A Novel Phage Cocktail Therapy of the Urinary Tract Infection in a Mouse Model.

Escherichia coli (E. coli) is a major bacterial pathogen associated with many cases of serious infections, such as urinary tract infections (UTI) and meningitis intestinal. The rapid emergence of antimicrobial multidrug-resistant bacteria occurring worldwide has been attributed to the overuse of antibiotics. Alternative strategies must be developed to overcome antibiotic resistance. A promising alternative for the treatment of infections is the use of phages as antibacterial agents. A total of 90 female albino mice were randomly divided into three groups (n=30) and used for the induction of UTI. The animals were acclimatized in their cages for 24 h before inoculation and allowed to access chow and water freely. For UTI induction, the peri-urethral area was sterilized with 70% ethanol, and bacterial inoculation was then injected into the bladder through the urethra using a 24-gauge sterile Teflon catheter with an outer diameter of 0.7 mm and length of 19 mm. A single phage and a phage cocktail preparation have been evaluated for their therapeutic activity in the mouse model of chronic UTI induced by transurethral injection of two isolates of the uropathogenic E. coli 8 and E. coli 302. The results of the transurethral and intra-peritoneal injection of phage(s) that prepared on day 10 after the establishment of the mouse chronic model showed no effect of a single phage PEC80 in the treatment of UTI, whereas both administration routes of the phage cocktail preparation resulted in the clearance of bacteria from mice urine and homogenates of the urinary bladders and kidneys of the sacrificed mice after 24 h following the administration of phage cocktail dose. The high activity of the phage cocktail in the treatment of mouse chronic model of UTI is attributed to the broader host range of the phage cocktail, compared to the very narrow host range of the phage PEC80. It is concluded that the phage therapy by using phage preparations as the 25 phages cocktail evaluated in this study is a highly promising and potential alternative therapy for human UTIs.

Development of partition coefficients, Vmax and Km values, and allometric relationships.

A knowledge of the methods used to obtain partition coefficients, Vmax, and Km values, and the use of allometric relationships is essential to understanding their role in physiologically based pharmacokinetic (PBPK) models. Vial equilibration methods for obtaining the partition coefficients of volatile and nonvolatile compounds were presented using the results from studies with p-chlorobenzotrifluoride (PCBTF) and isofenphos, respectively. Partition coefficients for volatile and nonvolatile compounds from published studies were included. Several published in vivo inhalation (gas uptake) studies and in vitro enzyme studies were presented to demonstrate several methods for obtaining Vmax and Km values. Allometric equations used in PBPK models for body weight scaling of respiration and cardiac rates between species were presented along with equations for within species body weight scaling of Vmax.

Development of in vitro Vmax and Km values for the metabolism of isofenphos by P-450 liver enzymes in animals and human.

The rate of metabolism of [14C]isofenphos (IFP) to isofenphos oxon (IFP-oxon), des N-isofenphos (d-N-IFP), and des N-isofenphos oxon (d-N-IFP-oxon) by rat, guinea pig, monkey, dog, and human liver microsomal P-450 enzymes was studied to obtain Vmax and Km values for Michaelis-Menten kinetics. The monkey had the highest Vmax value for the conversion of IFP to IFP-oxon (desulfuration), 162 nmol isofenphos hr-1 per 1.3 nanomoles P-450, followed by guinea pig (98), rat (66), dog (43), and human (14). The Km values for the desulfuration of isofenphos were 19.2, 7.4, 14.1, 23.3, and 18.4 microM, respectively, for the monkey, guinea pig, rat, dog, and human. The Vmax values for the dealkylation process (conversion of IFP to d-N-IFP) were 64.6, 17.2, 9.7, and 7.3 nmol isofenphos hr-1 per 1.3 nanomoles P-450 for the monkey, rat, dog, and human, respectively. For the dealkylation process, monkey had the highest Km value, 16.3 microM IFP, followed by human (11.2), rat (9.9), and dog (9.3). The rate of metabolism of IFP-oxon and d-N-IFP to d-N-IFP-oxon were separately studied. The Vmax and Km values obtained in this study for animal and human liver P-450 enzymes will be used to develop a PB-PK/PB-PD model to predict the fate and toxicity of isofenphos in animals and man.

Effect of inhaled dimethylselenide in the Fischer 344 male rat.

In this project, a total of 60 adult Fischer 344 male rats were exposed to dimethylselenide (DMSe) vapor at 1607, 4499, or 8034 ppm for 1 h (20 rats/group). In addition, 26 unexposed rats were used as controls. The exposed rats were observed frequently during the 7 d following exposure and appeared normal. The animals were sacrificed at either 1 or 7 d after inhalation and the major tissues were grossly examined and weighed. Selenium levels were found to be elevated only in the lung at d 1. At d 1, significant changes in organ weights were an increase in the lung weight at exposure levels of 1607 and 8034 ppm of DMSe and in liver weight at 4499 and 8034 ppm. At d 1, significant changes in the lung were an increase in protein at 1607 and 8034 ppm of DMSe, and an increase in RNA and a reduction in DNA at 4499 ppm DMSe. The only change in the liver was a reduction in DNA at 4499 ppm. At 7 d, the protein content and RNA content of spleen were increased. Lung, liver, kidney, spleen, thymus, lymph nodes, pancreas, and adrenal gland were examined microscopically and found to be normal. All of these observed responses were minor and did not severely impact the health of the rats. Overall, the data indicate that the inhalation of DMSe for 1 h has relatively low toxicity in rats even at high concentrations.

The lymphotoxic action of vanadate.

The acute toxicity of ammonium metavanadate (15.5 mg/kg) in mice was investigated to examine the induction of lymphoid necrosis to (1) verify the reproducibility of the lesions in the thymus, lymph nodes, and spleen; (2) determine whether the necrosis of lymphoid tissue previously observed during the first 3 days post-treatment but absent at 14 days was the result of differences in sensitivity of the mice or the result of recovery from the effects of vanadium; and (3) determine whether differences in the presence and the degree of necrosis between thymus and spleen were correlated with differences in the uptake of vanadium in these tissues. A timed sacrificed study was conducted in conjunction with a 48V tracer. In this study, BALB/C mice were injected subcutaneously (s.c.) with ammonium metavanadate solution (15.5 mg/kg). Groups of mice were sacrificed at 0.5, 1, 2, 3, 4, 6, 8, 10, 12, 14, 21, and 28 days postexposure. Lymphoid necrosis was found in the thymus, spleen, lymph nodes, and bone marrow, with the necrosis being most severe in the thymus. The necrosis was moderate at 0.5 days, most severe at 2 to 3 days, with recovery beginning at 4 days, and proceeding to full recovery at 14 to 28 days. At 0.5 days post-treatment, the concentration of vanadium in thymus and spleen was 4.4 and 8.3 micrograms/g, respectively. At all post-treatment periods, with the exception of the 1- and the 4-day periods, the concentration of vanadium in spleen was significantly higher than in the thymus, p less than 0.05. The treated animals showed neurological signs (ataxia, convulsion, dyspnea, and paralysis of hind legs) between 5 min and 54 hr post-treatment, but the concentration of vanadium in the brain was very low during this period (less than 5.2% of blood concentration).

Time and dose-response study of the effects of vanadate in rats: changes in blood cells, serum enzymes, protein, cholesterol, glucose, calcium, and inorganic phosphate.

A daily dosage of vanadate (0.9 mgV/kg) injected subcutaneously for 16 days to adult rats produced significant changes in blood cells and serum elements. The hematological changes included an increase in white blood cell count at two days after the last injection. At five days, red blood cell count (RBC), hemoglobin, and packed cell volume (PCV) were low. At 12 days, there were reductions in RBC, hemoglobin, PCV, and lymphocyte counts and an increase in polymorphonuclear cell (PMN) counts. At 25 days, RBC, hemoglobin, and PCV were still low. At 40 days, the only change was a reduction in RBC. Changes in the serum at two days posttreatment were a reduction in lactic dehydrogenase activity (LDH), alkaline phosphatase activity (AP), calcium, albumin, and total protein and an increase in cholesterol. At five days, glutamic-oxalacetic transaminase (GOT), lactic dehydrogenase (LDH), inorganic phosphate, and total protein were low and calcium was high. At 12 days, GOT, glutamic-pyruvic transaminase (GPT), and LDH were reduced, and the levels of calcium and cholesterol were elevated. At 25 days, there was a reduction in GPT and LDH and an increase in glucose, calcium, and albumin. At 40 days, the levels of GOT, LDH, AP, and inorganic phosphate were still low. Vanadate at lower dosage levels (0.3-0.6 mg V/kg per day for 16 days) also produced significant changes in blood cellular and serum elements but at lesser degrees of severity. These findings show that the exposure of rats repeatedly to low levels of Vanadate caused anemia, elevation in blood cholesterol levels, and a reduction in serum enzymes activities.

Time and dose-response study of the effects of vanadate on rats: morphological and biochemical changes in organs.

Vanadate at a dosage level of 0.9 mg V/kg per day produced acute toxic signs in rats when injected subcutaneously for 16 days. These signs were weakness, loss of appetite, dehydration, significant reduction in body weight, nose bleeding, and death. The pathological and biochemical changes were most severe in kidney tissue. The kidney lesions were bilateral and multifocal. At two days, degenerative and necrotic changes of the tubular and glomerular epithelium, thickening of glomerular membrane, vascular congestion, and edema were observed. At five days, proliferation of tubular epithelial and interstitial cells was observed. At 12 days, the cellular proliferation in both cortex and medulla was significantly greater. Fibrosis was observed at glomerular tuft, preglomeruli, pretubules, and interstitium (cortex and medulla). At 25 days, the collagen deposition reached the highest level in all regions, cellular proliferation decreased, and thickening of the arteriolar wall became prominent. The renal lesions were coupled with changes in the levels of protein, RNA, DNA, and hydroxyproline. At 40 days, the kidney showed signs of recovery. Blood urea nitrogen levels were significantly elevated at 2-25 days post-treatment. Stained tissue sections from liver, lung, heart, spleen, thymus, lymph nodes, testes, and adrenal glands of the treated rats were examined microscopically and appeared normal. Biochemically, significant changes (p less than .05) in protein, RNA, DNA, and hydroxyproline were also observed in these organs. At lower dosage (0.6 mg V/kg per day for 16 days), similar but less severe pathological and biochemical changes in kidneys and other organs were observed. At 0.3 mg V/kg per day for 16 days, the changes in the tissues were detected only at the biochemical level. These results indicate that the toxic effects of vanadium are cumulative and that vanadium-produced fibrosis in tissues is dose-dependent.

Acute toxicity of ammonium metavanadate in mice.

Acute toxicity of ammonium metavanadate solutions in normal saline (pH 6.7) or 0.1 M Tris-HCl-NaCl buffers (pH 7.2 or pH 7.8) was studied in BALB/c mice at 20 mg V/kg. Animals receiving these solutions subcutaneously started to show severe clinical signs 10-15 min postinjection and high mortality rates (45-73%) during the first 3 d. Animals dying because of vanadium toxicity did so only within the first 3 d after injection. NH4VO3-treated animals showed a tendency to increase their liver and spleen weights as compared to those receiving control solutions. Severe necrosis in lymphoid tissues (thymus, spleen, lymph nodes, and Peyer's patch), pulmonary hemorrhage, and renal acute tubular necrosis were commonly demonstrated in vanadium-treated animals. Toxicity of NH4VO3 solution in 0.1 M Tris-HCl-NaCl buffer (pH 7.8) was greatly reduced upon acidification with HCl to pH 6.1 or following boiling for 15 min (final pH of 7.7). Acidification of the solution reduced the mortality rate to 20 from 68%; however, the clinical signs were still severe. Boiling of the solution reduced the mortality rate to zero and moderated the severity of the clinical signs.