Determination of antibiotics using luminescent <Emphasis Type="Italic">Escherichia coli</Emphasis> and blood serum

The methodical bases for detecting antibiotics using a bioluminescent assay and blood serum are briefed. Antibiotics inhibit the luminescence of a genetically engineered Escherichia coli strain. The degree of inhibition depended on the type of antibiotic, its concentration, and the time of cell incubation with antibiotic. The highest cell sensitivity was recorded towards the aminoglycoside antibiotics, which amounted to 85 ± 10 ng/ml for gentamicin and streptomycin. The sensitivity of this system to a number of antibiotics essentially increased when the cells were previously activated with blood serum. The sensitivity of this method for gentamicin and streptomycin in the presence of blood serum amounted to 2.5 ± 0.5; for tetracycline, 45 ± 8 ng/ml. Use of the sera containing specific antibodies to the antibiotic detected provided a high sensitivity of the biosensor tested. Comparison of the luminescences of E. coli cells activated with normal and specific antisera upon incubation with an antibiotic allows the type of antibiotic and its quantitative content in the sample to be determined. Characteristic of the analysis of antibiotics with the help of recombinant E. coli are a high accuracy, sensitivity, specificity, simplicity, and a short time needed for measurement.     

Simultaneous radioimmunoassay of secretin and gastrin

Radioimmunoassay techniques for the measurement of gastrointestinal hormones have been modified to allow simultaneous assay of both secretin and gastrin in the same plasma samples. The system employs antibodies with high specificity for these two hormones and both ¹²⁵I-labeled secretin and ¹³¹I-labeled gastrin as markers. Separation of the free from the bound form of the hormones was achieved by plasma- and dextran-coated charcoal. The unique separation method has been found to be independent of the uniformity of plasma samples. The sensitivity and the reliability of the simultaneous assays were comparable to those of the single assays. The fasting levels of human plasma secretin and gastrin obtained by the simultaneous assay were 62.9 ± 3.6 and 43.5 ± 2.8 pg/ml respectively.     

Adiponectin Profile in Asian Patients Undergoing Coronary Revascularization and Its Association With Plaque Vulnerability: IDEAS‐ADIPO Study

Despite potent insulin‐sensitizing, anti‐inflammatory, and antiatherogenic effects in animal studies, the relationship between serum adiponectin level and coronary artery disease in patients remains unclear. We determined the adiponectin profile in a cohort of multiethnic Asian patients with coronary artery disease, and the association between serum adiponectin level and culprit lesion necrotic core (NC) content. Ninety‐four Asian patients (BMI, 25.3 ± 3.7 kg/m²) undergoing percutaneous coronary intervention were recruited. The serum adiponectin level was measured (n = 94), and the baseline virtual histology intravascular ultrasound examination was analyzed (n = 88). The median level of adiponectin was 3.7 µg/ml (interquartile range, 2.8–4.5 µg/ml). The serum adiponectin level was below 10 µg/ml in 90 patients (95.7%) and below 6 µg/ml in 80 patients (85.1%). There was a significant association between ethnicity and serum adiponectin level (P = 0.048). The median adiponectin level was highest among the Chinese, followed by the Malay and the Indians. Serum adiponectin levels were positively associated with culprit lesion NC content. A 1‐µg/ml increase in log adiponectin was associated with a 3.04% (95% confidence interval: 0.33–5.44) increase in culprit lesion NC content. This association remains significant after adjusting for age, sex, ethnicity, low‐density lipoprotein cholesterol, high‐density lipoprotein cholesterol, and procedural indication. We found a low serum level of adiponectin in Asian patients and a significant ethnic effect on serum adiponectin level. Increased serum adiponectin levels were independently associated with increased culprit lesion NC burden, suggesting a role for adiponectin in modulating coronary plaque vulnerability.     

HPLC determination of a chemically modified nonantimicrobial tetracycline: Biological implications

Chemically modified tetracycline (4-de-dimethylamino tetracycline), like commercially available tetracyclines, is known to inhibit experimentally induced pathologic collagen breakdown. A method for measurement of chemically modified tetracycline in small volumes (50 μl) of rat serum was developed using reversed-phase HPLC; this was necessary because this tetracycline analog lacks antimicrobial activity and, therefore, cannot be measured with standard bioassays. This method uses the same solution for extraction and elution thus providing a simple and rapid assay for both drugs. Using this technique, the concentration of chemically modified tetracycline and tetracycline were determined in rat serum at different times after oral administration. The serum concentration of chemically modified tetracycline was much higher than that for tetracycline, and its serum half-life was greater. The IC₅₀ of chemically modified tetracycline and tetracycline, as inhibitors of collagenase from rat polymorphonuclear leukocytes, was determined and found to be 4.1 × 10⁻⁸m (0.02 μg/ml) and 2.4 × 10⁻⁴m (120 μg/ml), respectively. Based on the serum levels of these drugs after oral administration, and their IC₅₀ values, chemically modified tetracycline is potentially a far more potent inhibitor of excess collagenase activity than tetracycline, during pathologic conditions, and may have the added advantage of not producing some of the typical complications of long-term antibiotic therapy.     

Evaluation of adrenocortical function in Florida manatees (Trichechus manatus latirostris)

The study objectives were to determine the predominant manatee glucocorticoid; validate assays to measure this glucocorticoid and adrenocorticotropic hormone (ACTH); determine diagnostic thresholds to distinguish physiological vs. pathological concentrations; identify differences associated with sex, age class, female reproductive status, capture time, and lactate; and determine the best methods for manatee biologists and clinicians to diagnose stress. Cortisol is the predominant manatee glucocorticoid. IMMULITE 1000 assays for cortisol and ACTH were validated. Precision yielded intra‐ and inter‐assay coefficients of variation for serum cortisol: ≤23.5 and ≤16.7%; and ACTH: ≤6.9 and ≤8.5%. Accuracy resulted in a mean adjusted R²≥0.87 for serum cortisol and ≥0.96 for ACTH. Assay analytical sensitivities for cortisol (0.1 µg/dl) and ACTH (10.0 pg/ml) were verified. Methods were highly correlated with another IMMULITE 1000 for serum cortisol (r=0.97) and ACTH (r=0.98). There was no significant variation in cortisol or ACTH with sex or age class and no correlation with female progesterone concentrations. Cortisol concentrations were highest in unhealthy manatees, chronically stressed by disease or injury. ACTH was greatest in healthy free‐ranging or short‐term rehabilitating individuals, peracutely stressed by capture and handling. Cortisol concentrations ≥1.0 µg/dl were diagnostic of chronic stress; ACTH concentrations ≥87.5 pg/ml were diagnostic of peracute stress. In healthy long‐term captive manatees, cortisol (0.4±0.2 µg/dl) and ACTH (47.7±15.9 pg/ml) concentrations were lower than healthy free‐ranging, short‐term rehabilitated or unhealthy manatees. Capture time was not significantly correlated with cortisol; ACTH correlation was borderline significant. Cortisol and ACTH were positively correlated with lactate. Zoo Biol 30:17–31, 2011. © 2010 Wiley‐Liss, Inc.     

Alpha-glucosidase inhibitory and antiplasmodial properties of terpenoids from the leaves of Buddleja saligna Willd

In our continuing search for biologically active natural product(s) of plant origin, Buddleja saligna, a South African medicinal plant, was screened in line with its traditional use for antidiabetic (yeast alpha glucosidase inhibitory) and antiplasmodial (against a chloroquine sensitive strain of Plasmodium falciparum (NF54)) activities. The hexane fraction showed the most promising activity with regards to its antidiabetic (IC₅₀?=?260?±?0.112?µg/ml) and antiplasmodial (IC₅₀?=?8.5?±?1.6?µg/ml) activities. Using activity guided fractionation three known terpenoids (betulonic acid, betulone and spinasterol) were isolated from this species for the first time. The compounds displayed varying levels of biological activities (antidiabetic: 27.31?µg/ml?≥?IC₅₀?≥?5.6?µg/ml; antiplasmodial: 14?µg/ml?≥?IC₅₀?≥?2?µg/ml) with very minimal toxicity.     

Detection and molecular characterization of carbendazim-resistant Colletotrichum truncatum Isolates causing anthracnose of soybean in Thailand

A loss of fungicide efficacy, particularly for carbendazim, was noted in soybean fields in Thailand and was considered to be due to the development of Colletotrichum truncatum resistance. The carbendazim sensitivity of C. truncatum populations isolated from various soybean fields in Thailand was thus evaluated with in vitro sensitivity assays and molecular characterization of mutations in the sequences of the ß₂-tubulin (TUB2) gene that confer carbendazim resistance in the pathogen. Among 52 isolates, 46 isolates were classified as highly resistant (HR) to carbendazim (EC₅₀ > 1,000 µg/ml). All HR isolates grew on PDA amended with carbendazim at 1,000 µg/ml. Six isolates were classified as carbendazim sensitive (S) (EC₅₀ < 1 µg/ml). Mycelial growth on PDA amended with 1 µg/ml carbendazim was inhibited by over 50% compared with growth on PDA alone. When a partial TUB2 gene from the isolates was amplified and analysed using predicted amino acid sequences, an alteration from glutamic acid to alanine at codon 198 (E198A) was found in 45 HR isolates for which the EC₅₀ was higher than 2000 µg/ml. This mutation resulted from a nucleotide substitution from adenine to cytosine (GA G → GC G). The other HR isolate, CtPhS_1, with EC₅₀ of 1,127 µg/ml, had an alteration at codon 200 (F200Y) (TT C → TA C).     

Distribution, diversity, and activity of sulfate-reducing bacteria in the water column in Gek-Gel lake, Azerbaijan

The distribution and activity of sulfate-reducing bacteria (SRB) in the water column of the alpine meromictic Gek-Gel lake were studied. Apart from traditional microbiological methods based on cultivation and on measuring the process rates with radioactive labels, in situ fluorescent hybridization (FISH) was used, which enables identification and quantification without cultivating organisms. The peak rate of sulfate reduction, 0.486 μg S 1⁻¹ day⁻¹, was found in the chemocline at 33 m. The peak SRB number of 2.5×10⁶ cells/ml, as determined by the most probable number method on selective media, was found at the same depth. The phylogenetic affiliation of the SRB, as determined by FISH, revealed the predominance of the Desulfovibrio spp., Desulfobulbus spp., and Desulfoarculus spp./Desulfomonile spp. groups. The numbers of spore-forming Desulfotomaculum spp. increased with depth. The low measured rates of sulfate reduction accompanied by high SRB numbers and the predominance of the groups capable of reducing a wide range of substrates permit us to assume utilization of electron acceptors other than sulfate as the main activity of the SRB in the water column. Original Russian Text ? O.V. Karnachuk, N.V. Pimenov, S.K. Yusupov, Yu.A. Frank, Ya.A. Puhakka, M.V. Ivanov, 2006, published in Mikrobiologiya, 2006, Vol. 75, No. 1, pp. 101–109.     

Nanoparticles combined with cefixime as an effective synergistic strategy against Salmonella enterica typhi

Enteric fever caused by Salmonella typhi has been the most crucial health issue in rural people, especially in Southeast Asia and Africa. Another disease, Salmonellosis, caused by a large group of bacteria of the genus Salmonella, cause substantial economic loss resulting from mortality and morbidity. Higher concentration and repeated use of antibiotics to treat these diseases will likely develop antibiotic resistance among the microbes. The nanoparticle has good penetration power and can kill microbes. Combining two strategies by using nanoparticles with antibiotics kills microbes and reduces the chances of the development of antibiotics resistance. Silver, Nickel, Copper, and Zinc oxide Nanoparticles were chemically synthesized and characterized in this study. Silver nanoparticles at a concentration of 10 µg/ml inhibit all the strains under study.In comparison, silver nanoparticles (16.90 µg/ml), Nickel nanoparticles (83 µg ml^?1), Copper nanoparticles (249 µg ml^?1), and Zinc oxide (1614 µg ml^?1) along with 50 µg/ml cefixime gave maximum zone of inhibition of 35 mm, 19 mm, 31 mm and 23 mm respectively. The antimicrobial assay showed that silver nanoparticles presented good antibacterial performance against all multi-drug-resistant pathogenic Salmonella sp alone as well as in combinations. The present study proved that silver nanoparticles at the lowest concentration along with cefixime could be a possible alternative to control the multi-drug-resistant pathogens.     

A radioimmunoassay for 11-oxotestosterone: Its application in the measurement of levels in blood sebum of rainbow trout (S. Gairdneri).

17β-Hyd.roxyandrost-4-ene-3,11-dione was linked via its 3-(O-carboxymethyl) oxime to bovine serum albumin to give a conjugate which was used to generate antiserum in rabbits. The antiserum, at an overall dilution of 1 in 16,000, together with [1,2-³H] 17β-hydroxyandrost-4-ene-3,11-dione synthesized from [1,2-³H] cortisone have been used to develop a radioimmunoassay for the parent steroid. The assay incorporates a purification step in which serum or plasma extracts are chromatographed on silica gel layers bound to plastic or aluminium sheets and the steroid, containing zones cut out and eluted directly with assay buffer. The cross-reactivities of several steroids with the antiserum and the specificity, sensitivity, accuracy and precision of the assay are described. Blood sera from Immature male rainbow trout contain $\text{ca}$ 0.2–0.4 μg/100 ml of 17β-hydroxyandrost-4-ene-3,11-dione. As male fish mature, serum levels rise sharply to reach values of 2 to >9 μg/100 ml. Levels in immature females rarely exceeded the assay sensitivity but serum from three ripe females showed low but detectable levels ($\text{ca}$ 0.2 μg/100 ml) of steroid. The assay has found application in sexing live fish for experimental purposes.     

In vitro comparative cytotoxic assessment of pristine and carboxylic functionalized multiwalled carbon nanotubes on LN18 cells

Multiwalled carbon nanotubes (MWCNTs) have been used in biomedical applications due to their ability to enter the cells. Carboxylic functionalization of MWCNT (MWCNT-COOH) is used to mitigate the toxicity of MWCNTs. Our study focuses on comparing the toxicity of MWCNT and MWCNT-COOH on the neuronal cells, LN18. Concentrations of 5, 10, 20, and 40 µg ml⁻¹ were used for the study, and cytotoxicity was determined at 0, 1, 3, 6, 12, 24, and 48 h of incubation. Cell viability was assessed by Trypan Blue, MTT, and Live dead cell assays, and the oxidative stress produced was determined by reactive oxygen species (ROS) and Lipid peroxidation assays. MWCNT-COOH showed higher cell viability than MWCNT for 20 and 40 µg ml⁻¹ at 24 and 48 h. This was also visually observed in the live dead cell imaging. However, at 48 h, the morphology of the cells appeared more stretched for all the concentrations of MWCNT and MWCNT-COOH in comparison to the control. A significant amount of ROS production can also be observed at the same concentration and time. Viability and oxidative stress results together revealed that MWCNT-COOH is less toxic when compared to MWCNT at longer incubation periods and higher concentrations. However, otherwise, the effect of both are comparable. A concentration of 5–10 µg ml⁻¹ is ideal while using MWCNT and MWCNT-COOH as the toxicity is negligible. These findings can further be extended to various functionalizations of MWCNT for wider applications.     

Quaternary ammonium compounds in soil: implications for antibiotic resistance development

Quaternary ammonium compounds (QACs) are surface-active, antimicrobial, high production volume (HPV) chemicals with a broad application in agriculture. This review provides a comprehensive overview of (1) predicted and measured concentrations of QACs in soils including their analysis, (2) sequestration mechanisms in soils based on their physicochemical properties and chemical structure, and (3) implications of concentrations and fate of QACs in soils for the proliferation of antibiotic resistance in the environment. Predicted environmental concentrations (PEC) for QACs that are applied to soils with manure are in the order of 3.5 mg kg^?1. Based on literature data, the median PEC of QAC in sewage sludge amended soils is 25 µg kg^?1. The positively charged QACs are mainly sorbed to clay minerals. We propose that QACs might be sequestered in the interlayer regions of layered silicates in clay-rich soils, reducing their acute toxicity, while increasing their persistence. The release of sequestered QACs from soil can still potentially maintain concentration levels that are sufficient to develop antibiotic resistance in the environment.     

Simultaneous determination of piroxicam and norfloxacin in biological fluids by high‐performance liquid chromatography with fluorescence detection at zero‐order emission mode

A new highly sensitive high‐performance liquid chromatographic method with fluorescence detection (HPLC–FLD) in zero‐order emission mode was developed for the first time for the simultaneous determination of piroxicam (PRX) and norfloxacin (NRF) in biological fluids. The fluorescence detector wavelengths were set at 278 nm for excitation and zero‐order mode for emission. The zero‐order emission mode produced greater sensitivity for the measurement of both drugs than a fixed emission wavelength (446 nm). The new developed method was validated according to International Conference of Harmonization (ICH) guidelines. Linearity was found to be over concentration ranges 0.001–20 μg/ml and 0.00003–0.035 μg/ml for PRX and NRF, respectively. The limits of detection were 4.87 × 10^?4 and 1.32 × 10^?5 μg/ml for PRX and NRF, and the limits of quantitation were 1.47 × 10^?3 and 4.01 × 10^?5 μg/ml, respectively. The current fluorescence method was found to be more sensitive than most commonly used analytical methods and was successfully applied for simultaneous determination of PRX and NRF in biological fluids (serum and urine) with recoveries ranging from 91.67% to 100.36% for PRX and from 96.00% to 101.43% for NRF.     

Rapid detection of resistance in Staphylococcus aureus by using Quicolor ES

Traditional microbiological methods are dependent on the growth of microorganisms, and hence require prolonged periods. The methods used to detect resistance in Staphylococcus aureus should have high sensitivity and specificity, yet provide results in a timely manner. The aim of this study was to evaluate the use of Quicolor (QC) ES^® agar for the rapid detection of resistance in S. aureus. We evaluated 100 clinical S. aureus isolates. Resistance detection was performed using traditional microbiological methods. Methicillin resistance detection was evaluated using traditional and molecular microbiological methods. Traditional antibiotic susceptibility testing methods, such as disc diffusion, were conducted using QC ES and Mueller–Hinton (MH) media. The plates were incubated at 36 °C for 5, 6 and 24 h. Rapid results obtained using QC ES agar after 5 h of incubation were consistent with those using the overnight procedure with MH agar for 83 of the 100 S. aureus (including methicillin-susceptible S. aureus) strains. However, the correlation for oxacillin between MH (24 h) and QC ES (5 h) was not satisfactory (r = 0.770). The total agreement between QC ES and MH agar was 83 % after 5 h, 89 % after 6 h, and 94 % after 24 h. The accurate and rapid detection of resistance in S. aureus is critical due to the associated therapeutic problems and infection control measures. We believe that the use of QC ES for S. aureus will reduce the delay in resistance detection, thus providing physicians and infection control practitioners with early information for better management.     

Na+-Dependent Transport of Amino Acids and Its Significance for Growth of a Lysine-producing Bacterium

A lysine-producing mutant Brevibacterium flavum HUT 8052, a threonine plus methionine (or threonine plus homoserine) auxotroph, grew rapidly as nearly as the wild strain in a medium supplemented with NaCl (60 µg/ml), threonine (100 µg/ml), and methionine (33.3 µg/ml). With NaCl concentrations less than 20 µg/ml, the mutant grew little or very slowly, The peculiar growth behavior of the mutant including the above phenomenon could be reasonably explained by the finding of Na⁺-dependent amino acids transport and the feedback inhibition of homoserine dehydrogenase by threonine in the bacterium.

The threonine transport was stimulated by Na⁺ and Li⁺. though the latter being less effective. The transport of threonine was inhibited by serine. The uptake of serine, isoleucine, leucine and valine was also stimulated by Na⁺     

Synergistic interactions between artocarpin-rich extract,lawsone methyl ether and ampicillin on anti-MRSA and their antibiofilm formation

Artocarpin-rich extract (ARE) was prepared using a green technology and standardized to contain 49·6% w/w artocarpin, while lawsone methyl ether was prepared using a green semi-synthesis. ARE, LME and ampicillin exhibited weak anti-MRSA activity with the MICs of 31·2–62·5 µg/ml. Based on the checkerboard assay, the synergistic interaction between ARE (0·03 µg/ml) and LME (0·49 µg/ml) against four MRSA isolates were observed with the fractional inhibitory concentration index (FICI) value of 0·008, while those of ARE (1·95–7·81 µg/ml) and ampicillin (0·49 µg/ml) as well as LME (0·49–1·95 µg/ml) and ampicillin (0·49 µg/ml) were 0·016–0·257. The time kill confirmed the synergistic interactions against MRSA with different degrees. The combination of ARE and LME as well as its combinations with ampicillin altered the membrane permeability of MRSA, which led to release of the intracellular materials. In addition, each compound inhibited the biofilm formation of standard MRSA (DMST 20654) and the clinical isolate (MRSA 1096). These findings suggested that cocktails containing ARE and LME might be used to overcome problems associated with MRSA. Additionally, the results implied that combination of either ARE or LME with available conventional antibiotic agents might be effective in countering these perilous pathogens.     

Toxic and inflammatory properties of two antibiotics: Muconomycin A and B

Toxicological investigations were conducted on two new antibiotics, Muconomycin A and Muconomycin B. These non-nitrogenous antibiotics were found to be highly toxic and capable of inducing profound inflammation in the peritoneal cavity of male albino rats. Either antibiotic produced large volumes (10–20 ml) of inflammatory exudate even when injected intraperitoneally in quantities of 1.6 × 10^?10 moles. An extensive profile of the electrolytes and proteins found in inflammatory exudates was developed. Simultaneous assays of the blood serum of treated rats provided a basis for comparing the concentrations of constituents of serum with those of the exudate. The results of these assays showed that the exudate contained lower concentrations of sodium and proteins, and greater amounts of potassium, calcium, and phosphorus than the serum. Chloride ion concentrations were variable. Since previous work showed that one of the manifestations of toxicity of these substances was the production of creatinuria, further studies were carried out with ATP/Creatine Phosphotransferase. These studies show that these antibiotics are potent in vitro inhibitors of the enzyme ATP/Creatine Phosphotransferase.     

Determination of S-phenylmercapturic acid by GC-MS and ELISA: a comparison of the two methods

Abstract

S-phenylmercapturic acid (PMA) is a specific urinary biomarker of benzene at exposure levels lower than 1?ppm. However, measuring PMA in urine is an expensive task by either GC or HPLC due to the necessity of extensive sample pretreatment. In the present study, a commercial chemiluminescence enzyme-linked immunosorbent assay (ELISA) test for PMA and GC-MS were used for screening urine samples of 60 workers employed in petrochemical settings. The ELISA results were evaluated by comparison with the GC-MS. Overall, the ELISA test proved sensitive (limit of detection?=?0.1?µg?l^?1), rapid, robust and reliable, affording results in good agreement with the GC-MS (54% of measurements) and no false-negatives. On the other hand, 46% of the ELISA assays were assigned as false-positives (arbitrarily established when ELISA >5?µg?l^?1, GC-MS <5?µg?l^?1) and a correlation coefficient of 0.687 was calculated between the two methods. It appears that urinary PMA routine biomonitoring on large numbers of samples is carried out in a cost-effective and rapid approach by preliminary screening with the ELISA assay followed by GC-MS confirmation of concentrations exceeding the biological exposure index for PMA.