Genomic Insights into and In Vitro Evaluation of Antimicrobial Combination Therapies for Carbapenem-Resistant Acinetobacter baumannii.

Background and Objectives: Acinetobacter baumannii (A. baumannii), particularly carbapenem-resistant A. baumannii (CRAB), represents a grave concern in healthcare settings and is associated with high mortality. This study aimed to conduct molecular, mutational, and phylogenetic analyses of specific genes in CRAB and evaluate the synergistic effects of selected antimicrobial combinations. Materials and Methods: Phenotypic characterization was performed on six CRAB strains by using the Modified Hodge Test (MHT) and IMP-EDTA Double-Disc Synergy Test (IMP-EDTA DDST). Carbapenemase- and metallo-beta-lactamase-encoding genes were amplified by using Polymerase Chain Reaction. Phylogenetic analysis using the MEGA 11 tool was used to determine the evolutionary relatedness of these genes. Mutational analysis was performed by using I-Mutant, MUPro, and PHD-SNP bioinformatics tools to predict mutations in the carbapenemase-encoding genes. Microdilution checkerboard titration assessed the synergistic effects of antimicrobial combinations (azithromycin-meropenem, rifampicin-meropenem, meropenem-colistin, and azithromycin-colistin) on these CRAB isolates. Results: The phenotypic characterization of six CRAB isolates revealed positive results for MHT and IMP-EDTA DDST. The molecular characterization revealed that carbapenemase- and MBL-encoding genes were present in all isolates with varying frequencies, including blaOXA-51 (100%) and blaIMP (0%). The sequence analysis revealed high evolutionary relatedness to sequences in the NCBI database. The mutational analysis identified 16 mutations, of which 1 mutation (P116L) in the blaOXA-58 gene predicted a change in the protein product, potentially contributing to carbapenem resistance. The checkerboard titration method did not reveal any synergism among the tested antimicrobial combinations against CRAB. Conclusion: This study's findings underscore the significant challenges posed by CRAB isolates harboring multiple resistant genes in treatment. This highlights the urgent need for novel antimicrobial agents, a crucial step towards reducing mortality rates not only in Pakistan but also globally.

Limosilactobacillus fermentum IKP 111 reduces pathogen load and improves immunity of broilers when challenged with Salmonella enteritidis.

In this study, we checked the effectiveness of L. fermentum IKP 111 in treating S. enteritidis infection in an in vivo study. Its oral administration to broiler chicks significantly reduced the colonization of S. enteritidis in the gut and there was a lower bacterial count of S. enteritidis in the droppings after infection. The administration of the probiotic L. fermentum IKP 111 also led to increase in weight gain in the broiler chicks as well as their immunomodulation against avian influenza virus (AIV) and Newcastle disease virus (NDV) as compared to the chicks challenged only with S. enteritidis. Our study provides evidence that the probiotic strain L. fermentum IKP 111 could be an alternate for controlling S. enteritidis infection while enhancing the gut health as well as the immune response of broiler chickens against viral infections.

Evaluation of humoral immunological response of newly prepared bovine multidrug-resistant Staphylococcus aureus mastitis vaccines in rabbits.

Multi-drugs resistant (MDR) Staphylococcus aureus is creating challenges to cure cow mastitis, resulting in massive economic loss globally. It necessitates the adoption of prevention and control systems such as vaccination. Plain (PMRSAV), Montanide oil adjuvanted (MMRSAV) and Aluminum hydroxide adjuvanted (AMRSAV) vaccines were prepared using a molecularly characterized isolate of MDR S. aureus from bovine origin. Immunogenicity of the selected isolate was evaluated in five groups of rabbits (A-E) at different concentrations by measuring GMT via IHA from serum samples after booster shot. The group E provoked significantly higher (P<0.05) antibody titer with peak at day 28 (64±0.5) and cumulative mean antibody titer (CMT) of rabbits was highest (45.6) followed by groups C (35.9), D (32.7), B (30.3) and A (24.5). The concentration yielding maximum antibody titer was used for vaccines preparation. Vaccines were evaluated in different rabbits groups by inoculating PMRSAV, MMRSAV, AMRSAV and Placebo. Serum samples evaluated through IHA revealed that rabbits injected with MMRSAV produced highest antibody titer reaching its peak at day 45 (90.51±0.23) with a slight decrease until day 60 (80.63±0.17) followed by AMRSAV and PMRSAV. Challenge protection assay revealed the survival rates of rabbits in groups PMRSAV, MMRSAV, AMRSAV and Placebo as 83.3%, 100%, 100% and 16.7%, respectively. The study concluded that MMSAV and AMSAV were safe, efficacious and immunogenic in experimental rabbits.

Activity of plant essential oils against antibiotic resistant Enterococcus faecalis isolated from diarrheic children.

Activity of plant essential oils and their fractions was evaluated against characterized isolates of antibiotic resistant Enterococcus faecalis recovered from diarrheic children. The isolates were confirmed by polymerase chain reaction (PCR) targeting 16S rRNA gene amplification followed by nucleotide sequencing and accession numbers retrieved were MW349990.1, MW349859.1, MW332122.1, MW356805.1, MW349975.1, MW349988.1, MW356790.1, MW356244.1, MW341593.1 and MW332549.1. These isolates were screened for antibiotic susceptibility to a wide range of antibiotic groups and mean zone of inhibition (ZOI) of all antibiotics were recorded. Antibacterial activity of plant essential oils (n=05) was checked against three antibiotic resistant isolates of E. faecalis. Three plant essential oils having higher ZOI including Cinnamomum verum, Syzygium aromaticum and Nigella sativa were used against resistant E. faecalis isolates to determine minimum inhibitory concentration (MIC). The lowest MIC observed was of S. aromaticum (11.39±3.94 mg mL-1). The S. aromaticum n-hexane plus chloroform fraction displayed higher mean ZOI (16.67±2.51 mm), while the lowest MIC was of n-hexane oil fraction. Based upon gas chromatography-mass spectrometry (GC/MS) analysis, the most effective fatty acid was eugenic acid which is present in higher proportion in both fractions. These fractions of essential oils proved safe for the treatment of antibiotic resistant diarrheic cases of children caused by E. faecalis.

Molecular markers for the detection of pathogenic and food poisoning potential of methicillin resistant Staphylococcus aureus isolated from wounds of hospitalized patients.

Pathogenic strains of Staphylococcus aureus are mostly resistant to methicillin and they can cause severe infections. The current study was planned to assess the food poisoning potential of pathogenic, methicillin resistant Staphylococcus aureus by molecular detection of enterotoxin A (Eta) gene. A total of 100 septic wound samples from patients admitted in surgical ward (n=50) and burn unit (n=50) of Mayo Hospital Lahore were collected aseptically. These samples were processed primarily for bacterial growth on nutrient agar and purified on mannitol salt agar where twenty (20) samples showed pin-point colonies with yellow discoloration of media. Moreover, isolates were further characterized on the basis of microscopic appearance and biochemical assays where fourteen (14) isolates were declared Staphylococcus. DNA of these isolates were subjected to 16S rRNA gene amplification and sequences of S. aureus were submitted to NCBI GenBank viz., MW344063.1, MW341438.1, MW344064.1, MW344065.1, MW341439.1, MW341440.1, MW345971.1, MW345972.1, MW345973.1, MW716458.1. All the isolates (n=10) demonstrated molecular confirmation of pathogenicity and methicillin resistance by amplification of Coa and mecA gene. Out of these ten isolates, three amplified enterotoxin A (Eta) gene were confirmed. It is concluded that enterotoxin A of S. aureus which causes food poisoning is present in pathogenic, methicillin resistant S. aureus isolated from various wounds infections.

Phytochemical composition and In-vitro activity of ethanolic extract of Eucalyptus globulus leaves against multidrug resistant poultry pathogens.

Aim of the present study was to determine the In-vitro antibacterial activity of ethanolic extract of E. globulus leaves against common multidrug resistant poultry pathogens. Phytochemical analysis through HPLC revealed that kaempeferol (7.315min) followed by querecetin (6.655min) and myrecetin (3.655min). Percent area of kaempeferol (6826.88%) was highest, followed by myrecetin (5516.22%) and querecetin (163.748%). Phytochemical investigation of ethanolic extract of E. globulus leaves through GCMS showed highest retention time (min) α-pinene (20.43) and α-terpineol (20.15) accompanied by spathulenol (11.97), piperitone (11.04). The ethanolic extracts of E. globulus leaves showed a highest zone of inhibition against S. pullorum SP6; 20.64± 2.08, E. coli SE 12; 19.75± 2.83, C. perfringens type A (CPM38-01); 19.46± 2.02. The highest level of MIC of E. globulus noted were against S. gallinarum S22; 133.37±53.294, S. gallinarum S1; 130.20±45.10, S. gallinarum S4; 129.47±24.182, S. gallinarum S3; 126.83±72.392. In conclusion, the study confirmed that the ethanolic extract of E. globulus is composed of active ingredients having antibacterial activity and can be referred as an alternate to antibiotics.

Activity of ethanolic extract of Eucalyptus globulus leaves against multi drug resistant poultry pathogens in broiler chicks.

The present study was designed to evaluate the antimicrobial activity of E. globulus leaves in broiler chicks. Total (n=255) day-old chicks were segregated into five groups i.e. Pathogenic E. coli, S. pullorum, S. gallinarum and C. perfringens type A and control negative group. Each bacterial challenged (1x 107 CFU) group was divided into control positive, antibiotic, probiotic and E. globulus group. Experimental birds were exposed to E. coli, S. pullorum, S. gallinarum and C. perfringens type A at different ages. At 35th day of experiment the log reduction for each group was determined. The highest log reduction in E. coli and C. perfringens Type A colonies count were found in E. globulus (3.26) (2.33) treated group followed by antibiotic (2.85) (1.59) and probiotic (2.84) (1.50) respectively. The log reduction in S. pullorum colonies count was highest in E. globulus (2.50) followed by probiotic (2.24) and antibiotic (2.16). The S. gallinarum colonies count log reduction was found highest for antibiotic (2.84) followed by probiotic (2.48) and E. globulus group. The results of in-vivo experiment revealed that ethanolic extract of E. globulus has antibacterial activity and it can be used as a replacement to low level of antibiotics added in poultry feed.

Distribution and antibiotic sensitivity pattern of Mycobacterium tuberculosis isolates from children, enrolled in a tertiary care hospital.

The present study was planned to assess the distribution of tuberculosis in children and evaluate the antimycobacterial sensitivity pattern of Mycobacterium tuberculosis (MTB) isolates from pediatric patients. A total number of 1718 pediatric patients suspected of Mycobacterium tuberculosis were enrolled in the Institute of Child Health and Children's Hospital, Lahore during 2016-17. Out of 1718, only 710 different types of samples were tested for MTB. The samples were processed using bacteriology and GeneXpert along with the chest X-ray and clinical picture of the patients. The sensitivity pattern of Streptomycin, Isoniazid, Rifampicin and Ethambutol (SIRE) was determined using BACTEC MGIT 960. Total patients were divided into four groups including group A (birth to 12 months), B (1 to 5 years), C (6 to 10 years), and D (11 to 15 years). Out of 710, 106 (55 females and 51 males) were declared positive and 604 negative for tuberculosis. Out of 106 positive cases, 89 (83.96%) were sensitive to Rifampicin and 17 (16.04%) were resistant. Only, 04 (3.77%) were resistant to both Rifampicin and Isoniazid and declared as multidrug-resistant (MDR). It was concluded that children of age 11 to 15 years were more prone to MTB and a minimum percentage of MDR isolates was recorded in age group A (birth to 12 months).

Appraisal of anti-mycobacterial potential against MDR-MTB in pediatric patients, cytotoxicity and mutagenicity of Aloe vera and Allium sativum.

The purpose of the studies was to evaluate an in-vitro anti-mycobacterial activity of Aloe vera and Allium sativum against MDR-MTB, their cytotoxicity and mutagenicity. Four extracts of Aloe vera and Allium sativum were prepared by Soxhlet apparatus and their minimum inhibitory concentrations (MIC's) were determined by BACTEC MGIT960 system against multi drug resistant Mycobacterium tuberculosis (MDR-MTB) isolates, collected from pediatric patients. Fractions of Aloe vera and Allium sativum extracts were separated using glass column chromatography, followed by evaluation of cytotoxicity and mutagenicity by tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and Ames test, respectively. Out of four extracts, ethanol extracts of Aloe vera and Allium sativum exhibited activity at MIC 5mg/mL to 7mg/mL and 3mg/mL to 5mg/mL, respectively and IC50 by MTT assay for combination of all fractions were 278.3mcg/100µL and 270.8mcg/100µL and in Ames assay M.I of TA98 were 0.14 and 0.07 and M.I of TA100 were 1.14 and 0.44, respectively. Aloe vera and Allium sativum extracts showed anti-mycobacterial activity against MDR-MTB isolates so, MIC of ethanol extracts of each plant and fractions of column chromatography had been checked. The MTT and Ames tests depicted that ethanol extracts of Aloe vera and Allium sativum were non-cytotoxic and non-mutagenic, and can be used in treatment of patients suffering from MDR-MTB.

In silico analysis of four structural proteins of aphthovirus serotypes revealed significant B and T cell epitopes.

Foot and Mouth disease (FMD) is economically devastating, highly contagious transboundry viral disease of livestock with 100% morbidity, rapid spread and severe production losses in animals. The FMDV has seven different serotypes. There is no vaccine that can protect animals from all serotypes. Hence, it is need of the day to develop a vaccine that protects animals from hetrologous challenge. In this study, we used immunoinformatics approach to find T and B-cell epitopes that will help to construct a universal vaccine for FMDV. For this purpose, first we constructed a consensus sequence for four structural proteins (VP1, VP2, VP3 and VP4) of aphthovirus for seven serotypes (A, O, C, Asia1, SAT1, SAT2 and SAT3). Various computational tools were used to perform multiple sequence alignment to identify the conserved regions, generation of consensus sequence through conserved regions, structures prediction and finally prediction of B and T cell epitopes. We predicted 5 B cell and 18 T cell epitopes. Finally a GPGPG spacer was used to join these epitopes to decrease binding affinity around the core binding regions. Hence, our study identified the epitopes which can be used to develop cross protective vaccines against all the fatal strains of Aphthovirus which can easily protect all the serotypes. Though, successful In vivo and In vitro studies are required to determine the genuine strength of our predicted epitopes against the fatal strains of Aphthovirus.

White Sesame Seed Oil Mitigates Blood Glucose Level, Reduces Oxidative Stress, and Improves Biomarkers of Hepatic and Renal Function in Participants with Type 2 Diabetes Mellitus.

OBJECTIVES: The study was designed to investigate the impact of white sesame seed oil (WSSO) consumption on fasting blood glucose (GLU), insulin (INS), glycosylated hemoglobin (HbA1c), and hepatic antioxidant enzymes. A secondary aim was to check the influence on serum biochemistry, hepatic, cardiac, and renal functions. METHODS: Forty-six participants with type 2 diabetes were recruited and randomly divided into two equal groups: diabetic control (DCON) and diabetic sesame oil (DSO). At baseline and 30, 60, and 90 days, blood samples were drawn and analyzed. Two-way repeated-measures analysis of variance was used to evaluate the difference between groups and across time. RESULTS: In both groups, GLU, INS, and HbA1c were not significantly different at baseline (mean 187.07 ± 5.63 mg/dl, mean 12.12 ± 1.03 µU/ml, and mean 7.55 ± 0.37%, respectively). At 90 days, GLU was significantly (p < 0.05) decreased in DSO (137.83 ± 3.16 mg/dl) when compared with DCON (218.13 ± 5.92 mg/dl), while INS was significantly increased in DSO (23.13 ± 1.15 µU/ml) as compared to DCON (7.93 ± 0.38 µU/ml). At 90 days, HbA1c was significantly lower (p < 0.05) in DSO as compared to DCON. Thiobarbituric acid reactive substances were significantly lower (p < 0.05) in DSO (1.08 ± 0.05 [MDA] nmol/ml) as compared to DCON (2.26 ± 0.07 [MDA] nmol/ml). In DSO, activities of hepatic antioxidant enzymes (superoxide dismutase, catalase, and glutathione peroxidase) increased while in DCON these activities decreased significantly (p < 0.05) across the time period. Biomarkers of liver, cardiac, and renal functions improved significantly in DSO as compared to DCON. CONCLUSION: WSSO as a functional food may play an important role in GLU regulation and against deleterious effects of diabetes in humans with type 2 diabetes.

Antiviral, embryo toxic and cytotoxic activities of Astragalus membranaceus root extracts.

Antiviral activity of Astragalus membranaceus aqueous and methanol root extracts was determined against Avian influenza H9 virus. Toxicity profile of extracts was evaluated using chicken embryos and BHK-21 cell line. Different concentrations (400, 200, 100, 50, 25. 12.5, 6.25 and 3.12µg/mL) of both aqueous and methanol extracts were mixed with standard virus inoculum (4HAunits) and incubated for 30minutes at 37°C prior to inject the chicken embryos. Chorioallantoic fluid harvested 72 hours post inoculation and evaluated for virus growth using hemagglutination assay. Same concentrations of both extracts without virus were injected in chicken embryos to evaluate embryo toxic activity as well. The cytotoxic activity of aqueous and methanol extracts was determined by MTT colorimetric assay using BHK-21 cells. Three concentrations (400, 200 and 100µg/mL) of aqueous and five concentrations (400, 200, 100, 50 and 25µg/mL) of methanol extract showed antiviral activity. None of the tested concentrations of aqueous and methanol A. membranaceus root extracts caused chicken embryo mortality. Cell survival percentage of aqueous extract was higher than 50 at all of the tested concentrations except 400µg/mL. Two concentrations (400 and 200µg/mL) of methanol extract showed cytotoxicity. It was concluded that aqueous and methanol roots extracts of A. membranaceus have antiviral activity and concentrations which were safe may be used for treatment of Avian influenza H9 virus infections.

Development and evaluation of vaccine against Staphylococcus aureus recovered from naturally occurring mastitis in she-camels.

The predominant Staphylococcus aureus (S. aureus), an etiological agent of camel mastitis is becoming drug resistant that invites prevention and control strategies. Vaccine production would have a valuable impact on public health. Therefore, in present study, inactivated vaccine with different adjuvants was prepared and evaluated against S. aureus. The vaccinal isolate recovered from camel subclinical mastitis was coagulase positive (PCR based), having expressed pseudocapsule, holding alpha-beta hemolysin characteristics, and multiple drug resistant. Inactivated alum precipitated S. aureus vaccine (APSV) and oil adjuvant S. aureus vaccine (OASV) were prepared after confirming its antigenicity in rabbits. Three groups of rabbits were randomly inoculated with APSV, OASV, and placebo (Unvaccinated, UV). Each group was further divided into two groups based on single and booster dose inoculation. Booster dose of vaccines in rabbits at day 15th of primary inoculation was given. Serum samples were taken on 15, 30, 45 and 60 days of primary inoculation from all rabbits. Analysis of variance was applied to compare geometric mean titer (GMT) of three groups, while t-test was applied to estimate the difference between single and booster dose response. The study found 1010 CFU/mL S. aureus as standard bacterial load for vaccines with higher and sustained antigenicity. The vaccines were safe from morbidity and mortality, and proved effective and stable for 7 and 4 months at 25 °C and 37 °C, respectively. The OASV produced significantly (p < 0.05) higher immune response followed by APSV throughout trial. The highest GMT by APSV and OASV vaccines with single dose inoculation was 37.92 and 69.92 at day 45th post primary inoculation, respectively. Similarly, 59.20 and 142.40 GMTs were noted with booster dose in case of APSV and OASV, respectively. The booster dose presented significantly (p < 0.05) higher GMT than that of single dose inoculation of vaccines. The study concluded APSV and OASV safe, effective, and stable with significant immunogenic results in experimental rabbits.

Role of Rab GTPases in HSV-1 infection: Molecular understanding of viral maturation and egress.

Most enveloped viruses exploit complex cellular pathways for assembly and egress from the host cell, and the large DNA virus Herpes simplex virus 1 (HSV-1) makes no exception, hijacking several cellular transport pathways for its glycoprotein trafficking and maturation, as well as for viral morphogenesis and egress according to the envelopment, de-envelopment and re-envelopment model. Importantly Rab GTPases, widely distributed master regulators of intracellular membrane trafficking pathways, have recently being tightly implicated in such process. Indeed, siRNA-mediated genetic ablation of specific Rab proteins differently affected HSV-1 production, suggesting a complex role of different Rab proteins in HSV-1 life cycle. In this review, we discuss how different Rabs can regulate HSV-1 assembly/egress and the potential therapeutic applications of such findings for the management of HSV-1 infections.

Association of textile industry effluent with mutagenicity and its toxic health implications upon acute and sub-chronic exposure.

Complex industrial discharges pose certain risks to the ecosystem. This study was aimed at identifying acute and sub-chronic toxicological effects of the textile industry wastewater. The textile wastewater was evaluated for the metals and organic pollutants by atomic absorption spectrophotometer and GC-MS respectively. In vitro genotoxicity and mutagenicity were assessed by Comet assay in peripheral lymphocytes isolated from Ovis aries and Ames test in Salmonella typhimurium strains TA-100 and 102 respectively. Physiological and behavioral changes along with systemic toxicity were determined in Rattus norvegicus albinus following acute and sub-chronic exposure. High amount of heavy metals such as Cr, Pb, Hg, As, and Cd were detected in textile wastewater. Organic pollutants such as 25-deacetoxy cucurbitacin-b, E-14-Hexadecenal, 11-Tricosene, and phthalates were also found. In vitro genotoxicity assessment in lymphocytes showed statistically significant DNA damaging potential of textile wastewater. Textile wastewater also showed significantly higher (p˂ 0.05) mutagenic potential in Salmonella TA-100 and TA-102 strains than sodium azide and 2-amino anthracycline. Acute exposure of textile wastewater to Rattus norvegicus was associated with several physiological changes and behavioral symptoms. Sub-chronic exposure of textile wastewater in Rattus norvegicus instigated the degeneration and necrosis of epithelial cells in renal tubules, hydropic degeneration and necrosis of hepatocytes, peri-bronchiolar infiltration and emphysema of the alveoli, and the degradation of myocardial cells. This study concludes that the textile wastewater may cause genotoxicity and mutagenicity, result in physiological and behavioral changes upon acute exposure, and inflict various pathological lesions upon sub-chronic exposure.

Anti-avian influenza virus H9N2 activity of aqueous extracts of Zingiber officinalis (Ginger) and Allium sativum (Garlic) in chick embryos.

In the present study, anti-Avian influenza virus H9N2 activity of aqueous extracts (5, 10, 15, 20, 25%) of Zingiber officinalis and Allium sativum was evaluated. Embryo-toxicity was evaluated by histopathological scoring of Chorio-allantoic membrane of chick embryos. Cytotoxicity of extracts was determined by MTT assay on Vero cells. Aqueous extract of ginger had antiviral activity at 10, 15, 20 and 25% while garlic had activity at 15, 20 and 25%. Histopathological scoring of chorio-allantoic membrane for aqueous extracts (5, 10, 15, 20, 25%) of ginger (0.66±0.57, 1.33±0.57, 1.66±0.57, 2.66±0.57, 3.66±0.57, respectively) and garlic (1.00±0.00, 1.33±0.57, 2.00±0.00, 2.33±0.57, 3.66±0.57, respectively) was concentration dependant. MTT assay revealed cytotoxicity of both plants was also concentration dependent. Extracts of ginger (5, 10, 15, 20, 25%) had lower cytotoxicity (71, 59, 28, 22, 0 % cell survival, respectively) as compared to garlic (61, 36. 20, 11, 3% cell survival, respectively). Overall results revealed that concentration of aqueous extract of ginger (10%), showing antiviral activity against H9N2, was less toxic to vero cells (> 50% cell survival). It is insinuated that ginger may have anti- Avian influenza virus H9N2 potential and its active compounds needs further investigations.

Toxicity Appraisal of Untreated Dyeing Industry Wastewater Based on Chemical Characterization and Short Term Bioassays.

Characterizing wastewaters only on a chemical basis may be insufficient owing to their complex nature. The purpose of this study was to assess toxicity of textile dyeing wastewater based on analytical techniques and short term toxicity based bioassays. In this study, screening of the fractionated wastewater through GC-MS showed the presence of phenols, phthalic acid derivatives and chlorpyrifos. Metal analysis revealed that chromium, arsenic and mercury were present in amounts higher than the wastewater discharge limits. Textile dyeing wastewater was found to be highly mutagenic in the Ames test. DNA damage in sheep lymphocytes decreased linearly with an increase in the dilution of wastewater. MTT assay showed that 8.3 percent v/v wastewater decreased cell survival percentage to 50 %. It can be concluded from this study that short term toxicity tests such as Ames test, in vitro comet assay, and cytotoxicity assays may serve as useful indicators of wastewater pollution along with their organic and inorganic chemical characterizations.

Toxinotyping and antimicrobial susceptibility of enterotoxigenic Clostridium perfringens isolates from mutton, beef and chicken meat.

A total of 300 meat samples comprising mutton, beef, and chicken meat (n = 100) collected from either local butcher shops or large meat outlets situated at various areas of Lahore City located in Punjab province of Pakistan were tested for the isolation of Clostridium perfringens. Prevalence of the organism was highest in the chicken (6 %) followed by mutton (5 %) and beef (1 %). Contamination level was high (10/150) in the samples collected from local butcher shops in comparison to the samples collected from large meat outlets (2/150). All of the raw meat samples were negative for the presence of alpha, beta and epsilon toxins of C. perfringens as detected through ELISA. Out of a total number of 12 isolates only half were capable of producing enterotoxins when cultured in trypticase glucose yeast (TGY) broth. Toxinotyping of the isolates showed that 3 were of type A while one each of the remaining three belonged to type B, C, and D. Antibiotic susceptibility testing of the toxin producing isolates revealed that C. perfringens were susceptible to chloramphenicol, ciprofloxacin, metronidazole, and ceftriaxone. All of the other drugs were relatively less effective with a least activity of amoxicillin against the isolates.

Evaluation of cytotoxicity and antiviral activity of ivermectin against Newcastle disease virus.

Cytotoxic and antiviral potential of ivermectin and ribavirin was evaluated. Cytotoxicity was checked on chick primary fibroblast cell line through MTT assay. Antiviral potential was determined against Newcastle disease virus on 9-day old chicken embryos. Six different concentrations (200, 100, 50, 25, 12.5 and 6.25µg/ml) of both the drugs were evaluated. The 100µg/ml concentration of ivermectin and higher were cytotoxic. The 25µg/ml concentration of ribavirin and higher were cytotoxic. Comparison of ivermectin and ribavirin showed that ivermectin was safe at 50µg/ml and lower concentrations. Ribavirin was protective for cell at 12.5µg/ml and 6.25µg/ml only. Comparison of antiviral activity indicated that ivermectin has strong antiviral potential at 100µg/ml and higher but same concentrations were cytotoxic. Ribavirin showed strong antiviral potential at all concentrations.

Preparation and in vitro evaluation of Nystatin micro emulsion based gel.

Nystatin is a polyene antimycotic obtained from Streptomyces noursei used in the treatment of topical and transdermal fungal infection. Nystatin is nearly insoluble in water (<0.1) and it is amphoteric in nature. The aim of the present study was to design and develop Nystatin micro emulsion based gel for efficient delivery of drug to the skin by water titration method. The Pseudoternary phase diagrams 1:2, 1:1 and 2:1 were constructed by water titration method. Micro emulsion based gel was prepared by using oleic acid, Tween 20, propylene glycol as an oil phase, surfactant and cosurfactant respectively. Cabopol 940 was used as a gelling agent. In vitro evaluation of micro emulsion based gel was done for pH, Viscosity, spreadability and droplet size. Micro emulsion based gel showed greater antifungal activity against Candida albicansas compared to control formulations. In vitro drug release studies were conducted for micro emulsion based gel and control formulation using Franz diffusion cell. Drug penetration through synthetic skin followed Zero order model as the values for R2 higher in case of zero order equation. The optimized micro emulsion based gel was found to be stable and showed no physical changes when exposed to different temperatures for a period of 4 week. The results indicated that the micro emulsion based gel system studied would be a promising tool for enhancing the percutaneous delivery of Nystatin.