Microbiological Assay Technique Research Articles

Residues of streptomycin antibiotic in commercial layers in Abeokuta and Ibadan metropolis

Old layers sold for human consumption in six open markets in Abeokuta and Ibadan metropolis of Ogun and Oyo States of Nigeria were analyzed for the presence of residues of streptomycin antibiotic. One hundred and twenty samples comprising of muscle (thigh and breast), liver and kidney tissues were subjected to microbiological assay technique for the detection of residues of the antibiotic. The assay was done on antibiotic medium 5 agar with Bacillus subtilis (ATCC 6633) organism. Results showed that 34.17% of the sampled birds were positive for the presence of streptomycin antibiotic. The concentration of the residues ranged between 0.18µg/g and 0.91µg/g. The mean residue levels recorded among the organs are not significantly different (P>0.05). Some of the muscle samples recorded residue concentration levels higher than the W.H.O recommended maximum residue level for streptomycin antibiotic in muscle tissue of food animals. In view of the importance of antibiotics in the treatment of bacterial diseases in both human and animals, it is advocated that the use of these drugs in food animals be done judiciously.

Pharmacokinetics of Cefquinome on Single Intravenous Administration in Marathwadi Buffalo Calves by Microbiological Assay Technique

Experiment was performed on six healthy Marathwadi buffalo calves of either sex of age above 6 months and weighing between 80 to 120 kg to study the different pharmacokinetic parameters after single intravenous administration @ 2 mg/kg body weight by microbiological assay technique. After intravenous administration of the drug, blood samples (4 ml each) of buffalo calves were collected from external jugular vein using disposable needles in clot activator tubes at different time intervals. The schedule of blood collection for pharmacokinetic studies after intravenous administration was at 0, 5, 10, 15, 30 min and 1, 2, 4, 6, 8, 10, 12 and 24 hrs. The serum levels of cefquinome were estimated by microbiological assay technique using large glass plate. Different Pharmacokinetic parameters were calculated as described by different scientists. The peak serum concentration, elimination half life, volume of distribution, total body clearance, absorption half life and area under curve values found were 1.74 ± 0.151 mcg/ml at 2.5 min of sampling time, 1.97±0.14 h, 3.97 ± 0.83 L/kg (Vd(B)) and 2.86 ± 0.34L/kg (Vdss), 1.11 ± 0.13 L/kg.h−1, 0.10 ± 0.02 h, and 1.93 ± 0.23 μg/ml/hr respectively. The bioavailability of cefquinome in buffalo calves was found to be 100 %. It may be concluded that the elimination half life of cefquinome was 2.54 h in Marathwadi buffalo calves indicating the repeating of doses at 12 to 15 h intervals in Marathwadi buffalo calves and the loading dose would be double than the maintenance dose of cefquinome after intravenous administration.

Pharmacokinetics of Cefquinome on Single Intramuscular Administration in Osmanabadi Goats by Microbiological Assay Technique

Pharmacokinetics of Cefquinome on Single Intramuscular Administration in Osmanabadi Goats by Microbiological Assay Technique

Effect of three anticoccidials on pharmacokinetics of tilmicosin in broiler chickens

The disposition kinetic of tilmicosin (25mg/kg) was studied following oral administration alone, pretreated with amprolium (240 ppm), pretreated with diclazuril (2.5 ppm) and pretreated with toltrazuril (25 ppm) in broiler chickens. The serum tilmicosin concentrations were determined by microbiological assay technique using Bacillus subtilis (ATCC 6633) as the test organism. Following oral administration of tilmicosin, the disposition curve was best described by two-compartment open model. The maximum serum concentration (Cmax) was 1.90 ± 0.11, 1.27 ± 0.13, 1.50 ± 0.14 and 1.41 ± 0.11µg/ml for tilmicosin alone and in the presence of amprolium, diclazuril and toltrazuril, respectively. The elimination half-life (T0.5 (el)) was significantly decreased (5.28 ± 0.30, 5.88 ± 0.33, 6.03 ± 0.25 h, respectively) in amprolium, diclazuril and toltrazuril pretreated broiler chicken compared to tilmicosin alone (7.30 ± 0.41 h). The outcomes illustrated a significant decrease in the interval between doses in amprolium, diclazuril and toltrazuril pretreated broiler chicken compared to tilmicosin alone. Amprolium diclazuril and toltrazuril, resulted in a significance decrease in AUC (12.02 ± 1.14, 15.50 ± 1.26 and 14.56 ± 1.46 µg.h.ml-1, respectively) compared to tilmicosin alone (21.98±1.83 µg.h.ml-1). It is concluded that the administration of amprolium, diclazuril and toltrazuril before tilmicosin would altered its pharmacokinetic profile in broiler chicken.

Comparative pharmacokinetics of cefoperazone following intravenous and intramuscular administration in goats

The pharmacokinetic profile of cefoperazone was studied in goats following intravenous and intramuscular administration of 20mg/kg body weight. Cefoperazone concentrations in serum were determined by microbiological assay technique using Escherichia coli (ATCC 10536) as test organism. Following i.v. administration, the cefoperazone serum concentration–time curve was best fitted in a two compartment open model. Cefoperazone has moderate distribution in the body of goats with Vdss of 0.44±0.03L/kg. The elimination half-life (T0.5(β)), area under curve (AUC) and total body clearance (Cltot) were 1.97±0.14h, 149.63±8.61μgml−1h−1, and 2.17ml/min/kg, respectively. Following i.m. administration, the drug was very rapidly absorbed, with an absorption half-life (T0.5(ab)) of 0.12±0.01h. The maximum serum concentration (Cmax) of 30.42±3.53μgml−1 was attained at (Tmax) 0.58±0.02h, with an elimination half-life (T0.5(el)) of 2.53±0.11h. The systemic bioavailability of cefoperazone in the goats after i.m. administration was 83.62% and in vitro protein binding was 20.34%. The serum concentrations of cefoperazone along 12h post i.m. injection in this study were exceeding the MIC of different susceptible micro-organisms responsible for serious disease problems. Consequently, a suitable intramuscular dosage regimen for cefoperazone was 20mg/kg repeated at 12h intervals in goats. The drug was detected in urine up to 12 and 18h following i.v. and i.m. administration, respectively.

Pharmacokinetic interaction of nitroxynil and cefoperazone in goats

The pharmacokinetic aspects of cefoperazone (CEFO) were investigated via intravenous (IV), and intramuscular (IM) injections in clinically healthy goats at a dose of 20 mg/kg b.wt. Goats were pre-treated with nitroxynil (25%) or nitroxynil (34%) subcutaneously in a dose 10 mg/kg b.wt, respectively. Cefoperazone was injected intramuscularly nine hours following anthelmintic administration, and blood samples were taken by jugular venipuncture at standardized intervals. Cefoperazone concentrations in serum were determined by microbiological assay technique. Goats pre-treated with nitroxynil (25%) or nitroxynil (34%) showed a significant decrease in serum cefoperazone level as compared to non-anthelmintic treated goats. The obtained data revealed that administration of nitroxynil in both concentrations negatively affected most of the cefoperazone parameters. In this respect, the elimination half-life T0.5(el), Cmax, AUC, and the systemic bioavailability (F %) were significantly decreased in both groups of nitroxynil-treated goats compared to non-anthelmintic treated goats. On the other hand, there were no significance differences between both nitroxynil (25% and 34%) treated goats. Concomitant administration of nitroxynil (25% and 34%) with cefoperazone resulted in significant alterations in the disposition kinetic of cefoperazone in goats. Consequently, the interaction between nitroxynil and cefoperazone could be of clinical significance and may require monitoring and adjustment of cefoperazone dosage.

Comparative Pharmacokinetics of Cefquinome (Cobactan 2.5%) following Repeated Intramuscular Administrations in Sheep and Goats

The comparative pharmacokinetic profile of cefquinome was studied in sheep and goats following repeated intramuscular (IM) administrations of 2 mg/kg body weight. Cefquinome concentrations in serum were determined by microbiological assay technique using Micrococcus luteus (ATCC 9341) as test organism. Following intramuscular injection of cefquinome in sheep and goats, the disposition curves were best described by two-compartment open model in both sheep and goats. The pharmacokinetics of cefquinome did not differ significantly between sheep and goats; similar intramuscular dose rate of cefquinome should therefore be applicable to both species. On comparing the data of serum levels of repeated intramuscular injections with first intramuscular injection, it was revealed that repeated intramuscular injections of cefquinome have cumulative effect in both species sheep and goats. The in vitro serum protein-binding tendency was 15.65% in sheep and 14.42% in goats. The serum concentrations of cefquinome along 24 h after injection in this study were exceeding the MICs of different susceptible microorganisms responsible for serious disease problems. These findings indicate successful use of cefquinome in sheep and goats.

Influence of flunixin on the disposition kinetic of cefepime in goats.

The pharmacokinetic profile of cefepime (10 mg/kg b.w.) was studied following intravenous and intramuscular administration of cefepime alone and coadministered with flunixin (2.2 mg/kg b.w.) in goats. Cefepime concentrations in serum were determined by microbiological assay technique using Escherichia coli (MTCC 443) as test organism. Following intravenous injection of cefepime alone and in combination with flunixin, there are no significant changes in the pharmacokinetic parameters. Following intramuscular injection of cefepime alone and in combination with flunixin, the maximum serum concentration was significantly increased in flunixin coadministered group compared with cefepime alone. However, no significant changes were reported in other pharmacokinetic parameters. The result of in vitro protein binding study indicated that 15.62% of cefepime was bound to goat's serum protein. The mean bioavailability was 92.66% and 95.27% in cefepime alone and coadministered with flunixin, respectively. The results generated from the present study suggest that cefepime may be coadministered with flunixin without change in dose regimen. Cefepime may be given intramuscularly at 12 h intervals to combat susceptible bacterial infections.

English

The effect of chloroquine phosphate on the pharmacodyanmic activity of ampicillin trihydrate against Staphylococcus aureus NCTC 6571 was investigated using physicochemical and microbiological assay techniques. The physicochemical method was thin layer chromatography and ultraviolet spectrophotometry in a condition simulating normal body temperature and pH. The microbiological assay compared the zones of inhibition of S. aureus NCTC 6571 by ampicillin trihydrate alone and in combination with chloroquine phosphate using the agar diffusion method. The kill kinetics was determined to give a dynamic assessment of the bactericidal activity of ampicilin trihydrate alone and in combination with chloroquine phosphate at various concentrations. The results showed that the zone of inhibition of S. aureus NCTC 6571 by ampicillin trihydrate in the presence of chloroquine was smaller than that produced by ampicillin alone. The kill kinetics revealed a significant increase in the percentage survivor of the organisms in the presence of chloroquine than with ampicillin alone. The results of the study suggest that there is no chemical interaction between the two drugs in vitro however, an antagonistic pharmacodynamic interaction exists between ampicilin trihydrate and chloroquine phosphate in vitro on S. aureus NCTC 6571. Key words: Ampicillin trihydrate, chloroquine phosphate, drug-drug interaction, antagonism, antimalarial, antibiotic.

Pharmacokinetic and Dosage Regimen of Gatifloxacin in Crossbred Cow Calves After Single Intravenous Administration

Pharmacokinetics of gatifloxacin (Gatiquin® - Cipla Ltd. Ahmedabad., India) in six cross bred cow calves following intravenous (IV) administration (4 mg/kg body weight). Estimation of gatifloxacin in plasma samples was analyzed by microbiological assay technique by using E. coli as test organism. Kinetic parameters of gatifloxacin were calculated by using two-compartment open model. Therapeutic concentration of gatifloxacin (0.2 µg/ml) was maintained up to 16 h and the drug was detectable up to 24 h. Distribution half life (t1/2 α), elimination half life (t1/2 β), mean residential time (MRT), Volume of distribution during area under curve (Vdarea) and total body clearance (ClB) of 0.125 ± 0.01 h, 13.17 ± 0.16 h, 18.54 ± 0.26 h, 1.81 ± 0.02 L/kg and 0.076 ± 0.01 L/kg/h, respectively were obtained for gatifloxacin. For maintaining therapeutic concentration of 0.2 µg/mL, a loading dose (D*) of around 0.8 mg/kg and maintenance dose (D0) of 0.4 mg/kg may be used at the dosage interval (τ) of 16 h for treating systemic infections. Key word: Pharmacokinetic, Gatifloxacin, Dosage regimen and Crossbred cow calves.

Paternal folate status at mating influences fetal brain IGF‐2 expression in rats

We investigated the effect of paternal folate status on placental and fetal whole brain folate content and DNA methylation, and fetal whole brain insulin‐like growth factor‐2 (IGF‐2) expression in rats. Rats were divided into four groups: paternal folate‐supplemented (PS) or folate‐deficient (PD), and maternal folate‐supplemented (MS) or folate‐deficient (MD) and were fed with folic acid‐supplemented (8mg/kg) and folic acid‐deficient (0mg/kg) diets. After 4 weeks, rats were mated (PS×MS, PS×MD, PD×MS and PD×MD) and placenta and fetal whole brain were isolated at day 20 of gestation. Folate content, DNA methylation and the expression of IGF‐2 in the placenta and brain analyzed by microbiological assay, ELISA and western blot technique, respectively. Compared to the PS×MS group, placental folate content (P < 0.0001) and DNA methylation in placenta (P < 0.0001) and brain (P < 0.0001) was significantly decreased in the PD×MS group as much as the PS×MD group. Brain folate content was decreased in the PD×MS group as much as the level of PD×MD group (P < 0.05). As compared to PS×MS group, the expression of IGF‐2 in the PD×MS group was decreased as much as the level of the PD×MD group (P < 0.0001). These results suggest that the effect of paternal folate deficiency could be as important to the growth and development of placenta and brain of the fetus as maternal folate deficiency.

Effect of Withania somnifera (Ashwagandha) on the Pharmacokinetics of Amikacin: A Future Antimicrobial Polypharmacy

The pharmacokinetic study of Amikacin with interaction of W. somnifera was investigated after single administration of 10 mg/kg of Amikacin intravenously and 500 mg/kg of W. somnifera orally in six non-descript healthy buffalo calves. Estimation of concentration of Amikacin in plasma was carried out by microbiological assay technique (Agar gel diffusion technique) by using Escherichia coli (ATCC 25922) as test organism. Following a single administration of 10 mg/kg of Amikacin intravenously and 500 mg/kg of W. somnifera orally in six non-descript healthy buffalo calves, plasma concentration of Amikacin, at 1 min of combined administration of these drugs were 17.05 ± 0.28 μg/ml. The effective therapeutic concentration of Amikacin (≥ 1.0 μg/ml) was maintained up to 24 h with mean value of 1.86 ± 0.038 μg/ml. The mean distribution half-life of phase 1 (t1/2 α1), phase 2 (t1/2 α2) and elimination half-life (t1/2 β) were calculated to be 0.060 ± 0.004, 3.99 ± 0.27 and 6.15 ± 0.21 h, respectively whereas the total body clearance (ClB) was ranged from 0.064 to 0.075 L/kg/h with a mean of 0.068 0.002 L/kg/h. A satisfactory intravenous dosage regimen of Amikacin in buffalo calves after combined administration would be 2.36 ± 0.095 mg/kg followed by 1.75 ± 0.10 mg/kg at 12 h interval supports the excellent clinical efficacy of Amikacin in buffalo calves.

Fabrication of novel biofibers by coating silk fibroin with chitosan impregnated with silver nanoparticles

Nanoparticle based agents often applied as coatings on biomaterials have shown promise in delivering the improved sterility against variety of microbes. In the present study, silk fibers (SF) were coated with chitosan impregnated with silver nanoparticles (Ag-C-SF). These Ag-C-SF fibers were characterized using scanning electron microscopy (SEM) with energy dispersive X-ray analysis, atomic force microscopy (AFM), Infra Red spectroscopy, Thermogravimetric Analysis and Microbiological assay techniques. AFM studies have confirmed the nano sized silver particles in chitosan solution; SEM pictures have exhibited the coating of chitosan along with silver nanoparticles on the silk fibroin. The modified fibers have also shown anti-microbial activity and improved thermal stability. The Ag-C-SF fibers may be explored as wound dressing and tendon reconstruction material in future.

The Influence of Silver Content on the Tribological and Antimicrobial Properties of ZrN/Ag Nanocomposite Coatings

ZrN/Ag nanocomposite coatings with varying silver contents were produced by co-deposition in a dual pulsed magnetron sputtering system. The coatings were characterised using scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), X-ray diffraction (XRD), scratch adhesion testing, thrust washer wear testing and nanoindentation. The hardness of the ZrN/Ag coatings and the friction coefficient running unlubricated against a steel counterface decreased with increasing silver content, whereas the coating-to-substrate adhesion increased for coatings with higher silver contents, compared to a 'pure' ZrN coating. The antimicrobial properties of the coatings were investigated using two well established microbiological assay techniques: zones of inhibition and a NBT (nitro-blue tetrazolium) redox dye. Zones of inhibition were used to determine the extent of silver ion release from the nanocomposite materials, and a NBT (nitro-blue tetrazolium) redox dye was used to determine the antimicrobial effectiveness of the surfaces following incubation. The microorganisms tested were Pseudomonas aeruginosa (P. aeruginosa), Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). Whilst no zones of inhibition were observed for S. aureus, on any of the surfaces, the diameter of the 'kill' zones generally increased with increasing silver content for the other microorganisms. For the NBT assays, after incubation, no P. aeruginosa colony forming units were observed on any surface and the number of viable cells of E. coli and S. aureus decreased with increasing silver content, compared to a 'pure' ZrN surface.

Age-related intramuscular pharmacokinetics of cefquinome in sheep

The pharmacokinetic profile of cefquinome was studied in one, six-months and one year old sheep following a single intramuscular doses of 1 and 10 mg kg −1 b.wt. Cefquinome concentrations in serum were determined by microbiological assay technique using Micrococcus luteus (ATCC 9341) as test organism. Following intramuscular administration of cefquinome, the absorption half-lives ( t 0.5(ab)) were 1.540, 1.037 and 0.664 h at a dose of 1 mg kg −1 b.wt. and 1.844, 1.290 and 1.605 h at a dose of 10 mg kg −1 b.wt. in the three ages, respectively. After the two doses, the maximum serum concentrations ( C max) of 0.732, 1.145, 1.205 and 3.525, 5.088, 4.576 μg ml −1 were attained after ( t max) of 3.812, 3.029, 2.174 and 3.785, 2.824, 3.095 h in the three ages, respectively. The elimination half-life ( t 0.5(el)) and MRT values of cefquinome were longer in one-month old sheep compared to six-months old and yearling sheep. The absorption and elimination processes were delayed in newborn sheep of one-month old in contrary to six-month and yearling animals. The in vitro serum protein-binding tendencies were 8.254%, 11.586% and 13.002%, for one, six-months and one year old sheep, respectively. Based on this study and economically, an optimal intramuscular dosage regimen of cefquinome would be 1 mg kg −1 once daily in one-month, six-months and one-year old sheep to achieve and maintain the therapeutic serum levels within safe limits.

Residues of tetracycline in imported frozen chickens in South West Nigeria

Imported old layer birds (100) sold for human consumption in open markets in Ogun, Lagos and Oyo States of Nigeria were analyzed for the presence of tetracycline using microbiological assay technique. The assay was done on antibiotic medium 2 agar seeded with Bacillus subtilis (NRRL-B-354). Results showed that 14% of the birds were positive for tetracycline residues deposition. The mean concentration of the residues was between 0.0316µg and 0.0513µg for thigh muscle samples while the concentration of the residues recorded from breast samples were between 0.0383µg and 0.0727µg. There was no significant difference (P>0.05) in the concentrations of tetracycline residues recorded in both muscle types. Location of purchase of the chickens also had no significant effect on residue deposition in the tissues. Keywords : residues, tetracycline, imported layers Tropical Veterinarian Vol. 23(1) 2005: 1-4

Subcutaneous pharmacokinetics and dosage regimen of cefotaxime in buffalo calves (Bubalus bubalis)

The pharmacokinetics and dosage regimen of cefotaxime following its single subcutaneous administration (10 mg/kg) were investigated in buffalo calves. Plasma and urine samples were collected over 10 and 24 h post administration, respectively. Cefotaxime in plasma and urine was estimated by microbiological assay technique using E. coli as test organism. The pharmacokinetic profiles fitted one-compartment open model. The peak plasma levels of cefotaxime were 6.48 ± 0.52 µg/ml at 30 min and the drug was detected upto 10 h. The absorption half-life and elimination half-life were 0.173 ± 0.033 h and 1.77 ± 0.02 h, respectively. The apparent volume of distribution and total body clearance were 1.17 ± 0.10 l/kg and 0.45 ± 0.03 l/kg/h, respectively. The urinary excretion of cefotaxime in 24 h, was 5.36 ± 1.19 percent of total administrated dose. A satisfactory subcutaneous dosage regimen for cefotaxime in buffalo calves would be 13 mg/kg repeated at 12 h intervals.

Monitoring the ingestion of anti-tuberculosis drugs by simple non-invasive methods

This investigation retrospectively assessed inexpensive non-invasive qualitative methods to monitor the ingestion of anti-tuberculosis drugs isoniazid, rifampicin and rifapentine. Results showed that commercial test strips detected the isoniazid metabolites isonicotinic acid and isonicotinylglycine as efficiently as the isonicotinic acid method in 150 urine samples. The presence of rifamycins in urine samples ( n = 1085) was detected by microbiological assay techniques and the sensitivity compared to the n-butanol extraction colour test in 91 of these specimens. The proportions detected by the two methods were significantly different and the sensitivity of the n-butanol procedure was only 63.8% (95% CL 51.2–76.4%) as compared to that of the superior microbiological method. Final validation ( n = 691) showed that qualitative assays measure isoniazid and rifamycin ingestion with an efficiency similar to high-performance liquid chromatography. The qualitative procedures may therefore be valuable in clinical trials and in tuberculosis clinics to confirm drug ingestion.

Development of a novel assay method for colistin sulphomethate

Increased use of colistin therapy for infections caused by Pseudomonas aeruginosa has indicated a need for a more robust microbiological assay technique. This report describes a quick and simple microbiological assay for quantifying levels of colistin sulphomethate in serum and urine samples from cystic fibrosis patients. The technique uses no specialised or costly equipment and is suitable for use in all routine diagnostic microbiology laboratories.

In vitro activity of propolis against Streptococcus pyogenes.

Propolis, a multifunctional substance used by bees to maintain the safety of their hives, is popular for its therapeutic potential against some micro-organisms. Ethanolic extracts of two propolis specimens, collected from different areas within a region in the north-west of Italy, were examined to evaluate their antimicrobial activity against 46 Streptococcus pyogenes strains. By both agar dilution and agar diffusion methods, the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were </= 234 microg ml-1, corresponding to a one in 512 dilution of the 12% (w/v) extracts. One of the two propolis samples was more active and this extract was shown to be richer in the flavonoids pinocembrin and galangin using HPLC. Therefore, with a simple microbiological assay technique, in particular the agar dilution method, it was possible to standardize the analysis of propolis samples to identify the quality parameters of this natural product before use for medical treatment.