Search

Objectives: To investigate the utilization pattern of Surgical antibiotic prophylaxis (SAP) for the prevention of surgical infections in most common abdominal and orthopedic surgical procedures. Methods : A prospective observational and non-intervention study was conducted at two teaching hospitals (PIMS and SIH) in Islamabad, Pakistan. Prescriptions records were reviewed to assess the utilization pattern of SAP during the nine-month duration. Data regarding most common surgical procedures, antimicrobial utilization, dose, route and administration timing of SAP were extracted for analysis. Observed practices were compared with standard treatment guidelines. Results : Out of total 1512 surgical procedures about, 56.9% (n=860) of patients performed abdominal followed by 652 (43.1%) orthopedic surgical procedures. A total of 212 (14%) surgical procedures were completely correct in all steps. SAP were given in 1474 (97.5%) of surgical procedures. In 712 of the participants (48.3%) were given Ceftriaxone following by Cefazolin (n=247, 16.7%). Abdominal surgeries were majorly managed with Ceftriaxone (59%) while the orthopedic surgeries managed with Ceftriaxone (31.3%) and Cefazolin (30%). Appropriate choice of SAP was observed in (n=275; 18.6%) procedures and about half (n=719, 49%) received antibiotics within optimal timing. The appropriate choice/selection of SAP according to the guidelines was greater in orthopedic surgical procedures (n=212; 14.8%) than abdominal surgeries (n=63; 4.3%); p≤0.001. Compliance with respect to timing was significantly lower in an orthopedic surgical procedure (n=301) as compared to abdominal surgeries (n=418); p≤0.001. Similarly, a statistically significant difference observed between PIMS and SIH with respect to SAP practices; p≤0.001. Conclusions : Inappropriate choice, the timing of administration and combination of SAP were observed in the current study. J Microbiol Infect Dis 2019; 9(3):104-111.

Bacillary diarrhea caused by Shigella flexneri is mediated by various virulence factors which make it the leading agent of diarrhea in developing countries. Previously, a high prevalence of S. flexneri, associated with diarrhea has been reported in Pakistan but no data is available on their virulence profile. The present study reports for the first time analysis of various virulence factors among S. flexneri serotypes isolated from clinical (diarrheal stool) and non-clinical (retail raw foods and drinking water) sources. A total of 199 S. flexneri (clinical: 155, raw foods: 22, water: 22) belonging to various serotypes were subjected to virulence genes detection and virulence profiling. The most frequent virulence gene was found to be ipaH (100%), followed by sat (98%), ial (71.3%), set1B (65.8%) and set1A (38.7%). A high level of virulence was detected in serotype 2b as compared to other serotypes as 32.3% of all serotype 2b have the entire set of five virulence genes including ipaH (100%), ial (100%), sat (37.7%), set1A (89.3%), and set1B (100%). Seven different virulence gene profiles (V1 - V7) were detected and the most frequently observed to be V1 (ipaH+, ial+, sat+, set1A+, set1B+) followed by V3 (ipaH+, ial+, sat+, set1B+). The predominant virulence gene pattern in serotype 2b isolated from clinical and non-clinical samples were V1 and V3. Furthermore, about 32% strains belonging to serotype 2b contain the complete set of five virulence genes isolated from patients with high disease severity. In conclusion, the current finding revealed for the first times that serotype 2b was the most virulent strains in both clinical and non-clinical samples in Pakistan. In addition, the virulence of serotype 2b was well correlated with high disease severity.

One of the main problems of the industrialized world is the accumulation of chromium (Cr) in soil, which is a serious threat to the crops. Complete removal of Cr from the contaminated soils poses a great challenge. However, this issue can be minimised by using plant growth promoting microbes as a bioremediation tool. In the present study, healthy plants established near the University campus in Mardan were selected for the isolation of Cr resistant endophytes. From the designated plants, 114 species of endophytic fungi were isolated. Among the 114 isolated strains, 4 strains have induced resistance in L. sativa against Cr. The strains were identified as Aspergillus fumigatus, Rhizopus sp., Penicillium radicum and Fusarium proliferatum based on ITS region (18 S rDNA) homology. The isolates have removed Cr from soil and culture media as well as bio-transformed it from highly toxic hexavalent to least toxic trivalent form, thus helped the Cr stressed L. sativa to restore its normal growth. The Rhizopus Sp. CUC23 has mainly accrued Cr and detoxified intracellularly, whereas A. fumigatus ML43 and P. radicum PL17 has detoxified up to 95% of Cr extracellularly. From the results, it is concluded that the selected endophytic strains might be used as biofertilizer for healthy and safe crop production in Cr contaminated soils.

Heavy metal (HM) pollution is of great concern in countries like Pakistan where a huge proportion of human population is exposed to it. These toxic metals are making their way from water bodies to soil where it not only interferes with plant growth and development but also initiates serious health issues in human consuming the produce of such soils. Bioremediation is one of the most viable and efficient solution for the problem. Purpose of the current study was to isolate endophytic fungi from plants grown on HM contaminated soil and screen them for their ability to tolerate multiple HM including chromium (Cr6+), manganese (Mn2+), cobalt (Co2+), copper (Cu2+) and zinc (Zn2+). Out of 27 isolated endophytes, only one strain (MHR-7) was selected for multiple heavy metals tolerance. The strain was identified as Mucor sp. by 18 S and 28 S ribosomal RNA internal transcribed spacer (ITS) 1 and 4 sequence homology. The strain effectively tolerated up to 900 µgmL−1 of these heavy metals showing no remarkable effect on its growth. The adverse effect of the heavy metals, measured as reduction of the fungal growth increased with increasing concentration of the metals. The strain was able to remove 60–87% of heavy metals from broth culture when supplied with 300 µg mL−1 of these metals. A trend of decline in bioremediation potential of the strain was observed with increasing amount of metals. The strain removed metals by biotransformation and/or accumulation of heavy metal in its hyphae. Application of Mucor sp. MHR-7 locked down HM in tis mycelium thereby making them less available to plant root reducing HM uptake and toxicity in mustard. Besides its bioremediation potential, the strain was also able to produce IAA, ACC deaminase and solubilize phosphate making it excellent phytostimulant fungus. It is concluded that MHR-7 is an excellent candidate for use as biofertilizer in fields affected with heavy metals.

Background Salmonella enteritidis infection is a frequently encountered zoonotic problem, occurring with concerning regularity in recent years on a worldwide basis. The study that we undertook for the first time detected S. enteritidis and associated antimicrobial resistance pattern in broiler chickens. Methods A total of 150 different poultry samples were first enriched and grown on selective media, and then processed for molecular detection of S. enteritidis by amplification of the spvb gene. Results The overall detection rate of S. enteritidis was 23.3% ( n =35), while an increased detection rate of S. enteritidis was found in the chicken breast tissue ( n =9; 30%). When antibiogram was tested for S. enteritidis against common antibiotics, increased resistance to ampicillin ( n =29; 82.2%), tetracycline ( n =28; 80%), augmentin ( n =27; 77.14%), and chloramphenicol ( n =19; 54.2%) was observed. Multidrug resistance was reported in 54.8% ( n =19) of the S. enteritidis isolates, while 20% ( n =07) of isolates were extensively drug resistant. Conclusion The present study for the first time reports S. enteritidis on the basis of spvb gene detection. The increased drug resistance in S. enteritidis is an emerging problem that could negatively impact efforts to prevent and treat broiler-transmitted S. enteritidis .

Background Pseudomonas aeruginosa is an emerging threat to public health worldwide due to their rapid development of drug resistance including beta-lactamases. The present study was designed to investigate the incidence of β-lactamases and genotypic pattern of CTX and OXA in the clinical isolate of multidrug resistant P. aeruginosa. Methods In this study a total of 102 MDR P. aeruginosa isolates obtained from Lady Reading Hospital, Peshawar, Pakistan were subjected to extended spectrum beta lactamase (ESBL), metallo beta lactamase (MBL) and plasmid mediated β-lactamase (AmpC) detection using phenotypic and molecular methods. Furthermore, sequencing of CTX and OXA gene was performed. Results Out of 102 MDR P. aeruginosa isolates, 71 (69.6%) were beta lactamase producers. The incidence of ESBL, MBL and AmpC in clinical isolates of P. aeruginosa was found to be 23.94%, 40.84% and 35.21% respectively. Co-production of ESBL and AmpC were also observed in some isolates. There were 14 (19.71%) CTX-M-15 harboring isolates which were ESBL (64.28%), MBL (21.42%) and AmpC (14.28%) producer. Co-production of ESBL/MBL (14.28%), ESBL/AmpC (14.28%) and MBL/AmpC (14.28%) were also observed in the CTX M-15 harboring isolates while 12.28% isolates were not ESBL, MBL or AmpC producer. OXA-10 was detected in 8 (11.26%) isolates which were ESBL (12.5%), MBL (37.5%) and AmpC (12.5%) producer. OXA 10 isolates also exhibit co-production of ESBL/AmpC (12.5%) and MBL/AmpC (12.5%). All CTX-M-15 carried the class A β-lactamase conserved domain while OXA-10 harbored conserved domain of class D β-lactamase. Conclusion The current study for the first time reported and characterized the CTX-M-15 and OXA-10 among MDR P. aeruginosa isolates from Pakistan. Further efforts are needed to understand the molecular mechanism of drug resistance with CTX and OXA harboring P. aeruginosa isolates.

Glucose-6-phosphate dehydrogenase (G6PD) is a key enzyme in the pentose phosphate pathway that ensures sufficient production of coenzyme nicotinamide adenine dinucleotide phosphate (NADPH) by catalyzing the reduction of NADP+ to NADPH. Noteworthy, the latter mediates the production of reactive oxygen species (ROS) by phagocytic cells such as neutrophils and monocytes. Therefore, patients with severe forms of G6PD deficiency may present impaired NADPH oxidase activity and become susceptible to recurrent infections. This fact, highlights the importance to characterize the immunopathologic mechanisms underlying the susceptibility to infections in patients with G6PD deficiency. Here we report the first two cases of G6PD deficiency with Bacille Calmette-Guerin (BCG) adverse effect, besides jaundice, hemolytic anemia and recurrent infections caused by Staphylococcus aureus. The qualitative G6PD screening was performed and followed by oxidative burst analysis using flow cytometry. Genetic and in silico analyses were carried out by Sanger sequencing and mutation pathogenicity predicted using bioinformatics tools, respectively. Activated neutrophils and monocytes from patients displayed impaired oxidative burst. The genetic analysis revealed the novel missense mutation c.1157T>A/p.L386Q in G6PD. In addition, in silico analysis indicated that this mutation is pathogenic, thereby hampering the oxidative burst of neutrophils and monocytes from patients. Our data expand the clinical and genetic spectrum of G6PD deficiency, and suggest that impaired oxidative burst in this severe primary immune deficiency is an underlying immunopathologic mechanism that predisposes to mycobacterial infections.

The interleukin (IL)-12/interferon(IFN)γ axis plays an important role in the control of mycobacterial diseases as demonstrated by the increased susceptibility to mycobacterial species in patients with an inborn error of the IL-12-dependent IFNγ immunity. Here, we report a novel mutation in the IL-12Rβ1 gene in a female Pakistani patient who was born in a consanguineous marriage and developed severe bacille Calmette–Guérin (BCG) infection and recurrent tuberculosis. After reviewing the patient’s clinical records, she was investigated for IL-12/IFNγ defects using enzyme-linked immunosorbent assay (ELISA), flow cytometry, and DNA genetic Sanger sequencing. Quantification of secretory cytokines from the patient’s peripheral blood mononuclear cells (PBMCs) revealed significantly reduced IFNγ production. Flow cytometric analysis revealed no surface expression of IL-12Rβ1 on PHA-activated T lymphocytes. In addition, IL-12-induced impaired STAT4 phosphorylation in the patient’s lymphocytes when compared with those from five healthy controls. The genetic analysis of IL-12Rβ1 gene identified a novel nonsense mutation c.199G>T/p.E67* within exon 3, which encodes part of the cytokine-binding region (CBR). In silico analysis indicates that this novel nonsense mutation generates a truncated protein with an apparent inactivating effect. Our data expand the genetic spectrum of IL-12Rβ1 deficiency. Moreover, our findings highlight the need for developing newborn screening for patients with primary immunodeficiency associated with mycobacterial infections in areas where BCG vaccination is mandatory in order to improve the treatment of patients, and consequently their quality of life.

Tuberculosis (TB), caused by Mycobacterium tuberculosis bacteria, is a leading infectious cause of mortality worldwide, including in Pakistan. Drug resistant M. tuberculosis is an emerging threat for TB control, making it important to detect the underlying genetic mutations, and thereby inform treatment decision making and prevent transmission. Whole genome sequencing has emerged as the new diagnostic to reliably predict drug resistance within a clinically relevant time frame, and its deployment will have the greatest impact on TB control in highly endemic regions. To evaluate the mutations leading to drug resistance and to assess for evidence of the transmission of resistant strains, 81 M. tuberculosis samples from Khyber Pakhtunkhwa province (North West Pakistan) were subjected to whole genome sequencing and standard drug susceptibility testing for eleven anti-TB drugs. We found the majority of M. tuberculosis isolates were the CAS/Delhi strain-type (lineage 3; n = 57; 70.4%) and multi-drug resistant (MDR; n = 62; 76.5%). The most frequent resistance mutations were observed in the katG and rpoB genes, conferring resistance to isoniazid and rifampicin respectively. Mutations were also observed in genes conferring resistance to other first and second-line drugs, including in pncA (pyrazinamide), embB (ethambutol), gyrA (fluoroquinolones), rrs (aminoglycosides), rpsL, rrs and giB (streptomycin) loci. Whilst the majority of mutations have been reported in global datasets, we describe unreported putative resistance markers in katG, ethA (ethionamide), gyrA and gyrB (fluoroquinolones), and pncA. Analysis of the mutations revealed that acquisition of rifampicin resistance often preceded isoniazid in our isolates. We also observed a high proportion (17.6%) of pre-MDR isolates with fluoroquinolone resistance markers, potentially due to unregulated anti-TB drug use. Our isolates were compared to previously sequenced strains from Pakistan in a combined phylogenetic tree analysis. The presence of lineage 2 was only observed in our isolates. Using a cut-off of less than ten genome-wide mutation differences between isolates, a transmission analysis revealed 18 M. tuberculosis isolates clustering within eight networks, thereby providing evidence of drug-resistant TB transmission in the Khyber Pakhtunkhwa province. Overall, we have demonstrated that drug-resistant TB isolates are circulating and transmitted in North West Pakistan. Further, we have shown the usefulness of whole genome sequencing as a diagnostic tool for characterizing M. tuberculosis isolates, which will assist future epidemiological studies and disease control activities in Pakistan.

Recent past years have witnessed the development of several artificial enzymes, using different materials to mimic natural enzymes with respect to their structure and functions. The nanozymes are nanomaterials possessing similar characteristics to the natural enzymes and have emerged recently as an innovative class of artificial enzymes. The nanozymes have got remarkable attention from the researchers and notable developments have been achieved owing to their unique properties compared with natural enzymes and classic artificial enzymes. In this regard, several nanomaterials have been scrutinized so far to mimic different natural enzymes for wider applications ranging from imaging, sensing, water treatment, pollutant removal, and therapeutics. The applications of nanozymes in biomedicine research are fast-growing and various nanozymes have been implicated in diagnostic medicine, targeted cancer therapy. Such abilities make them an appropriate alternative for the development of affordable, sustainable and safe diagnostic as well as therapeutic agents.

Chronic granulomatous disease (CGD) is a primary immunodeficiency caused by mutations in the five structural genes (CYBB, CYBA, NCF1, NCF2, and NCF4) that typically results in a decrease in function or inability to generate a respiratory burst, leading to defective killing of pathogens, including fungi and intracellular bacteria. Mutations in CYBB, encoding the gp91phox (also known as NOX2) result in X-linked CGD account for approximately 65% of CGD cases. Here, we aimed the characterization of a novel missense mutation c.1226C > A/p.A409E in the CYBB gene in a patient with X-linked CGD. Relevant clinical data of a male patient whose family was positive for XCGD was reviewed. Oxidative burst and NADPH protein expression was evaluated by flow cytometry, while Genetic analysis was performed by Sanger sequencing. Monocyte-derived macrophages (MDMs) were evaluated for their capacity for phagocytosis and growth suppression of the intracellular Mycobacterium tuberculosis (M. tuberculosis). We thus report the absence of an oxidative burst in the phagocytes of the patient. Flow cytometry evaluation revealed a normal expression of NADPH oxidase components in neutrophils and genetic analysis proved the existence of a novel missense c.1226C > A mutation in the CYBB gene resulting in p.A409E. Further, we have showed that the patient's MDMs were unhindered in their ability to take up mycobacteria normally. Instead, the MDMs failed to control the intracellular proliferation of M. tuberculosis, a phenotype that improved in the presence of recombinant human interferon-gamma (rhIFN-γ). This work expands the genetic spectrum of X-linked CGD and demonstrates improvement in macrophage function in X91+CGD patient by rhIFN-γ.

Objective: Chronic wound infections impose major medical and economic costs on health-care systems, cause significant morbidity, mortality and prolonged hospitalisation. The presence of biofilm producing bacteria in these wounds is considered as an important virulence factor that leads to chronic implications including ulceration. The undertaken study aimed to isolate and identify the biofilm aerobic bacterial pathogens from patients with chronic wound infections, and determine their antibiotics resistance profiles Method: During this study, swab specimens were collected from patients with chronic wounds at teaching hospitals of Peshawar, Pakistan between May 2013 and June 2014. The isolated aerobic bacterial pathogens were identified on the basis of standard cultural characteristics and biochemical tests. Antibiotics resistance profiles of biofilm producing bacteria against selected antibiotics were then determined. Results: Among the chronic wound infections, diabetic foot ulcers were most common 37 (37%), followed by surgical ulcers 27 (27%). Chronic wounds were common in male patients older than 40 years. Among the total 163 isolated bacterial pathogens the most prevalent bacterial species were Pseudomonas aeruginosa 44 (27%), Klebsiella pneumoniae 26 (16%), Staphylococcus species 22 (14%) and Streptococcus spp. 21 (13%). The isolation rate of bacterial pathogens was high among patients with diabetic foot ulcers 83 (50.9%). Among bacterial isolates, 108 (66.2%) were observed as biofilm producers while 55 (33.8%) did not form biofilm in our model. The investigated biofilm producing bacterial isolates showed comparatively high resistance against tested antibiotics compared to non-biofilm producing bacterial isolates. The most effective antibiotics were amikacine and cefepime against all isolates. Conclusion: Increased multidrug resistance in biofilm producing bacteria associated with chronic wounds was observed in this study. Judicious use of antibiotics is needed to control the wound associated biofilm associated pathogens.

Pesticides are one of the most potentially harmful chemicals introduced into the environment, and their adverse impacts on non-target organisms can be significant. The present study was conducted to shed light on effects of locally used insecticides chlorpyrifos (CPF) and lambda cyhalothrin (LCT) on oxidative stress biomarkers in human erythrocytes. The activity of catalase (CAT), superoxide dismutase (SOD), and protein contents as well as the levels of malondialdehyde (MDA) and osmotic fragility (OF) were measured in human erythrocytes exposed to CPF at concentrations of 0, 100, 500, 1000, and 2000 ppm and LCT at concentrations of 0, 100, 300, 600, and 800 ppm for 1 h and 3 h at 37°C. MDA levels and OF of erythrocytes were significantly higher in erythrocytes incubated with CPF and LCT at increasing concentrations of both insecticides and increased incubation time. However, erythrocyte CAT and SOD activities were decreased at all concentrations of CPF and LCT tested. Protein oxidation products were decreased at lower doses of CPF (100 and 500 ppm); at higher doses (1000 and 2000 ppm), total protein content was increased compared with control. In contrast LCT was associated with decreased in protein contents at all the concentrations. These results clearly demonstrated that CPF and LCT can induce oxidative stress in human erythrocytes ( in vitro).

Tuberculosis is a global health problem, and its early diagnosis is the ultimate strategy for prevention and control. The current study was undertaken to evaluate conventional and molecular diagnostic assays for the detection of mycobacteria in pulmonary tuberculosis (TB) patients from Khyber Pakhtunkhwa region of Pakistan. A total of 259 clinically suspected patients of TB were processed for Zeihl Neelsen (ZN) microscopy, BACTEC MGIT liquid culture and GeneXpert assay. Among 259 samples, 28 (10.81%) were positive for acid fast bacilli (AFB) on ZN microscopy. In liquid culture, the growth of mycobacterium species was obtained in 36 (13.89%) samples while the GeneXpert assay detected Mycobacterium tuberculosis (MTB) in 49 (18.91%) samples. Detection rate of MTB was significantly high (n = 49, p

Microbial proteolytic enzyme is one of the most important industrial enzymes that hydrolyze proteins. The applications of proteases under harsh industrial conditions like alkalinity, salinity, and temperature make them inactive and unstable. This suggests need for search for novel microbial sources for protease production having diverse properties. For this purpose, 54 bacterial strains were isolated from different salt mines of Karak, Pakistan and were investigated for their proteolytic activity on skim milk agar plates. The strain which showed maximum protease activity was characterized by 16S rRNA gene sequence analysis. Furthermore, growth and protease production was optimized for the characterized bacteria under different physical factors, i.e., pH, temperature and salinity. The isolate BLK-1.5 exhibited strong protease production and was identified as Bacillus subtilis based on biochemical characteristics and 16S rRNA gene sequence analysis. Maximum production of protease was recorded at pH 10, 37 °C and 7 % (w/v) NaCl. Molecular weight of proteases was estimated 38 kDa and its optimum activity was observed at pH 10, 50 °C and 2 % (w/v) NaCl. In conclusion, the protease produced by halo-tolerant Bacillus subtilis strain BLK-1.5 has diverse characteristics and could be useful in various industrial applications.

Objectives Drug-resistant tuberculosis (TB) is an emerging health problem. Rifampicin (RIF) is the major first-line drug against TB. RIF resistance can be used as a marker for the detection of multidrug-resistant TB (MDR-TB). The purpose of this study was to determine the RIF resistance pattern of Mycobacterium tuberculosis complex isolates among treated and untreated patients in Khyber Pakhtunkhwa, Pakistan. Methods A total of 349 drug-treated and untreated TB-diagnosed patients were enrolled in this study. RIF resistance was detected using a GeneXpert ® assay for amplification of the RIF resistance-determining region (RRDR) region of the rpoB gene. Results The overall prevalence of RIF resistance was 5.2% (18/349). Among 49 untreated TB patients, 3 samples (6.1%) were found resistant to RIF. Among 235 patients with a category 1 treatment regimen, 10 samples (4.3%) were resistant to RIF, whilst among 65 patients with a category 2 (Cat-2) treatment regimen, 5 samples (7.7%) were resistant to RIF. A comparison based on patient sex revealed high RIF resistance among male compared with female patients. RIF resistance was highest (4/21; 19.0%) in the 21–40 years age group among Cat-2 patients. Conclusions The overall prevalence of RIF resistance was high among treated and untreated TB patients. These findings will be helpful for better monitoring and management of RIF resistance in TB patients from Khyber Pakhtunkhwa, Pakistan.

Cinnamic acid (CA) is an allelochemical that inhibits the growth of root promoting soil microorganisms. To prevent the growth of soil microbes, CA modulates several metabolic pathways in host plants and soil microbes. The aim of the current study was to investigate the effect of CA on maize root growth, exudation of secondary metabolites and its interaction with beneficial endophyte Pz11. The endophyte Pz11 was isolated from the roots of drought stressed Asphodelus tenuifolius (wild onion). The Pz11 strain was identified as Fusarium culmorum by homology of the internal transcribed spacer (ITS) region of 18 S rDNA sequence. The F. culmorum Pz11 produced phytostimulants and signaling compounds, such as indole-3-acetic acid (IAA), flavonoids and sugars. Moreover, the strain have effectively colonized the roots of maize and subsequently enhanced the growth of its host plants. On the contrary, application of CA has reduced root growth in maize seedlings as well as root colonization ability of F. culmorum Pz11. Also, maize seedlings exposed to CA exude low quantities of flavonoids and polyphenols. In conclusion, CA reduces the maize root growth and exudation of secondary metabolites, which may affects its ability to attract plant growth promoting endophytic fungi.

Background Medicinal plants have been founded as traditional herbal medicine worldwide. Most of the plant’s therapeutic properties are due to the presence of secondary metabolites such as alkaloids, glycosides, tannins and volatile oil. Methods The present investigation analyzed the High-Pressure Liquid Chromatography (HPLC) fractions of Glycyrrhiza glabra (Aqueous, Chloroform, Ethanol and Hexane) against multidrug resistant human bacterial pathogens (Escherichia coli, Acinetobacter baumannii, Staphylococcus aureus and Pseudomonas aeruginosa). All the fractions showed antibacterial activity, were subjected to LC MS/MS analysis for identification of bioactive compounds. Results Among total HPLC fractions of G. glabra (n = 20), three HPLC fractions showed potential activity against multidrug resistant (MDR) bacterial isolates. Fraction 1 (F1) of aqueous extracts, showed activity against A. baumannii (15 ± 0.5 mm). F4 from hexane extract of G. glabra showed activity against S. aureus (10 ± 0.2 mm). However, F2 from ethanol extract exhibited activity against S. aureus (10 ± 0.3 mm). These active fractions were further processed by LC MS/MS analysis for the identification of compounds. Ellagic acid was identified in the F1 of aqueous extract while 6-aldehydo-isoophiopogonone was present in F4 of hexane extract. Similarly, Liquirtigenin was identified in F2 of ethanol. Conclusions Glycyrrhiza glabra extracts HPLC fractions showed anti-MDR activity. Three bioactive compounds were identified in the study. 6-aldehydo-isoophiopogonone and Liquirtigenin were for the first time reported in G. glabra. Further characterization of the identified compounds will be helpful for possible therapeutic uses against infectious diseases caused by multidrug resistant bacteria.

E2F4 is an important basal transcription factor with the potential to promote tumor growth. Its upregulation in various types of cancer has been linked to numerous genetic factors; however, the nature of the involvement of epigenetic mechanisms, including DNA methylation, remains elusive. In the present study, E2F4 expression profiles were determined in 100 paired breast tumor and control samples, through RT-qPCR using the SYBR® green method. Furthermore, the E2F4 promoter methylation status in each of these samples was assessed using methylation specific PCR, in order to evaluate its impact on gene expression. A two-fold increase in E2F4 gene expression was observed in the breast tumors compared with in their respective controls (P=0.022); of these tumors, ~72% were under-methylated. The change in methylation status was also significantly higher (P45 and ≤45 years) had comparable methylation profiles, though they had significantly decreased methylation status compared with controls. Various histo-pathological types also have different methylation profiles, indicating the presence of a tissue specific methylation signature. The results of the present study demonstrated that E2F4 methylation status can have a notable influence on its expression, and that it may have prognostic value in breast carcinogenesis.

Severe combined immunodeficiency (SCID) is a potentially fatal primary immunodeficiency (PID) that is caused by mutations in genes such as IL2RG, JAK3, IL7RA, RAG1, RAG2, and ADA. The products of these genes are involved in the development of several immune cells such as T, B and natural killer (NK) cells. Most of the SCID forms are autosomal recessive with the exception of IL2RG defects that cause an X-linked SCID. Among the different SCID types, there is a rare SCID form called leaky SCID, which is less severe when compared to the other classical SCID phenotypes. Leaky SCID can be caused by hypomorphic mutations in RAG1 and RAG2 that result in only partial loss of enzymatic function of the proteins respectively encoded by these genes. Here we report a novel missense mutation (c. 307C > T/p.H103Y) in the RAG1 gene in a patient with leaky SCID. In addition, we characterize the clinical and immunological features of this patient that developed along with other severe and recurrent infections such as mycobacterial diseases (BCGitis and pulmonary tuberculosis), the first occurrence of Chromobacterium violaceum in a patient with SCID. Understanding the increased susceptibility to mycobacteria presented by the patient, in which a functional investigation of IL-12/IFN-γ axis was performed, which demonstrated reduced production of IFN-γ in the supernatans of peripheral blood mononuclear cell cultures from the patient compared with those from healthy subjects. In conclusion, our data expands the molecular and clinical spectrum associated with the leaky SCID phenotype.

Olive plant produces a variety of bioactive molecules and thus has important medicinal value in folk medicine. In this study, different leaf and fruit extracts of Pakistani wild variety of Olea europaea was tested for their phytochemical content, antimicrobial activity and mass spectrometric analysis. Olive leaves and fruit samples were extracted with five different solvents to obtain the crude extract and screened for various kinds of phytochemicals. Phytochemicals were further confirmed through Fourier Transmission Infra-red Spectroscopy (FTIR).The plant extract showed significant antimicrobial activity against all the strains tested. Methanol, ethanol and ethyl acetate extracts were found more effective against most of the pathogenic bacteria with high zone of inhibition. Gas Chromatography-Mass Spectrometry (GC-MS) analysis revealed that olive fruits have Oleic acid, Palmitic acid, Linoleic acid, Octadecadienoic acid, Stearic acid, Palmitoleic acid and Tridecanoic acid as oil contents. In this work, the antimicrobial potential and phytochemical contents were explored which may further pave the way for the bio-industrial applications. Keywords: Olea europaea; Phytochemical content; Antimicrobial activity; GC-MS analysis http://dx.doi.org/10.19045/bspab.2017.60032

Microorganisms are commonly used for the production of different types of enzymes, among which amylases are extensively used for different industrial purposes like food industry, paper industry, textile industry and detergents etc. In this study, extremophilic bacteria were isolated from salt mines of Karak, Pakistan which have the ability to grow and express amylases at diverse environmental conditions. In Total, 43 out of the 53 bacteria were found positive for amylase production. Furthermore, microscopic, biochemical and phylogenetic analysis inferred from 16S rRNA gene sequencing identified that amylase producing strain SBA-5 belong to Bacillus subtilis. The strain SBA-5 identified as Bacillus subtilis exhibited high amylaseproduction at pH 9 and temperature 37°C. The present study revealed that the salt mines of Karak have high population of halotolerant microorganism feasible for use under diverse environmental conditions, which can be of great importance for various bio-industrial applications. Keywords: Amylase producing bacteria; Protein purification; Salt mines of Karak http://dx.doi.org/10.19045/bspab.2017.60038

Hepatitis C Virus is a causative agents of liver cirrhosis, having highly diversified genome. Genotype study has important role in clinical setup and prognosis of hepatitis C virus infection. The aim of this was to investigate the prevalence and risk factors associated with hepatitis C virus genotypes in district Kohat, Khyber Pakhtunkhwa, Pakistan. Hepatitis C virus pre-infected patients (n = 600) were analyzed to assess circulating HCV genotypes, using reverse transcriptase approach. Moreover serum ALT levels were determined followed by correlation with genotypes. In current study cohort, majority of patients (62%) were, having elevated ALT (> 50 U/L). similarly patients with mixed HCV genotypes, 3a/3b have relatively higher ALT levels (~62) compared to 2a/3a (~43) and others. We found significant correlation (p = 0.002) of genotype 2a in connection with ALT (49.48) level. Similarly 3a, 3b, mixed as well as un-typeable genotypes correlations were also found highly significant (p

Background Pseudomonas aeruginosa causes common infections in immunocompromised and cystic fibrosis patients. However, drug resistance capability and release of virulence factors play a key role in bacterial pathogenicity. Methods Beta-lactamase-producing clinical isolates of P. aeruginosa were screened for biofilm formation and pigment production. Subsequently, all the isolates were subjected to the detection of six virulence genes ( OprI , OprL , LasB , PlcH , ExoS , and ToxA ). Results Among beta-lactamase-producing isolates ( n =54), about 85.18% ( n =46) were biofilm producers. Pigment production was observed in 92.59% ( n =50) isolates. Clinical samples were subsequently screened for detection of virulence factors. Among them, 40.74% ( n =22) isolates were found to be OprI positive, while 29.62% ( n =16) were OprL producers. In the case of LasB and PlcH , 24% ( n =13) and 18.5% ( n =10) isolates produced these virulence genes, respectively. Among the isolates, 37.03% ( n =20) and 33.33% ( n =18) expressed virulence factors ExoS and ToxA , respectively. Furthermore, 42.59% ( n =23) isolates coproduced more than one type of virulence factors. Conclusion In the current study, pigment display, biofilm formation, and virulence genes were detected in P. aeruginosa clinical isolates. Such factors could be crucial in the development of drug resistance.

Pseudomonas aeruginosa is an important pathogen of both community and hospital acquired infections, and a major threat to public health for continuous emergence of multi-drug resistance. Current prevalence and pattern of multidrug resistance in the clinical isolates of P. aeruginosa is reported here. Samples were collected from September 2013 to January 2014 tertiary care hospital, Peshawar. Samples were subjected to phenotypic and molecular based detection of P. aeruginosa and were further processed for multidrug resistance pattern. Out of 3700 samples, 102 were identified as MDR P. aeruginosa. Prevalence of MDR isolates were found in pus (34.3%), wounds (28.4%), urine (19.6%), blood (14.7%) and sputum (2.9%) respectively. Isolates were more resistant to Sulphamethoxazole/Trimethoprim (98.04%), Amoxycillin/Clavulanic acid, Doxycycline and Chloramphenicol (95.1%) each, while least resistant to Imipenem (43.1%), Cefoperazone/Sulbactam (50.98%) and Amikacin (53.9%). Extensive MDR pattern was observed in P. aeruginosa was found as (n = 17, 16.6%) isolates were resistant to all four classes of antibiotics. Increased burden of MDR P. aeruginosa was documented in the study. Moreover, some isolates were even resistant to four classes of antibiotics. Findings of the study will be helpful to devise an appropriate antibiotic treatment strategy against MDR P. aeruginosa to cope the chances of evolving resistant pathogens.

Background/aim: Extended spectrum beta lactamase (ESBL) production among E. coli is one of the principal mechanisms that augment resistance to antibiotics. In the current study the molecular detection of class A beta lactamases among uropathogenic Escherichia coli was evaluated. Materials and methods: A total of 355 urine samples were collected from a tertiary care hospital in Peshawar. The ESBL production among E. coli isolates was detected by using the disc synergy diffusion method. Moreover, the molecular detection of bla TEM-1 and bla CTX-M-1 ESBLs, the antibiotic resistance pattern, and the minimum inhibitory concentrations (MICs) were also documented. Results: Among the 355 urine samples, 157 isolates were E. coli, and 23.56% of the isolates were ESBL E. coli. Among phenotypic ESBL producers, bla CTX-M-1 and bla TEM-1 were found in 59.45% and 40.54% of the isolates, respectively. A high resistance rate was observed against aztreonam (97.29%), while the lowest resistance was observed against imipenem (2.7%). The MICs of ESBLs E. coli for ceftriaxone, ciprofloxacin, and gentamicin was >512 μg/mL, 4 μg/mL to 128 μg/mL, and 1 μg/mL to 14 μg/mL respectively. Conclusion: The present study showed that bla CTX-M-1 ESBL production is more prevalent in our clinical E. coli isolates. More often the ESBLs were resistant to commonly used antibiotics.

Objective To investigate the epidemiological status of dengue viral infection and associated risk factors in Khyber Pakhtunkhwa, Pakistan. Methods A total of 302 clinically suspected cases of dengue were enrolled in this study. All the serum samples were subjected to ELISA for the detection of anti-dengue immunoglobulin G antibody. Results Dengue prevalence was significantly detected in male patients (n = 149, 87.13%) as compared to female patients (n = 22, 12.87%) (P = 0.000 4). Patients in the age group of 20–39 years (n = 114, 66.67%) were more prone to dengue, but less prevalent in the age group more than 60 years. Gender, age category and area were significant factors in the dengue prevalence (P = 0.034 8). Conclusions The overall prevalence of anti-dengue immunoglobulin G was 56.60% (n = 171) in referred dengue-suspected patients. Stagnant water reservoirs were found responsible for the spread of the disease favoring mosquito breeding sites.

Purpose: Purpose: To determine TEM-116 beta-lactamase gene prevalence in drug-resistant Pseudomonas aeruginosa isolates from Pakistan. Methods: Sequence analysis of TEM beta-lactamase isolates and their antibiotic susceptibility patterns were carried out. Quantitative bacteriostatic concentrations for commonly used antibiotics were measured against TEM-116 beta-lactamase isolates. Results: Among the 102 isolates of P. aeruginosa , 23 (22.5 %) were TEM beta-lactamase producers. Sequence analysis of TEM gene from selected isolates showed homology with TEM-116. Two mutations at positions 84 (p.Val84Ile) and 184 (p.Ala184Val) were documented. The TEM-116 isolates exhibited 100 % resistance to sulphamethoxazole/trimethoprim, amoxycillin/clavulanic acid and doxycycline, but showed sensitivity to levofloxacin, norfloxacin and amikacin. One TEM-116 P. aeruginosa (PA11) isolate was resistant to all available antibiotics. Conclusion: These results reveal increased antibiotic resistance in the TEM-116 P. aeruginosa isolates studied, a phenomenon which will be helpful in understanding the molecular mechanisms of antibiotic resistance in P. aeruginosa . Keywords: P. aeruginosa , Clinical isolates, Sequencing, TEM-116, Antibiotic susceptibility

Staphylococcus aureus is an increasing problem in clinical practice because of reduced susceptibility to available antibiotics. The objective of the study was to determine the frequency of Methicillin-resistant S. aureus (MRSA) in Peshawar, Pakistan. Clinical isolates of S. aureus were subjected to determination of antibiotic resistance, MICs and inducible clindamycin resistance (ICR). Out of total 280 S. aureus isolates, the frequency of MRSA was 36.1 % (n = 101). MRSA infection was found higher among the age group 50–59 years (60.71 %, OR 3.09), followed by 20–29 years (47.5 %, OR 1.74). Frequency of MRSA in female and male was 39.8 and 34 % respectively. MRSA was more frequent in blood specimens (48.7 %, OR 2.14). The frequency of community and hospital acquired MRSA was 42 and 34.8 % respectively. MRSA showed high resistance (100 %) to penicillin and cefoxitin followed by erythromycin (99 %). While MRSA exhibited 100 % susceptibility to vancomycin and linezolid. We have also found 7 vancomycin intermediate sensitive S. aureus (VISA) isolates. ICR was observed in 15.84 % (n = 16) of MRSA isolates. It is concluded that MRSA is potential threat to public health in Peshawar. Vancomycin and linezolid could be prescribed as a drug of choice in treating MRSA associated infections. In addition, ICR should be routinely checked to avoid clindamycin treatment failure.