Punicalagin, a pomegranate polyphenol sensitizes the activity of antibiotics against three MDR pathogens of the Enterobacteriaceae.

BACKGROUND: Multidrug resistance (MDR) in the family Enterobacteriaceae is a perniciously increasing threat to global health security. The discovery of new antimicrobials having the reversing drug resistance potential may contribute to augment and revive the antibiotic arsenal in hand. This study aimed to explore the anti-Enterobacteriaceae capability of bioactive polyphenols from Punica granatum (P. granatum) and their co-action with antibiotics against clinical isolates of Enterobacteriaceae predominantly prevalent in South Asian countries. METHODS: The Kandhari P. granatum (Pakistani origin) extracts were tested for anti-Enterobacteriaceae activity by agar well diffusion assay against MDR Salmonella enterica serovar Typhi, serovar Typhimurium and Escherichia coli. Predominant compounds of active extract were determined by mass spectrometry and screened for bioactivity by agar well diffusion and minimum inhibitory concentration (MIC) assay. The active punicalagin was further evaluated at sub-inhibitory concentrations (SICs) for coactivity with nine conventional antimicrobials using a disc diffusion assay followed by time-kill experiments that proceeded with SICs of punicalagin and antimicrobials. RESULTS: Among all P. granatum crude extracts, pomegranate peel methanol extract showed the largest inhibition zones of 25, 22 and 19 mm, and the MICs as 3.9, 7.8 and 7.8 mg/mL for S. typhi, S. typhimurium and E. coli, respectively. Punicalagin and ellagic acid were determined as predominant compounds by mass spectrometry. In plate assay, punicalagin (10 mg/mL) was active with hazy inhibition zones of 17, 14, and 13 mm against S. typhi, S. typhimurium and E. coli, respectively. However, in broth dilution assay punicalagin showed no MIC up to 10 mg/mL. The SICs 30 µg, 100 µg, and 500 µg of punicalagin combined with antimicrobials i.e., aminoglycoside, ß-lactam, and fluoroquinolone act in synergy against MDR strains with % increase in inhibition zone values varying from 3.4 ± 2.7% to 73.8 ± 8.4%. In time-kill curves, a significant decrease in cell density was observed with the SICs of antimicrobials/punicalagin (0.03-60 µg/mL/30, 100, 500 µg/mL of punicalagin) combinations. CONCLUSIONS: The P. granatum peel methanol extract exhibited antimicrobial activity against MDR Enterobacteriaceae pathogens. Punicalagin, the bacteriostatic flavonoid act as a concentration-dependent sensitizing agent for antimicrobials against Enterobacteriaceae. Our findings for the therapeutic punicalagin-antimicrobial combination prompt further evaluation of punicalagin as a potent activator for drugs, which otherwise remain less or inactive against MDR strains.

Revealing novel cytb and nad5 genes-based population diversity and benzimidazole resistance in Echinococcus granulosus of bovine origin.

Cystic echinococcosis (CE) is a neglected zoonotic disease caused by Echinococcus granulosus (sensu stricto). The parasite affects a wide range of livestock and wild animals. In this study, the population diversity of the Echinococcus species was investigated based on mitochondrial cytochrome b (cytb) and NADH dehydrogenase subunit 5 (nad5) genes. In addition to this, ß-tubulin gene isoforms of Echinococcus granulosus were amplified to determine the resistance against benzimidazoles. For this purpose, 40 cyst samples from cattle (n = 20) and buffaloes (n = 20) were collected from the main abattoir of Sialkot. DNA extraction was performed using Qiagen Blood and Tissue Kits. Amplification was performed through PCR. Each amplicon was confirmed by GelRed™ stained agarose gel (2%). Samples were sequenced in a DNA analyzer and viewed for any misread nucleotide by using MEGA (v.11). Corrections in nucleotide sequence and multiple sequence alignment were made through the same software. NCBI-BLAST was used for sample specific sequences to identify them as belonging to a particular species. Diversity indices were estimated using DnaSP (v.6) while phylogenetic analysis was inferred using the Bayesian method using MrBayes (v.1.1). ß-tubulin gene isoforms sequence analysis was performed to find out the candidate gene causing benzimidazole resistance. All 40 isolates were found positive for E. granulosus. BLAST-based searches of sequences of each isolate for each gene (nad5 and cytb) confirmed their maximum similarity with the G1 genotype. Overall, high haplotype diversity (Hd nad5 = 1.00; Hd cytb = 0.833) and low nucleotide diversity (π nad5 = 0.00560; π = cytb = 0.00763) was identified based on diversity indices. For both the genes, non-significant values of Tajima's D (nad5 = -0.81734; cytb = -0.80861) and Fu's Fs (nad5 = -1.012; cytb = 0.731) indicate recent population expansion. Bayesian phylogeny-based results of nad5 and cytb sequences confirmed their genotypic status as distinct from other Echinococcus species. This study shed light on the status of benzimidazole resistance in Echinococcus granulosus for the very first time from Pakistan. The findings of this study will significantly add in the information available on genetic diversity of Echinoccous granulosus based on cytb and nad5 genes sequences.

Burden of Tobacco in Pakistan: Findings From Global Adult Tobacco Survey 2014.

Introduction: The Global Adult Tobacco Survey (GATS) is the global standard for systematically monitoring adult tobacco use and tracking key tobacco control indicators. Methods: Using a multistage stratified cluster design, 9856 households were sampled, and one individual was randomly selected from each household. Standard GATS questionnaire was used to collect information on tobacco use, cessation, second-hand smoke, knowledge, attitudes, and perceptions. Data were analyzed per standard GATS protocol. Results: Of 9856 individuals, 7831 individuals completed the interview. The response rate was 81%. Overall, 19.1% adults were currently using tobacco products and among them, 12.4% smoked tobacco, and 7.7% smokeless tobacco. Exposure to second-hand smoke was seen in 86% in a restaurant while it was 76% on public transportation. A total of 24.7% smokers made a quit attempt in the past 12 months. Anticigarette smoking information was observed by 37.7% adults, while 29.7% current smokers thought about quitting after reading health warning labels on cigarette packages. Most (85%) adults favored no smoking in public places, and 74.8% favored increasing taxes on tobacco products. Current cigarette smokers spent Pakistani Rupees 767.3 per month (7.78 USD) on manufactured cigarettes and consumed 4500 cigarette sticks (225 packs) annually. Conclusions: Besides 19.1% tobacco users, the majority (86%) were exposed to second-hand smoke at public places indicating that ban on tobacco use in public places is not being followed. A quarter of current smokers wants to quit smoking who may be provided assistance to reduce tobacco burden. Implications: This study provides national-level data about tobacco use and its burden and also indicates weak implantation of tobacco control laws. There is need to devise a strategy for proper implementation of these laws to reduce the tobacco burden in the country.

Secondhand smoke exposure at home among one billion children in 21 countries: findings from the Global Adult Tobacco Survey (GATS).

OBJECTIVE: Children are vulnerable to secondhand smoke (SHS) exposure because of limited control over their indoor environment. Homes remain the major place where children may be exposed to SHS. Our study examines the magnitude, patterns and determinants of SHS exposure in the home among children in 21 countries (19 low-income and middle-income countries and 2 high-income countries). METHODS: Global Adult Tobacco Survey (GATS) data, a household survey of people 15 years of age or older. Data collected during 2009-2013 were analysed to estimate the proportion of children exposed to SHS in the home. GATS estimates and 2012 United Nations population projections for 2015 were also used to estimate the number of children exposed to SHS in the home. RESULTS: The proportion of children younger than 15 years of age exposed to SHS in the home ranged from 4.5% (Panama) to 79.0% (Indonesia). Of the approximately one billion children younger than 15 years of age living in the 21 countries under study, an estimated 507.74 million were exposed to SHS in the home. China, India, Bangladesh, Indonesia and the Philippines accounted for almost 84.6% of the children exposed to SHS. The prevalence of SHS exposure was higher in countries with higher adult smoking rates and was also higher in rural areas than in urban areas, in most countries. CONCLUSIONS: A large number of children were exposed to SHS in the home. Encouraging of voluntary smoke-free rules in homes and cessation in adults has the potential to reduce SHS exposure among children and prevent SHS-related diseases and deaths.

Prevalence and risk factors for diabetes mellitus in a selected urban population of a city in Punjab.

OBJECTIVE: To estimate the prevalence of diabetes mellitus (diagnosed and undiagnosed), impaired fasting glucose and possible risk factors for diabetes mellitus among Pakistani population. METHODS: This cross sectional study was performed in Rawalpindi which is one of the cities in Northern Punjab of Pakistan in July 2008. An area was selected in Rawalpindi city, with mixed population representative of almost all provinces with different socioeconomic groups. Three hundred and thirteen houses were selected through systematic random sampling technique and fasting blood glucose was obtained and subjects were labeled to have diabetes according to WHO criteria of diagnosing diabetes mellitus. The statistical analysis was performed by using Stata version 10. RESULTS: There were 1091 respondents who were selected after cleaning the data, among them 293 were males and 798 were females. Of the total 15.41% of the males and 12.31% of females were found to have diabetes mellitus. Thus making a total prevalence of 13.14%. Impaired fasting glucose (IFG) was found in 5.14% males and 5.78% females making a total prevalence of 5.61%. Over all (DM & IFG) was found to be 20.55% in males and 18.09% in females. The main risk factors identified were obesity, family history, hypertension and increasing age. CONCLUSION: There is an increased prevalence of Type 2 diabetes in Pakistan and main risk factors identified were obesity, overweight, family history of diabetes mellitus, and hypertension.