin (chromogenic cephalosporin) test. Human isolates of P. gingivalis (91), P. endodontalis (2), and P. asaccharolytica (1) were tested, with four closely related Porphyromonas spp. of animal origin and four reference strains. The in vitro susceptibility of 64 P. gingivalis strains was investigated on Brucella blood agar by the E test. None of the human Porphyromonas isolates tested produced β-lactamase, but one Porphyromonas strain of animal origin, most closely resembling P. endodontalis , produced β-lactamase. P. gingivalis was susceptible to almost all of the drugs tested: benzylpenicillin, ampicillin, cefaclor, cefuroxime, erythromycin, clindamycin, tetracycline, doxycycline, metronidazole and ciprofloxacin; all strains were inhibited at 0.016 μg/ml, 0.023 μg/ml, 0.315 μg/ml, 0.064 μg/ml, 0.19 μg/ml, 0.016 μg/ml, 0.094 μg/ml, 0.047 μg/ml, 0.023 μg/ml, and 0.75 μg/ml of these drugs, respectively. Cotrimoxazole exhibited variable efficacy against P. gingivalis ; the range of MICs was 0.1095-32.0 μg/ml. The results indicate that β-lactamase production is currently not a problem amongst clinical isolates of P. gingivalis and strains are susceptible to most antimicrobial agents.     

Activity of the diastereoisomers of 13,23-dimethylpenta-triacontane, the sex pheromone of Glossina pallidipes, and comparison with the natural pheromone

ABSTRACT. All three synthetic diastereoisomers of 13,23-dimethyl-pentatriacontane, the pheromone of Glossina pallidipes (Austen), have been tested in the G. pallidipes sex pheromone bioassay, together with one of the four diastereoisomers of 13,17-dimethylpentatriacontane, originally proposed as a candidate pheromone of G. pallidipes. Of the three isomers of 13,23-dimethylpentatriacontane only the (13R,23S) isomer was active with an ED-50 of 4.47±1.09/μg (mean±SD). The (13R,17R)-13,17-dimethylpentatriacontane was inactive. The diastereo-isomeric mixtures of 13,17- and 13,23-dimethylpentatriacontane were active with ED-50 values of 17.88±1.25/μg and 8.88±1.15μg respectively. The natural pheromone was active with an ED-50 of 8.07±1.17 μg indicating it to be a diastereoisomeric mixture.     

Relationship between clinical characteristics and survival of gastroenteropancreatic neuroendocrine neoplasms: A single-institution analysis (1995–2012) in South China

In addition to stimulating linear growth in children, growth hormone (GH) influences metabolism and body composition. These effects should be considered when individualizing GH treatment as dose-dependent changes in metabolic markers have been reported. Hypothesis: There are different dose-dependent thresholds for metabolic effects in response to GH treatment. A randomized, prospective, multicentre trial TRN 98-0198-003 was performed for a 2-year catch-up growth period, with two treatment regimens (a) individualized GH dose including six different dose groups ranging from 17–100 μg/kg/day (n=87) and (b) fixed GH dose of 43 μg/kg/day (n=41). The individualized GH dose group was used for finding dose–response effects, where the effective GH dose (ED 50%) required to achieve 50% Δ effect was calculated with piecewise linear regressions. Different thresholds for the GH dose were found for the metabolic effects. The GH dose to achieve half of a given effect (ED 50%, with 90% confidence interval) was calculated as 33(±24.4) μg/kg/day for Δ left ventricular diastolic diameter (cm), 39(±24.5) μg/kg/day for Δ alkaline phosphatase (μkat/L), 47(±43.5) μg/kg/day for Δ lean soft tissue (SDS), 48(±35.7) μg/kg/day for Δ insulin (mU/L), 51(±47.6) μg/kg/day for Δ height (SDS), and 57(±52.7) μg/kg/day for Δ insulin-like growth factor I (IGF-I) SDS. Even though lipolysis was seen in all subjects, there was no dose–response effect for Δ fat mass (SDS) or Δ leptin ng/ml in the dose range studied. None of the metabolic effects presented here were related to the dose selection procedure in the trial. Dose-dependent thresholds were observed for different GH effects, with cardiac tissue being the most responsive and level of IGF-I the least responsive. The level of insulin was more responsive than that of IGF-I, with the threshold effect for height in the interval between.     

Endogenous profiles of indoleamines: serotonin and melatonin in different tissues of Coffea canephora P ex Fr. as analyzed by HPLC and LC-MS-ESI

Endogenous indoleamine profiles in various ex vitro and in vitro tissues of commercially important Coffea canephora were analyzed by using a high performance liquid chromatography and further confirmed with electrospray ionization mass spectrometry. High content of serotonin (SER) (98.54 ± 5 μg/g) and melatonin (MEL) (115.25 ± 6 μg/g) were found in freshly harvested seeds of C. canephora followed by zygotic embryo (65.25 ± 4 and 96.54 ± 5 μg/g fresh weight) and endosperm (34.08 ± 2 and 51.08 ± 4 μg/g fresh weight) of ripened fruits. Similarly endogenous pools of SER and MEL were moderate in in vitro tissues of C. canephora, i.e. callus (25.85 ± 2 and 75.74 ± 4), somatic embryos (31.88 ± 2 and 19.30 ± 2 μg/g fresh weight) and in vitro regenerated plant stalk (15.78 ± 1 and 38.25 ± 3 μg/g fresh weight), respectively. In view of significant levels of both SER and MEL in various tissues and beans of Coffea, further investigations on their physiological role needs to be investigated.     

Mechanism of Action of the Gonadal Steroids Producing Diminution of Growth of Staphylococcus Aureus

S ummary . Studies were undertaken to characterize the mechanism of action of the gonadal steroids responsible for decreasing growth of Staphylococcus aureus in vitro. Progesterone or testosterone at 20 μg/ml significantly increased the leakage of ¹⁴C activity from staphylococci pre-loaded with ¹⁴C-glucose. This enhancement of leakage was not detected with Gram negative micro-organisms. Hormonal diminution of total uptake of alanine was relatively independent of temperature and of the phase of culture. Anaerobiosis increased the steroidal diminution of alanine uptake c. 2-fold. Fraction-ation of staphylococci following exposure to various ¹⁴C-substrates in the presence of progesterone at 40 μg/ml did not reveal any distinctive influences on macromolecular syntheses. Entry of the labels into cellular pools, however, was altered for 8 of the 10 substrates tested. Exchange experiments detailed the effects of steroids on the efflux of internal alanine and lysine. With progesterone at 40 μg/ml, alanine effluxed from the internal pool 3 times as fast as from the corresponding controls. The opposite effect occurred with lysine and progesterone depressed its exit rate. The stepwise removal of cellular constituents indicated a preferential binding of hormones to cell wall components. Using ¹⁴C-progesterone or ¹⁴C-diethylstilbestrol, 24% and 29%, respectively, of the added hormone was firmly bound to mucopeptide preparations, compared to 1–5% bound to whole cells or isolated cell walls. We suggest that the hormones interfere with the integrated functioning of membrane-associated processes.     

Azadirachtin: its effect on gut motility, growth and moulting in Locusta

ABSTRACT. The biological effect of azadirachtin on fifth instar nymphs of Locusta migratoria migratorioides (R & F) have been studied in detail. Azadirachtin injection at the beginning of the instar results in a dose-dependent range of developmental aberrations. Low concentrations ( c. 1.7/μg/g body weight) result in adults with curled wing tips and reduced longevity; higher concentrations ( c. 2.9μg/g) result in death during the imaginal moult; doses of c. 6.5μg/g cause death immediately prior to the moult; and doses of c. 7.3μg/g induce a greatly extended instar. Such doses are related to a proportionately slower growth rate of the insect and a significantly reduced food intake, as assessed by wet weight and faeces production. Doses of 80μg/g result in death within 24 h. Experiments in vivo and in vitro demonstrate a significant reduction with azadirachtin treatment in the rate of passage of food through the gut, and in gut motility. The significance of this direct effect on gut motility is discussed in relation to the mode of action of azadirachtin on growth and moulting.     

Effects of cadmium chloride on the development of rainbow trout Oncorhynchus mykiss early life stages

The sub-chronic (28–56 days) effects of exposure to low concentrations of cadmium (Cd; 0·05, 0·25, 0·50 and 2·50 μg l⁻¹) shortly following fertilization on embryos, larvae and juvenile rainbow trout Oncorhynchus mykiss were examined. Premature hatching occurred at lower concentrations (0·05 and 0·25 μg l⁻¹ Cd), however, delayed hatching was seen in the 2·50 μg l⁻¹ Cd group, with >90% of hatching occurring on the last day of the hatching period. Larval growth was negatively affected by Cd exposure in a concentration-dependent manner. Larvae exposed to 2·50 μg l⁻¹ Cd were 13·9 ± 0·8% shorter in total length ( L _T) and weighed 22·4 ± 3·5% (mean ± s . e .) less than controls at the end of the exposure period. Plasma sex steroid concentrations (oestradiol in juvenile females and 11-ketotestosterone in juvenile males) were elevated (four- to 10-fold over controls) in exposed fish in both males and females, following 28 days of exposure to 0·05, 0·25 and 0·50 μg l⁻¹ Cd, respectively. These results suggest that environmentally realistic concentrations (in the μg l⁻¹ range) of Cd can affect the development of O. mykiss impacting embryos, larvae and juvenile fish.     

Enhancement of Bacillus thuringiensis with monosodium glutamate against larvae of obliquebanded leafroller (Lep.:Tortricidae)

Abstract:  Monosodium glutamate, a taste enhancer widely used in food industry, was tested in the laboratory to determine its phagostimulatory effects on larvae of Choristoneura rosaceana (Harris). Larvae fed apple leaves treated with 50–700  μ g/l monosodium glutamate increased leaf tissue consumption by approximately 40%. The stimulatory effect of monosodium glutamate (at 675  μ g/l concentration) was maintained throughout 10 days of continuous exposure. Adding 675  μ g/l monosodium glutamate to commercial formulation of Bacillus thuringiensis ssp. kurstaki, (DiPel^®2X DF), lowered LC₅₀ from 450 to 150  μ g/l (P < 0.05, Lethal Ratio Significance Test), indicating good potential for monosodium glutamate to enhance B. thuringiensis -based formulations.