Antibacterial activity of anacardic acid and totarol, alone and in combination with methicillin, against methicillinresistant Staphylococcus aureus

The inhibitory and bactericidal activities of anacardic acid and totarol, alone and in combination with methicillin, were investigated against methicillin-resistant Staphylococcus aureus (MRSA). The growth of two MRSA strains was inhibited by 6·25 μg ml^-1 of anacardic acid and 0·78 μg ml^-1 of totarol. The time-kill curve study showed that these two compounds were bactericidal against MRSA. Anacardic acid killed MRSA cells more rapidly than totarol, and no viable cells were detected after being exposed to 6·25 μg ml^-1 of anacardic acid for 6 h. Anacardic acid showed bactericidal activity against MRSA at any stage of growth, and also even when cell division was inhibited by chloramphenicol. In the combination studies, the minimal inhibitory concentration (MIC) of methicillin was lowered from 800 to 1·56 μg ml^-1 for MRSA ATCC 33591, and from 800 to 6·25 μg ml^-1 for MRSA ATCC 33592, by combining with 1/2 X MIC of anacardic acid. The time-kill curves demonstrated synergistic bactericidal activities for these combinations.     

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 .     

Antivibrio compounds produced by <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. W3: characterisation and assessment of their safety to shrimps

In order to explore compounds naturallly inhibitory to shrimp pathogenic vibrios, a culture filtrate of Pseudomonas sp. W3 at a pH of 2 was extracted with ethyl acetate (EtOAc) to produce 82.15 mg/l of a yellow–brown extract (EtOAc-W3) that had MIC values of 225-450 μg/ml against the growth of 18 shrimp pathogenic Vibrio harveyi strains. The MIC of EtOAc-W3 against the most pathogenic strain PSU 2015 was 450 μg/ml and this strain had the lowest LD₅₀ (50% lethal dose) to pacific white shrimp (Litopenaeus vannamei, PL 21). At this MIC value, EtOAc-W3 in artificial sea water (ASW) killed strain PSU 2015; however in natural sea water, only a partial growth inhibition was observed. The toxicity to pacific white shrimp and antivibrio activity of the EtOAc-W3 were investigated by conducting an experiment with 4 sets; native control (commercial ASW), EtOAc-W3 control (MIC/10, 45 μg/ml), challenge (inoculation 6.0 × 10⁶ c.f.u./ml PSU 2015) and treatment (6.0 × 10⁶ c.f.u./ml PSU 2015 + 45 μg/ml EtOAc-W3). The same experiment was repeated by increasing the dose of EtOAc-W3 to 90 μg/ml (MIC/5). Both concentrations of EtOAc-W3 tested had no toxicity to postlarval shrimps. A significant decrease in shrimp mortality was observed over a 72 h period as approximately 80% of the shrimps died in each challenge set but only 63 and 23% died in the presence of 45 and 90 μg/ml EtOAc-W3. The major component of EtOAc-W3 was supposed to be 2-heptyl-4-quinolone (HHQ) by FAB-MS and ¹H-NMR analyses of the purified fraction.     

Characterization of the rpsL and rrs genes of streptomycin-resistant clinical isolates of Mycobacterium tuberculosis in Japan

Mutations in the rpsL and rrs genes associated with streptomycin resistance in Mycobacterium tuberculosis clinically isolated in Japan were characterized. The rpsL genes of 172 clinical isolates were amplified by PCR and classified into two groups on the basis of Mbo II restriction digestion. Thirty-three out of 54 (61·1%) streptomycin-highly resistant isolates (MIC > 200 μg ml⁻¹) were not digested by Mbo II. By contrast, the remaining 21 of 54 (38·9%) streptomycin-highly resistant isolates, all of 41 isolates with streptomycin resistance at a lower level (20 μg ml⁻¹ < MIC ≤ 200 μg ml⁻¹), and all of 77 streptomycin-sensitive isolates, were restricted. Thus, all isolates resistant for Mbo II digestion showed a high level of resistance to streptomycin. Subsequently, the sequence for the rpsL and rrs genes from the 46 isolates were analysed. Eighteen out of 19 (94·7%) streptomycin-highly resistant isolates carried a mutation in any rpsL gene at position 43 or 88, or the rrs gene ; 10 out of 17 (58·8%) streptomycin-resistant isolates at a lower level were confirmed to exhibit the mutation of either the mutated rpsL gene at position 88, or the rrs gene. In the total 36 streptomycin-resistant isolates, the mutation of the rpsL or rrs gene was observed in 28 streptomycin-resistant isolates, corresponding to 77·8%, whereas none of the streptomycin-sensitive isolates had mutations in either the rpsL or rrs gene.     

Antibiotic Susceptibility of Helicobacter pylori Clinical Isolates: Comparative Evaluation of Disk-Diffusion and E-Test Methods

Antimicrobial susceptibility of 25 Helicobacter pylori strains isolated from patients with acid peptic diseases were tested for in vitro sensitivity to commonly used antibiotics using disk-diffusion and E-test, methods. All strains tested were susceptible to tetracycline by E-test, with the minimum inhibitory concentration (MIC) values being <0.125 μg/ml for all strains except for 6 (<0.023 μg/ml). However 1 strain was resistant by disk-diffusion method. One strain was resistant to clarithromycin both by disk diffusion and E-test (MIC <48 μg/ml), and 1 strain was resistant only by disk diffusion. Only one strain was resistant to amoxicillin by disk diffusion and E-test (MIC >256 μg/ml). For ciprofloxacin, three strains were resistant by disk diffusion and two by E-test (MIC <32 μg/ml). Sixteen strains were resistant to metronidazole by disk diffusion and E-test (MIC ≥ 8 μg/ml), and 1 was resistant only by E-test (MIC <48 μg/ml). Overall, 64% of the strains were resistant to metronidazole. The MIC for metronidazole was also tested by agar-dilution method, and metronidazole resistant strains had an, MIC >8 μg/ml. The disk-diffusion method showed excellent correlation with E-test results; there was 100% agreement for amoxicillin a other antibiotics showed 90% to 95% accuracy. Disk diffusion is cheaper than E-test (approximately 2.6 cents vs. US$2.60), is easy to perform, and is a reliable method for testing H. pylori susceptibility to antimicrobial agents in the clinical microbiology laboratory.     

Effects of Polyethylene Glycol 400 Monolaurate and Sodium Chloride Mixtures on the Antibacterial Activity of Solubilized Trichlorocarbanilide

S ummary . Polyethylene glycol 400 monolaurate (PGM), a nonionic surfactant, affected the solubility of trichlorocarbanilide (TCC), the amount solubilized being directly proportional to the concentration of PGM in the system. The bacteriostatic activity of TCC against Staphylococcus aureus was affected by high but not low concentrations of PGM; proportions of PGM to the germicide of up to 10: 1 did not interfere with the minimum inhibitory concentration (MIC) of TCC (0·08 μg/ml). The bactericidal activity against Staph. aureus of systems consisting of TCC and different proportions of PGM with and without 0·5% of NaCl was much higher than that of saturated aqueous solutions of TCC alone. However, TCC solutions containing NaCl and PGM were more bactericidal than corresponding systems without NaCl; the death rates in TCC-PGM-NaCl systems were 2–4 times that in TCC-PGM systems. The marked increase in the death rate was obtained with systems containing 2·5% of PGM.     

The Removal of Nitrate from Solution by Floc-forming Bacteria on Decomposing Cellulose Particles

S ummary : Cellulose particles in aerated liquid medium inoculated with activated sludge quickly became enveloped in floccular microbial growth (cellulose floc) able to assimilate nitrate rapidly from solution. Sedimenting the floc removed assimilated nitrogen, excess cellulose and biomass. At 18 and 22°, nitrate was removed from solution at 1·76 and 1·83 μg of nitrate-N/ml/h, respectively. Similar results were found with floc formed by a cellulose decomposing isolate and some noncellulolytic floc-forming bacterial contaminants. Washed preformed cellulose floc removed nitrate from dilute solution at 0·89 μg of nitrate-N/ml/h at pH 7·1–8·6. The C : N ratio of the supernatant fluid changed rapidly as nitrate became exhausted; the significance of this is considered in relation to complete removal of C and N by further biological oxidation.     

Immunodot detection of nisin Z in milk and whey using enhanced chemiluminescence

A highly specific antisera was produced in New Zealand white rabbits against nisin Z, a 3400 Da bacteriocin produced by Lactococcus lactis ssp. lactis biovar. diacetylactis UL 719. A dot immunoblot assay was then developed to detect nisin Z in milk and whey. As few as 1·5 10⁻¹ international units per ml (IU ml⁻¹), corresponding to 0·003 μg ml⁻¹ of pure nisin Z, were detected in carbonate-bicarbonate buffer within 6 h using chemiluminescence. When milk and whey samples were tested, approximately 0·155 μg ml⁻¹ (7·9 IU ml⁻¹) of nisin Z was detected. The detection limit obtained was lower than that of traditional methods including microtitration and agar diffusion.     

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.     

The relation between bacterial carbon and dissolved humic compounds in oligotrophic lakes

Abstract The mean annual biomass of planktonic bacteria showed large variations both within and between lakes. The lowest bacterial biomass was found in acidified lakes (7.8–12.1 μg C · 1⁻¹), and tended to increase with increasing water colour (up to 44.1 μg C · 1⁻¹). The highest recorded bacterial biomass was 138 μg C · 1⁻¹. The mean annual bacterial biomass equalled 23–45% of the algal biomass. Zooplankton biomass was high, compared to algal biomass (40–50%). Multiple regression analysis of 10 variables showed a strong positive correlation between bacterial biomass and humic content ( r = 0.74, P < 0.001), while other parameters, except pH, showed no correlation. The observation thus strongly supports the role of humic compounds in aquatic secondary production.     

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.     

Application of bioluminescence-based microbial biosensors to the ecotoxicity assessment of organotins

The toxicity of two common organotin pollutants and their initial breakdown products (tributyltin, dibutyltin, triphenyltin and diphenyltin) were assessed using two different bioluminescent microbial biosensors: Microtox and lux -modified Pseudomonas fluorescens pUCD 607. The organotins were made up as standards, and tested both in double-deionized water and in extracted soil solution, the latter representing a realistic matrix for terrestrial contamination. Microtox was especially sensitive to the organotins, with 50% effective concentration (EC₅₀) values (15 min) for tributyltin as low as 21·9 μg l⁻¹ in pure water, and 0·118 μg l⁻¹ in soil extract. The sensitivity of Microtox was increased by an order of magnitude in soil extract. The Ps. fluorescens was less sensitive, with EC₅₀ values (30 min) of around 800 μg l⁻¹ in pure water. The toxicity to Ps. fluorescens was decreased by around an order of magnitude in soil extract. The two biosensors showed different response patterns, with Microtox being more sensitive to the triorganotins and Ps. fluorescens being more sensitive to the diorganotins.     

Seasonal patterns of nitrogen fixation in termites

1. Termite nitrogenase activity was highest in autumn and spring (≈ 3 μg N₂ fixed termite fresh mass (g)^–1 day^–1) and lowest in winter and summer (≈ 0·8 μg N₂ fixed termite fresh mass (g)^–1 day^–1).
2. The nitrogenase activity of worker termites was significantly higher than all other castes (1·58 ± 0·27 μg N₂ fixed termite fresh mass (g)^–1 day^–1).
3. Worker termites constituted the largest proportion of all the castes throughout the study period (≈ 90%).
4. The localized input of fixed nitrogen by termites may reach 15·3 mg N log^–1 day^–1 and 5·6 g N log^–1 year^–1.     

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.     

Toxicity versus avoidance response of golden shiner, Notemigonus crysoleucas, to five metals

Avoidance thresholds and 96-h LC50 values were determined for golden shiners, Noiemigonus crysoleucas , for five individual elements: chromium, Cr; copper, Cu; cadmium, Cd; arsenic, As; selenium, Se. The avoidance concentrations were 73, 26 and 28 μgl^-1 for Cr-VI, Cu and As-III, respectively. Cadmium and Se were not avoided at experimental concentrations up to 68 and 3489 μg1^-1, respectively. Acute flow-through 96-h LC50 values were Cr-VI 55·0, Cu 84·6, and As-III 12·5 mg 1^-1, which were more than two orders of magnitude above avoidance concentrations. The acute flow-through 96-h LC50 values for Cd and Se were 2·8 and 11·2 mg 1^-1, respectively. These concentrations are 31 and 2·2 times the highest concentration employed in the avoidance tests, neither of which were avoided by the test organisms. Thus, simple toxicity tests do not identify the environmental hazard of some elements, and the most toxic elements may not elicit a behavioural response. When used in concert with tests of organism function, more realistic indicators of environmental hazard or safety may be determined.     

Delta-endotoxin proteins associated with spherical parasporal inclusions of the four Lepidoptera-specific Bacillus thuringiensis strains

Four Lepidoptera-specific reference strains of Bacillus thuringiensis , belonging to serovars sumiyoshiensis (H3a:3d), fukuokaensis (H3a:3d:3e), darmstadiensis (H10a:10b) and japonensis (H23), which produce spherical parasporal inclusions, were examined for comparative characterization of δ-endotoxins. SDS-PAGE profiles of the alkali-solubilized parasporal inclusions revealed the presence of single major protein bands of 130 kDa in the four strains. Chymotrypsin and trypsin treatment of the proteins gave profiles different from those of the strains HD-1 (serovar kurstaki , H3a:3b:3c) and T84 A1 (serovar sotto , H4a:4b). Also, minor variations were observed in proteolysis profiles among the four strains. The LC₅₀ values of purified parasporal inclusions for the silkworm ( Bombyx mori ) larvae were 7·35, 6·45, 3·08 and 2·63 μg g⁻¹ diet, respectively, showing that their toxicity levels were 5–15 times lower than that of the strain HD-1 (0·49 μg g⁻¹ diet). Analysis by immunodiffusion and immunoblotting with polyclonal antisera revealed that parasporal inclusion proteins of the four strains are highly related, whereas they shared few or no common antigens with those of the strains HD-1, T84 A1 and Buibui (serovar japonensis ).     

In vitro antibacterial activity of laboratory grown culture of Spirulina platensis

The hexane, ethyl acetate, dichloromethane, methanol extracts and spent media (extracellular substances) were tested in vitro for their antibacterial activity for which one Gram-positive bacterium (Staphylococcus aureus) and four Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, and Klebsiella pneumoniae) were used as test organisms. The methanol extract showed more potent activity than other organic extracts, spent medium of the culture exhibited little activity against E. coli only. No inhibitory effect was found against Klebsiella pneumoniae.The broth microdilution assay gave minimum inhibitory concentrations (MIC) values ranging from 1 to 512 μg/ml. The MIC of methanol extract against S. aureus and E. coli were 128 μg/ml and 256 μg/ml, respectively.     

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.     

Soil microbial carbon uptake characteristics in relation to soil management

Abstract The kinetics of glucose uptake by soil microbial communities in 16 different soild (7 under monocultures and 9 under crop rotations) differing in microbial biomass content, % C_org, pH and clay content were investigated at 22°C. The V _max value of microbial bimasses under monoculture, was o.27 μg C_gluc · μg⁻¹ C_mic · h⁻¹ (range 0.18–0.44), twice as high as the mean value of V _max of microbial biomasses under rotations (0.13 μg C_gluc, range 0.07–0.19). Mean values of K _m were 714 μg C_gluc and 290 μg C_gluc · g⁻¹ soil, respectively.
These differences were highly significant ( P =0.001, based on SE) and could not be relate to particle size distribution of the soils, pH or C_org. A Michaelis-Menten type uptake response was apparent over the total range of glucose concentrations used (45.4–1453.3 μg C_gluc · g⁻¹ soil) for microbial biomasses under rotation while the majority of microbial biomasses under monocultures showed a similar response only at low glucose concentrations. A different uptake mechanism appeared to be involved at higher glucose concentrations (similar to diffusion) in monoculture soils.     

Growth and Differentiation of Primary Cultures of Mouse Mammary Epithelium Embedded in Collagen Gel

Mammary glands from BALB/cfC3H midpregnant (9–11 days) mice were dissociated with collagenase and pronase, separated on a Percoll gradient, and the epithelial cells were cultured inside collagen gel. The cell number increased three-to five-fold when cultured for 6–8 days in DME/F12 (1: 1) medium containing 3% swine serum, insulin (10 μg/ml), cortisol (1.0 μg/ml), prolactin (10 μg/ml), transferrin (10 μg/ml), and epidermal growth factor (0.01 μg/ml). The casein level, as determined by radioimmunoassay, at the end of this growth phase, was much lower than that present in freshly dissociated cells. In order to stimulate casein production, the gels were released from the sides of the plastic dish and allowed to float for eight days in Waymouth's medium, containing insulin (10 μg/ml), cortisol (5 μg/ml), prolactin (10 μg/ml), and 0.25% bovine serum albumin. The casein level at the end of this differentiation phase was found to be comparable to that seen in the original freshly dissociated cells. Cells grown in DME/F12 (1: 1) medium containing 3% swine serum, insulin (10 μg/ml), and transferrin (10 μg/ml) were still capable of undergoing casein production, indicating that the presence of exogenous lactogenic hormones such as cortisol and prolactin, as well as exogenous growth factors such as epidermal growth factor, is not necessary during the growth phase for subsequent casein production during the differentiation phase. Two factors that seemed more important for subsequent casein stimulation were: (1) releasing collagen gels at the beginning of the differentiation phase, and (2) switching to'differentiation' medium. This present two-step protocol has allowed primary cultures of dissociated midpregnant mouse mammary epithelial cells to undergo several rounds of division inside a collagen gel matrix and to be subsequently stimulated to produce the mammary-specific protein, casein.