Expression and functional characterization in yeast of an endoglucanase from Bacillus sonorensis BD92 and its impact as feed additive in commercial broilers.

Some ingredients used in poultry feed formulation contain carbohydrate polymers which are difficult to digest and thus hinder nutritional feed value. Toward overcoming this limitation, exogenous enzymes have been added to poultry feed to improve its nutritive value. The present study was designed to provide first enzymatic characterization of endoglucanase (BsEgl) from the genome of B. sonorensis BD92 expressed in Pichia pastoris. Further, we tested its impact alone and in combination with a ß-glucosidase (Bteqßgluc) on growth in commercial broilers as feed additive. The expressed enzyme displayed features of GH5 family and had optimum activity against carboxymethyl cellulose at pH 5 and 50 °C. The BsEgl was stable at a range of pH from 4 to 8 for 60 min and at 50 °C for 180 min. Supplementing broilers diet with BsEgl alone or in combination with Bteqßgluc resulted in better feed conversion ratio among treatments during a five weeks testing period. Moreover, meat percentage was also highest for this treatment, and all treatments with recombinant enzymes increased intestinal length in birds compared to treatment control group. Blood parameters and serum biochemistry profile showed non-significant difference among groups. These results support that recombinant cellulolytic enzymes supplement high fiber diets improve their nutritional performance.

First-in-Human Trial of the Oral Ataxia Telangiectasia and RAD3-Related (ATR) Inhibitor BAY 1895344 in Patients with Advanced Solid Tumors.

Targeting the ataxia telangiectasia and RAD3-related (ATR) enzyme represents a promising anticancer strategy for tumors with DNA damage response (DDR) defects and replication stress, including inactivation of ataxia telangiectasia mutated (ATM) signaling. We report the dose-escalation portion of the phase I first-in-human trial of oral ATR inhibitor BAY 1895344 intermittently dosed 5 to 80 mg twice daily in 21 patients with advanced solid tumors. The MTD was 40 mg twice daily 3 days on/4 days off. Most common adverse events were manageable and reversible hematologic toxicities. Partial responses were achieved in 4 patients and stable disease in 8 patients. Median duration of response was 315.5 days. Responders had ATM protein loss and/or deleterious ATM mutations and received doses ≥40 mg twice daily. Overall, BAY 1895344 is well tolerated, with antitumor activity against cancers with certain DDR defects, including ATM loss. An expansion phase continues in patients with DDR deficiency. SIGNIFICANCE: Oral BAY 1895344 was tolerable, with antitumor activity in heavily pretreated patients with various advanced solid tumors, particularly those with ATM deleterious mutations and/or loss of ATM protein; pharmacodynamic results supported a mechanism of action of increased DNA damage. Further study is warranted in this patient population.See related commentary by Italiano, p. 14.This article is highlighted in the In This Issue feature, p. 1.

Identification and functional characterization of a ß-glucosidase from Bacillus tequelensis BD69 expressed in bacterial and yeast heterologous systems.

BACKGROUND: The identification and characterization of novel ß-glucosidase genes has attracted considerable attention because of their valuable use in a variety of industrial applications, ranging from biofuel production to improved digestibility of animal feed. We previously isolated a fiber-degrading strain of Bacillus tequelensis from buffalo dung samples, and the goal of the current work was to identify ß-glucosidase genes in this strain. We describe the cloning and expression of a new ß-glucosidase gene (Bteqßgluc) from Bacillus tequelensis strain BD69 in bacterial and yeast hosts. The recombinant Bteqßgluc were used to characterize specificity and activity parameters, and candidate active residues involved in hydrolysis of different substrates were identified through molecular docking. METHODS: The full length Bteqßgluc gene was cloned and expressed in Escherichia coli and Pichia pastoris cultures. Recombinant Bteqßgluc proteins were purified by immobilized metal affinity or anion exchange chromatography and used in ß-glucosidase activity assays measuring hydrolysis of ρ-nitrophenyl-ß-D-glucopyranoside (pNPG). Activity parameters were determined by testing relative ß-glucosidase activity after incubation under different temperature and pH conditions. Candidate active residues in Bteqßgluc were identified using molecular operating environment (MOE) software. RESULTS: The cloned Bteqßgluc gene belongs to glycoside hydrolase (GH) family 4 and encoded a 54.35 kDa protein. Specific activity of the recombinant ß-glucosidase was higher when expressed in P. pastoris (1,462.25 U/mg) than in E. coli (1,445.09 U/mg) hosts using same amount of enzyme. Optimum activity was detected at pH 5 and 50 °C. The activation energy (E a) was 44.18 and 45.29 kJ/mol for Bteqßgluc produced by P. pastoris and E. coli, respectively. Results from other kinetic parameter determinations, including pK a for the ionizable groups in the active site, Gibbs free energy of activation (ΔG ‡), entropy of activation (ΔS ‡), Michaelis constant (K m) and maximum reaction velocity (V max) for pNPG hydrolysis support unique kinetics and functional characteristics that may be of interest for industrial applications. Molecular docking analysis identified Glu, Asn, Phe, Tyr, Thr and Gln residues as important in protein-ligand catalytic interactions.

An update on carbohydrases: growth performance and intestinal health of poultry.

Poultry is an imperative domesticated livestock species that provides high quality protein and micronutrients as meat and eggs. In poultry production, feed is the single major input constituting 70-75% of total production cost. Feed mainly consists of cereal grains, those provide energy to the birds. However, these grains contain different levels of anti-nutritional factors such as non-starch polysaccharides (NSP). These NSP are indigestible by poultry birds due to the lack of vital endogenous enzymes (carbohydrases) thus increase intestinal viscosity which slower the migration and absorption of nutrients. Consequently, these NSP may also increase the chances for infection by inducing competition within gut microbiota for digestible nutrients. This affects bird's health and increases the production cost. Therefore, there is a need to find efficient and effective solutions for these problems. Carbohydrases supplementation have an important role in poultry diets with high NSP contents. Feed enzymes are being used from years to enhance growth performance and digestibility but have limited activity for selective ingredients. New generation carbohydrases with a board range of activity and stability help to degrade the complex substrates and improve growth performance of poultry. Present review summarizes the updated literature on the use of carbohydrases to improve bird's performance and intestinal health.

Statistical based experimental optimization for co-production of endo-glucanase and xylanase from Bacillus sonorensis BD92 with their application in biomass saccharification.

Endo-glucanase (cellulase) and xylanase have high industrial demand due to their vast application in industrial processes. This study reports statistical based experimental optimization for co-production of endo-glucanase and xylanase from Bacillus sonorensis BD92. Response surface methodology (RSM) involving central composite design (CCD) with full factorial experiments (23) was applied to elucidate the components that significantly affect co-production of endo-glucanase and xylanase. The optimum co-production conditions for endo-glucanase and xylanase were as follows: carboxymethyl cellulose (CMC) 20 g/L, yeast extract 15 g/L, and time 72 h. The maximum endo-glucanase and xylanase production obtained was 1.46 and 5.69 U/mL, respectively, while the minimum endo-glucanase and xylanase production obtained was 0.66 and 0.25 U/mL, respectively. This statistical model was efficient because only 20 experimental runs were necessary to assess the highest production conditions, and the model accuracy was very satisfactory as coefficient of determination (R2) was 0.95 and 0.89 for endo-glucanase and xylanase, respectively. Further, potential application of these enzymes for saccharification of lignocellulosic biomass (wheat bran, wheat straw, rice straw, and cotton stalk) was also investigated. The results revealed that the biomass was susceptible to enzymatic saccharification and the amount of reducing sugars (glucose and xylose) increased with increase in incubation time. In conclusion, Bacillus sonorensis BD92 reveals a promise as a source of potential endo-glucanase and xylanase producer that could be useful for degrading plant biomass into value-added products of economic importance using precise statistically optimized conditions.

Comparison of vitamin D levels with bone density, calcium, phosphate and alkaline phosphatase - an insight from major cities of Pakistan.

OBJECTIVE: To compare vitamin D levels with bone mineral density, serum calcium, phosphorous and alkaline phosphatase. METHODS: The cross-sectional multicentre study was conducted at Pakistan Health Research Council centres in Islamabad, Lahore, and Karachi,and comprised subjects coming for either vitamin D testing or bone mineral density examination. Information related to demography, height/weight, skin colour, smoking, use of sun screen, daily milk intake, sun exposure and exercise was taken along with biochemical tests like serum calcium and phosphorous, alkaline phosphatase, and 25 hydroxy vitamin D.Bone mineral density was done using dual-energy X-ray absorptiometry scan. SPSS 15.0 was used for data analysis. RESULTS: Of the 291 subjects, 100(34.3%) each were enrolled from Islamabad and Lahore, while 91 (31.2%) were from Karachi. Overall, 245(84%) had insufficient vitamin D which was significantly associated with age and dark skin colour (p<0.05 each). Besides, 137(48%) cases had a reduced bone density, and there was a significant difference between age groups, gender and skin colour (p<0.05 each).Vitamin D deficiency did not have any effect on bone mineral density (p>0.05) Serum calcium and phosphorus levels were also similar regardless of vitamin D level (p>0.05). CONCLUSIONS: Vitamin D deficiency did not have any direct impact on bone mineral density and serum calcium, phosphorous and alkaline phosphatase.

Salmonella Infection Amongst Food Workers In Lahore.

This descriptive observational study was conducted at Pathology lab Jinnah hospital, Lahore, January 2013 to October 2015. We include all the food workers in different restaurants, slaughters, inn, recreation centre and bakeries of Lahore for the purpose to identify the salmonella infection, we used Typhoid IgGI/IgM Rapid test device methods (Serum/Plasma), is a lateral flow chromatographic immunoassay technique were calculated. We analysed 1025 individuals, 62 (6%) were found positive for the said diseases out of which 14 (1.3%) IgG positive, 18 (1.8%) IgG IgM positive and 30 (2.9%) were IgM positive. All the individuals who were found positive were without signs and symptoms. Salmonellosis continues to be an important global cause of infectious intestinal disease and spread readily by means of food personnel, zoonotic and contaminated equipments and surface. The screening of the individuals who are silent carriers, help in controlling the spread of salmonella infection in the public.

Dermatological evaluation of counter irritant effect of methanol leaf extract of Rumexvesicarius Linn. in rabbits.

OBJECTIVE: To assess the counter-irritant and anti-inflammatory activity of Rumexvesicarius in dermatological use. METHODS: The animal-based experimental study was conducted at the Royal Institute of Medical Sciences, Multan, Pakistan, in November 2014. Sodium lauryl sulfate, phenol, histamine and sandpaper irritation models were used. Irritation was induced by the clockwise frictional movement of fine sandpaper to the ear of rabbits and then applying sodium lauryl sulfate, histamine and phenol single topical application onto the ear of the rabbit. The counter-irritant effect was determined by calculating the mean decrease in redness and erythema with those of control and standard dexamethasone. RESULTS: There were 20 rabbits in the study with a mean weight of 1.50±0.033 kg. R. vesicarius (100 and 150mg/mL) showed excellent counter-irritant effect when compared with control and standard groups. Both the doses depicted counter-irritant effect with the highest inhibition (94.42%) in sandpaper group, followed by sodium lauryl sulphate (90%), phenol (94.23%) and histamine (88.46%) irritation models respectively. CONCLUSIONS: Methanol leaf extract of R. vesicarius countered the effect of irritation in experimental animals. It showed significant effect in terms of dose and counter-irritancy time.

In vitro and in vivo toxicological evaluations of methyl ferulate, methyl p-coumarate, and pulegone 1,2-epoxide.

CONTEXT: Toxicological screening of natural compounds for medicinal purposes. OBJECTIVES: The objective of this study is to evaluate the toxicity of methyl ferulate (MF), methyl p-coumarate (MpC), and pulegone 1,2-epoxide (PE) with in vitro and in vivo assays. MATERIALS AND METHODS: The in vitro toxicity of MF, MpC, and PE was assessed at a concentration of 10 mg/ml with the Ames assay using two strains of Salmonella typhimurium TA98 and TA100. Human red blood cells (RBC) were used to determine the hemolytic activity of these compounds. The cytotoxicity of above compounds was determined with brine shrimp lethality bioassay (BSLB) at the concentrations of 0.1-20 mg/ml. While dermal and ocular irritation studies were conducted on healthy rabbits (n = 8) for 96 and 12 h post-topical application of test compounds, respectively. RESULTS: PE produced 6-8% hemolysis of RBCs at all the tested concentrations while MF and MpC produced 10-5% hemolysis up to 20 mg/ml, and 50-85% hemolysis at concentrations of 40 and 80 mg/ml, respectively. The Ames assay indicated that MF, MpC, and PE were non-mutagenic as the test values were not significantly higher as compared with background values of the assay. BSLB suggested the lethal concentration (LC50) values of MF, MpC, and PE as 4.38, 6.74, and 25.91 mg/ml, respectively. In vivo ocular and dermal irritation scores of MF, MpC, and PE were comparable with ethanol (control) in rabbits indicating the non-irritant nature of these natural compounds. CONCLUSION: The present studies suggest that these compounds are non-toxic/non-irritant and might be used for medicinal purposes.

Dengue knowledge and its management practices among physicians of major cities of Pakistan.

OBJECTIVE: To assess knowledge and practices related to dengue management among physicians. METHODS: The cross-sectional study was conducted at hospitals in Islamabad, Lahore, Faisalabad, Peshawar, Quetta and Karachi between June and December 2012Physicians from public and private sectors filled a self-administered questionnaire about dengue knowledge and its management practices. A maximum score of 100 was assigned to the knowledge portion. Data was analysed using SPSS 15. RESULTS: A total of 400 subjects participated in the study; 200(50%) each from public and private hospitals. Of them, 223(56%) were males; 268(67%) were in the 21-30 years age bracket. The highest score was recorded in Quetta 67 followed by 65 in Karachi, 62 in Lahore, Faisalabad, Peshawar and 59 in Islamabad. Of the total, 200 (50%) were not aware that leucopenia is a criterion for diagnosing probable dengue. Similarly 140 (35%) did not know the criteria for diagnosing dengue haemorrhagic fever and warning signs of severe dengue. Total of 204 (51%) were not aware of the criteria for discharging of the admitted cases. There was no significant difference between dengue knowledge of the physicians belonging to public and private sectors (p>0.05). CONCLUSIONS: Quite a large number of physicians lacked knowledge of probable diagnosis of dengue and appropriate time to discharge the patients.

Economic burden of dengue in four major cities of Pakistan during 2011.

OBJECTIVE: To assess the economic burden of dengue infection by calculating cost per patient and disability adjusted life years lost. METHODS: The cross-sectional study was conducted in Islamabad, Lahore, Faisalabad and Karachi from July 2012 to March 2013. Residential addresses and telephonic numbers of dengue patients were taken from the records of Pakistan Institute of Medical Sciences, Islamabad, Mayo and Ganga Ram Hospital, Lahore, Civil Hospital, Karachi, and Allied Hospital, Faisalabad. A total of 250 dengue confirmed cases - 50 from each hospital - were randomly selected. Information regarding duration of illness and out-of-pocket expenses were collected to estimate the direct cost, while indirect cost (number of work days missed by the patient) was calculated from disability adjusted life years using Murray's formula. RESULTS: Overall, there were 162(65%) men and 88(35%) with a mean age of 30.4±13.5years. More than half 138(55%) were below 30 years of age. Socio-economically, 145(58%) belonged to low, 70(28%) middle and 35(14%) to high socioeconomic groups. Of the total, 210(84%) cases had dengue fever followed by 32(12.8%) dengue haemorrhagic fever and 8(3.2%) dengue shock syndrome cases. Average duration of illness was 32±7.1 days. Overall direct cost per patient was Rs.35,823 (US$358) and average pre-hospitalisation, hospitalisation and post-hospitalisation was Rs.6154, Rs.21,242 and Rs.8,427 respectively. The overall disability adjusted life years per million population was 133.76. CONCLUSIONS: Although the government had provided free treatment for dengue in public-sector hospitals, still patients had to pay Rs.21,242 during hospital stay, resulting in substantial burden which needs to be addressed.

Role of C-reactive protein as a marker for microalbuminuria in type 2 diabetics.

BACKGROUND: One of the most important complications of Type 2 diabetes mellitus is diabetic nephropathy which is reported as the leading cause of premature deaths due to renal failure. This study was being conducted to see the role of C-reactive protein a marker in type 2 diabetic patient with micro-albuminuria. METHODS: This cross sectional study was carried out in the Diabetic Clinic of Pakistan Medical Research Council, Research Centre Lahore. Personal history about the disease was collected on a questionnaire. Blood and urine sample of 50 type 2 diabetic patients between the ages of 40-65 years were collected to analyze C-reactive protein and micro-albuminuria respectively. Data were analyzed using SPSS-15. The Pearson correlation coefficient test was used to determine correlation between urine albumin and C-reactive protein. RESULTS: There were 22 micro- albuminuria positive cases out of total 50 diabetic cases. Of these 14 (63%) showed raised C-reactive protein levels (p < 0.00). CONCLUSION: There is significant association between the level of serum C- reactive proteins and micro-albuminuria in type 2 diabetics.

A retrospective analysis of dengue fever case management and frequency of co-morbidities associated with deaths.

BACKGROUND: Dengue epidemic in Lahore (2011) resulted in hundreds of deaths and affected thousands. As most of the studies were focused on its diagnosis and treatment, scanty data is available on associated diseases/co-morbidities in these patients that could have contributed to a higher mortality. There were no local guidelines available on recording, reporting and management of these co-morbidities. The objective of this study was to analyze the initial presentations of dengue cases and to estimate the frequency of co-morbidities in dengue patients. METHODS: Data of 556 dengue cases was retrieved from 2 major public sector tertiary-care hospitals for patients who were admitted during 2011 epidemic and a case record analysis was done. Data was retrieved from patient's information reports which included demography, signs and symptoms and the laboratory investigations. In addition verbal autopsy of deceased cases was also done from their relatives using standardized WHO verbal autopsy form after making modifications as per needed. RESULTS: Of 556 cases studied, 390 (70%) were males. The mean age was 36 years and 30% of the cases were between 20-29 years. Average duration of the hospital stay was 6 days. Out of the total, 435 (78%) were dengue fever (DF) cases followed by dengue hemorrhagic fever (DHF) in 95 (17%) and dengue shock syndrome (DSS) in 26 (4%) cases. A total of 40 cases died and among them 17 were diagnosed with DSS, 13 DF and 10 DHF. Further the verbal autopsy from relatives of deceased cases showed 29 (60%) deceased had co-morbid diseases which included hypertension, diabetes etc. DSS was common in patients who had hypertension (27) either alone or associated with other illnesses. CONCLUSIONS: Co-morbidities with dengue infection were seen in 60% deceased cases indicating the reasons for high dengue related complications and death.

Virulence profile of different phylogenetic groups of locally isolated community acquired uropathogenic E. coli from Faisalabad region of Pakistan.

BACKGROUND: Uropathogenic E.coli (UPEC) are among major pathogens causing urinary tract infections. Virulence factors are mainly responsible for the severity of these emerging infections. This study was planned to investigate the distribution of virulence genes and cytotoxic effects of UPEC isolates with reference to phylogenetic groups (B2, B1, D and A) to understand the presence and impact of virulence factors in the severity of infection in Faisalabad region of Pakistan. METHODS: In this study phylogenetic analysis, virulence gene identification and cytotoxicity of 59 uropathogenic E.coli isolates obtained from non-hospitalized patients was studied. RESULTS: Among 59 isolates, phylogenetic group B2 (50%) was most dominant followed by groups A, B1 (19% each) and D (12%). Isolates present in group D showed highest presence of virulence genes. The prevalence hlyA (37%) was highest followed by sfaDE (27%), papC (24%), cnf1 (20%), eaeA (19%) and afaBC3 (14%). Highly hemolytic and highly verotoxic isolates mainly belonged to group D and B2. We also found two isolates with simultaneous presence of three fimbrial adhesin genes present on pap, afa, and sfa operons. This has not been reported before and underlines the dynamic nature of these UPEC isolates. CONCLUSIONS: It was concluded that in local UPEC isolates from non-hospitalized patients, group B2 was more prevalent. However, group D isolates were most versatile as all were equipped with virulence genes and showed highest level of cytotoxicity.

Molecular profiling of antimicrobial resistance and integron association of multidrug-resistant clinical isolates of Shigella species from Faisalabad, Pakistan.

Bacillary dysentery, common in developing countries, is usually caused by Shigella species. A major problem in shigellosis is the rapid emergence of multidrug-resistant strains. This is the first detailed molecular study on drug resistance of Shigella isolates from the Faisalabad region of Pakistan. Ninety-five Shigella isolates obtained after screening of 2500 stool samples were evaluated for in vitro resistance to commonly used antimicrobial agents; the presence or absence of 20 of the most relevant drug resistance genes; and the prevalence of integrons 1, 2, and 3. Shigella flexneri was found to be the most prevalent and most resistant species. Collectively, high resistance was found towards ampicillin (96.84%), tetracycline (93.68%), streptomycin (77.89%), and chloramphenicol (72.63%). Significant emerging resistance was detected towards the modern frontline drugs ciprofloxacin (12.63%), cefradine (17.89%), ceftriaxone (20.00%), cefoperazone (22.10%), and cefixime (28.42%). Prevalence rates for bla(TEM), bla(CTX-M), gyrA, gyrB, qnrS, aadA1, strAB, tetA, tetB, catA, and catP were 78.94%, 12.63%, 20.00%, 21.05%, 21.05%, 67.36%, 42.10%, 12.63%, 53.68%, 33.68%, and 25.26%, respectively. Class 2 integrons (42.10%) were more common in the local isolates. Simultaneous detection of class 1 and 2 integrons in some isolates and a rapidly emerging resistance to modern frontline drugs are the major findings of this study.

Diagnostic utility of p63 (Ab-1) and (Ab-4) tumor markers in the squamous cell carcinomas of head and neck.

P63 is a gene product required in cell cycle regulation which plays vital roles in tumor differentiation. Aims of the present study were to assess the frequency, pattern, sensitivity and specificity of two p63 protein clones P63 4A4 and P63 4A4+Y4A3 in squamous cell carcinomas (SCCs). Thirty cases of head and neck region SCC diagnosed on the basis of HandE staining were examined along with 60 cases of head and neck region biopsies other than squamous cell carcinoma, negative on HandE staining, were taken as control. Immunostaining was performed on slides according to the Thermo Scientific UltraVision LP detection System. P63 4A4+Y4A3 clone is more sensitive 96.6% in comparison to 86% in P63 4A4 with having greater NPV of 98.3%. The results signify the importance of P63 4A4+Y4A3 marker over the old markers and may be used as a confirmatory marker of squamous cell carcinoma.

Multiple drug resistance patterns in various phylogenetic groups of uropathogenic E. coli isolated from Faisalabad region of Pakistan

The objective of this work was the phylogenetic characterization of local clinical isolates of uropathogenic E. coli with respect to drug resistance. A total of 59 uropathogenic E. coli responsible for community acquired urinary tract infections were included in this study. A triplex PCR was employed to segregate each isolate into four different phylogenetic groups (A, B1, B2 and D). Drug resistance was evaluated by disc diffusion method. The drugs used were ampicillin, aztreonam, cefixime, cefoperazone, ceftriaxone, cephradine among â-lactam group; amikacin, gentamicin, and streptomycin among aminoglycosides; nalidixic acid and ciprofloxacin from quinolones; trimethoprim-sulfomethoxazole, and tetracycline. Among 59 uropathogenic E. coli isolates majority belonged to phylogenetic group B2 (50 percent) where as 19 percent each belonged to groups A and B1, and 12 percent to group D. All the isolates were multiple drug resistant (MDR). Most effective drugs against Group A, B1, and B2 were gentamicin, amikacin and cefixime; ceftriaxone and quinolones; and ceftriaxone and amikacin, respectively. Group D isolates were found to be highly resistant to all drugs. Our results have shown emergence of MDR isolates among uropathogenic E. coli with dominance of phylogenetic group B2. However, it was found that group D isolates were though less frequent, more drug resistant as compared with group B2. Groups A and B1 were relatively uncommon. Amikacin, ceftriaxone and gentamicin were the most effective drugs in general.

Efficacy of fix dose combination (atorvastatin and amlodipine) in treatment of uncontrolled hypertension and dyslipidemia.

BACKGROUND: The fixed-dose combination containing the antihypertensive agent amlodipine and the statin, atorvastatin, is the first combination of its kind designed to treat two risk factors for cardiovascular disease (CVD), i.e., hypertension and dyslipidemia. In this study, blood pressure and lipid lowering effects of combination of amlodipine and atorvastatin were evaluated in uncontrolled hypertensive patients. METHODS: Thirty patients both male and female in the age group 35-60 years attending the Hypertensive Clinic of PMRC FJMC suffering from uncontrolled hypertension were selected. Baseline blood pressure was checked after half hour rest in sitting and standing position using mercury sphygmomanometer. Blood sample was collected from all patients after overnight fasting for assessment of serum cholesterol, triglycerides, LDL and HDL cholesterol levels. They were prescribed with fixed dose combination of 5 mg amlodipine and 10 mg atorvastatin. Patients were followed for their blood pressure measurement after every 4 weeks up to 12 weeks. At the end of 12 weeks their fasting blood sample was taken again for determination of serum cholesterol, triglyceride, LDL and HDL cholesterol levels. RESULTS: Systolic blood pressure after 4, 8 and 12 weeks was significantly lower at all intervals from baseline. When systolic blood pressure after 8 and 12 weeks was compared with 4 weeks, the effect was again significant (p=0.024, p=0.002 respectively).There was no significant reduction seen in 8 versus 12 weeks (p=0.493). Diastolic blood pressure at 4, 8 and 12 weeks was significantly lower from baseline. Diastolic blood pressure after 4 and 8 weeks when compared with 8 and 12 weeks was not significantly low (p=0.99 and 0.91 respectively). Lipid profile of the patients was significantly reduced from baseline after twelve weeks of fixed dose combination of treatment (p<0.000). CONCLUSION: Combination therapy proved to be effective in controlling hypertension and dyslipidemia than single pill. It also improved patient's compliance. It is suggested that polypill should be prescribed instead of multiple drugs.

Multiple drug resistance patterns in various phylogenetic groups of uropathogenic E.coli isolated from Faisalabad region of Pakistan.

The objective of this work was the phylogenetic characterization of local clinical isolates of uropathogenic E. coli with respect to drug resistance. A total of 59 uropathogenic E. coli responsible for community acquired urinary tract infections were included in this study. A triplex PCR was employed to segregate each isolate into four different phylogenetic groups (A, B1, B2 and D). Drug resistance was evaluated by disc diffusion method. The drugs used were ampicillin, aztreonam, cefixime, cefoperazone, ceftriaxone, cephradine among ß-lactam group; amikacin, gentamicin, and streptomycin among aminoglycosides; nalidixic acid and ciprofloxacin from quinolones; trimethoprim-sulfomethoxazole, and tetracycline. Among 59 uropathogenic E. coli isolates majority belonged to phylogenetic group B2 (50%) where as 19% each belonged to groups A and B1, and 12% to group D. All the isolates were multiple drug resistant (MDR). Most effective drugs against Group A, B1, and B2 were gentamicin, amikacin and cefixime; ceftriaxone and quinolones; and ceftriaxone and amikacin, respectively. Group D isolates were found to be highly resistant to all drugs. Our results have shown emergence of MDR isolates among uropathogenic E. coli with dominance of phylogenetic group B2. However, it was found that group D isolates were though less frequent, more drug resistant as compared with group B2. Groups A and B1 were relatively uncommon. Amikacin, ceftriaxone and gentamicin were the most effective drugs in general.

Effects of ampicillin, gentamicin, and cefotaxime on the release of Shiga toxins from Shiga toxin-producing Escherichia coli isolated during a diarrhea episode in Faisalabad, Pakistan.

The Shiga toxin-producing Escherichia coli (STEC) is an emerging foodborne pathogen. The proportion of cases attributed to STEC in an episode of diarrhea in the Faisalabad region of Pakistan was investigated. In addition, as increase in Shiga toxin (Stx) release after exposure to various antimicrobial agents is widely reported, we also elucidated the in vitro effects of three commonly used antibiotics (ampicillin, gentamicin, and cefotaxime) on Stx release. Isolation and detection of STEC was done using enzyme-linked immunosorbent assay and polymerase chain reaction, followed by phenotypic characterization. In vitro Stx release from isolated STEC was determined using enzyme-linked immunosorbent assay, and Stx-induced verocytotoxicity was quantified using cytotoxicity detection assay. STEC was detected in 5 (21.7%) of 23 patients. Exposure to minimum inhibitory concentration (MIC) of ampicillin, gentamicin, and cefotaxime resulted in a considerable decrease in toxin release and level of cytotoxicity in most of the STEC isolates when compared with control (without antibiotic exposure). Exposure to sub-MIC of ampicillin resulted in a relative increase in Stx release and cytotoxicity (p