In vitro study: methylene blue-based antibacterial photodynamic inactivation of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is one of the most antibiotic-resistant and opportunistic pathogens in immunocompromised and debilitated patients. It is considered the cause of most severe skin infections and is frequently found in hospital burn units. Due to its high antibiotic resistance, eliminating P. aeruginosa from skin infections is quite challenging. Therefore, this study aims to assess the novel in vitro antibacterial activity of methylene blue using a 635-nm diode laser to determine the effective power and energy densities for inhibition of P. aeruginosa. The strain was treated with various concentrations of methylene blue and 635-nm diode laser at powers of 300 mW/cm2 and 250 mW/cm2. The diode laser's potency in the photo-destruction of methylene blue and its degradation through P. aeruginosa were also evaluated. Colony-forming unit (CFU)/ml, fluorescence spectroscopy, optical density, and confocal microscopy were used to measure the bacterial killing effect. As a result, the significant decrease of P. aeruginosa was 2.15-log10, 2.71-log10, and 3.48-log10 at 60, 75, and 90 J/cm2 after excitation of MB for 240, 300, and 360 s at a power of 250 mW/cm2, respectively. However, a maximum decrease in CFU was observed by 2.54-log10 at 72 J/cm2 and 4.32-log10 at 90 and 108 J/cm2 after 300 mW/cm2 of irradiation. Fluorescence images confirmed the elimination of bacteria and showed a high degree of photo-destruction compared to treatment with methylene blue and light alone. In conclusion, MB-induced aPDT demonstrated high efficacy, which could be a potential approach against drug-resistant pathogenic bacteria. KEY POINTS: • Combination of methylene blue with 635-nm diode laser for antibacterial activity. • Methylene blue photosensitizer is employed as an alternative to antibiotics. • aPDT showed promising antibacterial activity against Pseudomonas aeruginosa.

Prevalence and abundance of antibiotic-resistant genes in culturable bacteria inhabiting a non-polar passu glacier, karakorum mountains range, Pakistan.

Natural pristine environments including cold habitats are thought to be the potent reservoirs of antibiotic-resistant genes and have been recurrently reported in polar glaciers' native bacteria, nevertheless, their abundance among the non-polar glaciers' inhabitant bacteria is mostly uncharted. Herein we evaluated antibiotic resistance profile, abundance of antibiotic-resistant genes plus class 1, 2, and 3 integron integrases in 65 culturable bacterial isolates retrieved from a non-polar glacier. The 16S rRNA gene sequencing analysis identified predominantly Gram-negative 43 (66.15%) and Gram-positive 22 (33.84%) isolates. Among the Gram-negative bacteria, Gammaproteobacteria were dominant (62.79%), followed by Betaproteobacteria (18.60%) and Alphaproteobacteria (9.30%), whereas Phyla Actinobacteria (50%) and Firmicutes (40.90%) were predominant among Gram-positive. The Kirby Bauer disc diffusion method evaluated significant antibiotic resistance among the isolates. PCR amplification revealed phylum Proteobacteria predominantly carrying 21 disparate antibiotic-resistant genes like; blaAmpC 6 (100%), blaVIM-1, blaSHV and blaDHA 5 (100%) each, blaOXA-1 1 (100%), blaCMY-4 4 (100%), followed by Actinobacteria 14, Firmicutes 13 and Bacteroidetes 11. Tested isolates were negative for blaKPC, qnrA, vanA, ermA, ermB, intl2, and intl3. Predominant Gram-negative isolates had higher MAR index values, compared to Gram-positive. Alignment of protein homology sequences of antibiotic-resistant genes with references revealed amino acid variations in blaNDM-1, blaOXA-1, blaSHV, mecA, aac(6)-Ib3, tetA, tetB, sul2, qnrB, gyrA, and intI1. Promising antibiotic-resistant bacteria, harbored with numerous antibiotic-resistant genes and class 1 integron integrase with some amino acid variations detected, accentuating the mandatory focus to evaluate the intricate transcriptome analysis of glaciated bacteria conferring antibiotic resistance.

Isolation and molecular characterization of Adenovirus in suspected acute flaccid paralysis patients: A preliminary report from Pakistan.

Human adenoviruses (HAdVs) usually cause asymptomatic or mild infection, but infrequently, they are responsible for various severe syndromes including neurological disorders. Various research studies have investigated the association of HAdVs with acute flaccid paralysis (AFP). The purpose of this study was to investigate the genetic diversity of HAdVs and their association with AFP. Stool samples from patients ≤ 12 years of age with suspected AFP were collected from all over Pakistan within the framework of poliovirus surveillance. Poliovirus- and enterovirus-negative samples were screened for HAdVs. For virus isolation, the human epithelial cell line HEp-2c was used, culture-positive samples were screened by nested PCR assay, and partial hexon gene sequences were used for genotype identification. Out of 172 samples, 94 were positive by virus isolation, 89 were positive by PCR, and 32 isolates were genotyped successfully. Phylogenetic analysis showed that the HAdVs belonged to species A (HAdV-A12 and A31), B (HAdV-B3 and B7), C (HAdV-C1 and C6), D (HAdV-D19 and D93), and F (HAdV-F41), showing 99-100% nucleotide sequence identity and 98.3-100% amino acid sequence identity). Most of these genotypes have been reported previously in AFP cases, but this is the first report of the detection of HAdV-D93 in stool samples from AFP cases. The detection of a significant fraction of the HAdVs genotypes indicates that these genetically distinct genotypes are circulating in Pakistan and suggests their possible role in the pathogenesis of AFP.

Positivity, diagnosis and treatment follow-up of cutaneous leishmaniasis in war-affected areas of Bajaur, Pakistan.

This study was conducted to explore the frequency of positivity of cutaneous leishmaniasis (CL) in the tribal district Bajaur located near the Pak-Afghan border. The present study was conducted at the Leishmaniasis Center of Headquarter Hospital Khar District Bajaur, Pakistan. In total, 646 patients were recruited and included in the study after ethical approval and consent from the patients. CL was confirmed by taking blood samples from the sides of the lesion and observing them under a microscope using Giemsa staining. Information about demographic factors was collected from the study participants with a questionnaire and analyzed by SPSS. It was found that 73.8% of suspected patients were positive and 26.2% were negative for CL. There were 51.9% male and 48.1% female patients. The most frequently affected site was the face (42.6%), and most of the patients (85.8%) had only one lesion. The positivity of CL was higher among those under age 15 years. The area of most positivity, with 45.2% of the cases, was Tehsil Mamund. Most of the patients (46.6%) lived in stone houses, with 98.6% of patients having domestic animals in their houses. Approximately 198 patients were treated with intramuscular and intralesional injections of meglumine antimoniate, and their weekly follow-up revealed that 48% of patients recovered, while the remaining patients left the course of treatment at different stages of therapy. The positivity of CL is high in this area and is confirmed by the detection of Leishmania amastigotes in the blood collected from their lesions. Socioeconomic factors are the main underlying causes of the rapid spread of this disease and meglumine antimoniate is an effective drug.

Degradation of lignin by Bacillus altitudinis SL7 isolated from pulp and paper mill effluent.

Lignin is a major by-product of pulp and paper industries, and is resistant to depolymerization due to its heterogeneous structure. Degradation of lignin can be achieved by the use of potential lignin-degrading bacteria. The current study was designed to evaluate the degradation efficiency of newly isolated Bacillus altitudinis SL7 from pulp and paper mill effluent. The degradation efficiency of B. altitudinis SL7 was determined by color reduction, lignin content, and ligninolytic activity from degradation medium supplemented with alkali lignin (3 g/L). B. altitudinis SL7 reduced color and lignin content by 26 and 44%, respectively, on the 5th day of incubation, as evident from the maximum laccase activity. Optimum degradation was observed at 40 °C and pH 8.0. FT-IR spectroscopy and GC-MS analysis confirmed lignin degradation by emergence of the new peaks and identification of low-molecular-weight compounds in treated samples. The identified compounds such as vanillin, 2-methyoxyhenol, 3-methyl phenol, oxalic acid and ferulic acid suggested the degradation of coniferyl and sinapyl groups of lignin. Degradation efficiency of B. altitudinis SL7 towards high lignin concentration under alkaline pH indicated the potential application of this isolate in biological treatment of the lignin-containing effluents.

Bacterial community characterization of Batura Glacier in the Karakoram Range of Pakistan.

High-altitude cold habitats of the Karakoram are rarely explored for their bacterial community characterization and metabolite productions. In the present study, bacterial communities in ice, water, and sediments of Batura Glacier were investigated using culture-dependent and culture-independent methods. Twenty-seven cold-adapted bacterial strains (mostly psychrotrophic) were isolated using R2A, Tryptic Soy Agar (TSA), and Luria-Bertani (LB) media, at 4 °C and 15 °C. Most of the isolates exhibited growth at a wide range of temperature (4-35 °C), pH (5-12), and salinity (1-6%). Among the bacterial isolates, 52% were identified as Gram-positive and the remaining 48% represented as Gram-negative. The results of phylogenetic analysis indicated that all the culturable bacteria belonged to 3 major phylogenetic groups, i.e., Actinobacteria (48%), Bacteroidetes (26%), and Proteobacteria (22%), while Flavobacterium (26%), Arthrobacter (22%), and Pseudomonas (19%) were represented as the dominant genera. Similarly, Illumina amplicon sequencing of 16S rRNA genes after PCR amplification of DNA from the whole community revealed dominance of the same phylogenetic groups, Proteobacteria, Actinobacteria, and Bacteroidetes, while Arthrobacter, Mycoplana, Ochrobactrum, Kaistobacter, Janthinobacterium, and Flavobacterium were found as the dominant genera. Among the culturable isolates, 70% demonstrated activity for cellulases, 48% lipases, 41% proteases, 41% DNases, and only 7% for amylases. Most of the glacial isolates demonstrated antimicrobial activity against other microorganisms including the multiple-drug-resistant strains of Candida albicans, Klebsiella pneumoniae, Acinetobacter sp., and Bacillus sp. 67% of Gram-negative while 46% of Gram-positive glacial bacteria were resistant to trimethoprim/sulfamethoxazole. Resistance against methicillin and vancomycin among the Gram-positive isolates was 23% and 15%, respectively, while 11% of the Gram-negative isolates exhibited resistance against both colistin sulfate and nalidixic acid.

Rotavirus gastroenteritis in Pakistan, 2018: updated disease burden.

OBJECTIVE: Rotavirus A (RVA) is a significant cause of severe diarrheal illness and one of the common causes of death in children under the age of five. This study was aimed at detecting the prevalence of RVA in Pakistan after rotavirus vaccines were introduced. Fecal samples were obtained from 813 children from different hospitals in Rawalpindi and Islamabad, Pakistan, from January 2018 to December 2018. To obtain additional information from the parents / guardians of the children, a standard questionnaire was used. RESULTS: Using an enzyme-linked immunosorbent assay kit (ELISA), rotavirus antigen was detected and ELISA positive samples were subjected to reverse transcription PCR (RT-PCR). The findings showed 22% prevalence of RVA in children with acute gastroenteritis (AGE) via ELISA and 21% prevalence via RT-PCR in children with AGE. There was no statistically significant difference between gender, age and RVA infections. The winter, spring and fall/autumn seasons were statistically significant for RVA prevalence. CONCLUSION: The present study will provide post vaccine prevalence data for the health policy makers. The implementation of rotavirus vaccines, along with adequate nutrition for babies, clean water supply and maternal hygienic activities during infant feeding, is recommended. Furthermore, continuous surveillance is mandatory in the whole country to calculate the disease burden caused by RVA.

Genome Annotation of Poly(lactic acid) Degrading Pseudomonas aeruginosa, Sphingobacterium sp. and Geobacillus sp.

Pseudomonas aeruginosa and Sphingobacterium sp. are well known for their ability to decontaminate many environmental pollutants while Geobacillus sp. have been exploited for their thermostable enzymes. This study reports the annotation of genomes of P. aeruginosa S3, Sphingobacterium S2 and Geobacillus EC-3 that were isolated from compost, based on their ability to degrade poly(lactic acid), PLA. Draft genomes of the strains were assembled from Illumina reads, annotated and viewed with the aim of gaining insight into the genetic elements involved in degradation of PLA. The draft genome of Sphinogobacterium strain S2 (435 contigs) was estimated at 5,604,691 bp and the draft genome of P. aeruginosa strain S3 (303 contigs) was estimated at 6,631,638 bp. The draft genome of the thermophile Geobacillus strain EC-3 (111 contigs) was estimated at 3,397,712 bp. A total of 5385 (60% with annotation), 6437 (80% with annotation) and 3790 (74% with annotation) protein-coding genes were predicted for strains S2, S3 and EC-3, respectively. Catabolic genes for the biodegradation of xenobiotics, aromatic compounds and lactic acid as well as the genes attributable to the establishment and regulation of biofilm were identified in all three draft genomes. Our results reveal essential genetic elements that facilitate PLA metabolism at mesophilic and thermophilic temperatures in these three isolates.

Characterization of Bacillus nealsonii strain KBH10 capable of reducing aqueous mercury in laboratory-scale reactor.

The environmental release of mercury is continuously increasing with high degree of mobility, transformation and amplified toxicity. Improving remediation strategies is becoming increasingly important to achieve more stringent environmental safety standards. This study develops a laboratory-scale reactor for bioremediation of aqueous mercury using a biofilm-producing bacterial strain, KBH10, isolated from mercury-polluted soil. The strain was found resistant to 80 mg/L of HgCl2 and identified as Bacillus nealsonii via 16S rRNA gene sequence analysis. The strain KBH10 was characterized for optimum growth parameters and its mercury biotransformation potential was validated through mercuric reductase assay. A packed-bed column bioreactor was designed for biofilm-mediated mercury removal from artificially contaminated water and residual mercury was estimated. Strain KBH10 could grow at a range of temperature (20-50 °C) and pH (6.0-9.0) with optimum temperature established at 30 °C and pH 7.0. The optimum mercuric reductase activity (77.8 ± 1.7 U/mg) was reported at 30 °C and was stable at a temperature range of 20-50 °C. The residual mercury analysis of artificially contaminated water indicated 60.6 ± 1.5% reduction in mercury content within 5 h of exposure. This regenerative process of biofilm-mediated mercury removal in a packed-bed column bioreactor can provide new insight into its potential use in mercury bioremediation.

Biodegradation of Poly(3-hydroxybutyrate) and Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by Newly Isolated Penicillium oxalicum SS2 in Soil Microcosms and Partial Characterization of Extracellular Depolymerase.

A fungus, designated as strain SS2 able to degrade aliphatic polyesters, poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), was isolated from soil. Strain SS2 was identified through rDNA gene sequencing and showed maximum closeness to Penicillium oxalicum. The newly isolated P. oxalicum strain SS2 had completely degraded PHB and PHBV both in emulsion and films form within 36-48 h at 30 °C. Furthermore, P. oxalicum SS2 degraded PHB and PHBV films in soil environment in lab-built soil microcosms within 1 week. The polymer films were evaluated for changes after degradation through scanning electron microscopy (SEM), nuclear magnetic resonance (NMR), differential scanning calorimetry (DSC) and Fourier Transform Infrared spectroscopy (FTIR). The PHBV depolymerase enzyme was purified to homogeneity through column chromatography and molecular mass was found approximately 36 kDa. The depolymerase was stable over a wide range of temperature (15-60 °C) and pH (3.0-8.0) with optimum 40 °C and pH 5.0. The enzyme activity was significantly affected by various metal ions and surfactants. The enzyme activity was strongly enhanced in the presence of divalent cationic metal Cu2+ while inhibited by Zn2+ and non-polar detergents Tween 20 and Tween 60. Finally, it is concluded that P. oxalicum strain SS2 has profound degradation capabilities, and can be applied for the treatment of plastic-contaminated environments.

Antioxidative and Radioprotective Properties of Glycosylated Flavonoid, Xanthorhamnin from Radio-Resistant Bacterium Bacillus indicus Strain TMC-6.

A radio-resistant bacterium labeled as strain TMC-6 was isolated from Thal desert, Pakistan and identified through 16S rRNA gene sequencing as Bacillus indicus strain TMC-6 (MN721293). The isolate was found to be resistant to UV radiation dose of 6.780 × 103 J/m2 and showed 50% survivability to mitomycin C (6 µg/ml) and H2O2 (30 mM). The bacterium showed yellowish orange coloration when grown on tryptone yeast glucose (TGY) medium. The cellular metabolite was extracted in methanol and purified through solid phase extraction with C18 column cartridge. The compound was characterized through UV/Visible spectrophotometry, Fourier Transform Infra-Red (FT-IR) spectroscopy and Liquid Chromatography Mass Spectrometry (LC-MS). The LC-MS analysis of the compound revealed a molar mass of 769 [m/z]- that matched the chemical formula C34H42O20 and identified as a glycosylated flavonoid xanthorhamnin. The compound showed significant antioxidant (77.05%) and metal chelation (79.80%) activities. Xanthorhamnin showed promising oxidative damage inhibitory actions in bovine serum albumin (65.32%) and mice liver lipids (71.61%) and prevented DNA strand breaks from oxidative stress. Cytotoxicity in brine shrimp larvae was observed when compared with mitomycin C indicating its effect toward cancerous cells. These findings concluded that xanthorhamnin from radio-resistant Bacillus indicus strain TMC-6 has high antioxidant, radioprotective, and antitumor properties against UV-mediated oxidative damages.

Preparation and characterization of resistant starch type III from enzymatically hydrolyzed maize flour.

Polysaccharides including resistant starch are categorized as dietary fiber and are used as an important prebiotic. Similar to soluble fibers, resistant starch also has a number of physiological effects that have been shown to be beneficial for health. Starch hydrolyzing enzymes, most importantly amylases, play essential roles in the production of resistant starch. This study aimed to develop α-amylase-treated maize flour with slow digestibility and unique physicochemical characteristics compared to native maize flour. In the current study, resistant starch type III from maize flour was prepared using α-amylase obtained from indigenously isolated Bacillus licheniformis. The α-amylase gene from B. licheniformis was amplified and cloned into the pET-24(a) vector, expressed in E. coli BL21 (DE3) cells and purified by metal ion affinity chromatography. The purified enzyme enhanced the yield of resistant starch 16-fold in maize flour. Scanning electron microscopy revealed that the granular structure of maize flour was disrupted into a dense network with irregular structure, and X-ray diffractograms confirmed the transformation from an amorphous to a crystalline structure upon α-amylase treatment. Thermogravimetric analysis revealed increased amylose content of α-amylase-treated maize flour. Moreover, α-amylase-treated maize flour resulted in a significant enhancement of the desired properties of maize flour, such as resistant starch content, amylose, milk absorption capacity, and iodine and fatty acid complexing ability, and a reduction in swelling power, water binding, oil absorption capacity, and in vitro digestibility compared to untreated maize flour. Resistant starch type III showed low digestibility and increased complexing ability with iodine and fatty acid and therefore could be a safe and beneficial alternative as a coating material for the delivery of active, sensitive ingredients to the colon.

Antiproliferative, antioxidant and binding mechanism analysis of prodigiosin from newly isolated radio-resistant Streptomyces sp. strain WMA-LM31.

Streptomyces genus are filamentous Gram positive bacteria, of great intrest, producing biologically active compounds. Recent market and consumer curiosity in natural products have forced scientist and industry for the development of new products with therapeutic potential. This study focuses on evaluation of antioxidant and anticancerous properties of prodigiosin from radio-resistant Streptomyces sp. strain WMA-LM31. A molecular docking approach was adopted to understand theoretical binding mechanism and affinity for anticancer targets. A radio-resistant bacterium, labelled as strain WMA-LM31, was isolated from desert soil and screened for its radio-resistant potential and prodigiosin production. 16S rRNA gene sequencing showed that the bacterium clusters to genus Streptomyces and found resistant to ultraviolet radiation (dosage of 2 × 103 J/m2). Strain WMA-LM31 produced a red color pigment in tryptone glucose yeast (TGY) medium.The LC-MS analysis of the purified compound showed a molar mass of 324 [m/z]+ matched the chemical formula C20H25N3O, identified as prodigiosin. The compound showed strong antioxidant (62.51%) activities along with significant inhibitory action against oxidative damages to bovine serum albumin (BSA) and mice liver lipids in comparison to standard ascorbic acid. IC50 values of HepG2 and HeLa cell lines was found at 12.66 and 14.83 µg/mL of prodigiosin concentration, respectively. Furthermore, molecular docking was performed with two different cancers macromolecular targets: [2O2F (Bcl-2) and 1DI8 (CDK-2)], and BSA (PDB id: 3V03). The results indicated that the binding affinity of prodigiosin to its target molecules is due to the presence of terminal pyrrole rings. It is concluded from the results that prodigiosin from Streptomyces sp. strain WMA-LM31 has strong antioxidant, anticancer and apoptotic properties. The knowledge of binding mechanisms and interactions of prodigiosin could provide future directions in designing potent target specifc drugs.

Prevalence and antifungal susceptibility of Candida species in a tertiary care hospital in Islamabad, Pakistan.

OBJECTIVE: To determine the prevalence and antifungal susceptibility pattern of Candida species. METHODS: This prospective, cross-sectional study was conducted at the Quaid-e-Azam International Hospital, Islamabad, Pakistan, from January 2014 to February 2015, and comprised different clinical samples which were analysed for various types of microbial infections. Species differentiation was confirmed by biochemical and molecular methods. Antifungal susceptibility against amphotericin B, fluconazole and voriconazole was determined by Clinical and Laboratory Standards Institute M44-A disk diffusion method. RESULTS: Of the 219 Candida isolates, majority of them were isolated from urine 78(35.6%) and vaginal swabs 59(26.9%). Moreover, 144(65.8%) samples were of females and 75(34.2%) were of males. Candida albicans 128(58.45%) was the most predominant species followed by Candida glabrata 30(13.69%), Candida tropicalis 26(11.87%), Candida krusei 17(7.76%), Candida parapsilosis 12(5.47%), Candida dubliniensis 3(1.37%) and Candida lusitaniae 3(1.37). All isolates were least susceptible to amphotericin B with a susceptibility rate of 213(97.26%). The highest resistance was found for voriconazole 40(18.26%) compared to fluconazole 32(14.61%). CONCLUSIONS: Candida species possessed high resistance rate against various antifungal agents.

Rifampicin resistance mutations in the 81 bp RRDR of rpoB gene in Mycobacterium tuberculosis clinical isolates using Xpert MTB/RIF in Khyber Pakhtunkhwa, Pakistan: a retrospective study.

BACKGROUND: Multi-drug resistant tuberculosis (MDR-TB) is a major public health problem especially in developing countries. World Health Organization (WHO) recommends use of Xpert MTB/RIF assay to simultaneously detecting Mycobacterium tuberculosis (MTB) and rifampicin (RIF) resistance. The primary objective of this study was to determine the frequency of MDR-TB in patients suspected to have drug resistance in Khyber Pakhtunkhwa. The frequency of probes for various rpoB gene mutations using Xpert MTB/RIF assay within 81 bp RRDR (Rifampicin Resistance Determining Region) was the secondary objective. METHODS: A total of 2391 specimens, received at Programmatic Management of Drug Resistant TB (PMDT) Unit, Lady Reading Hospital (LRH) Peshawar, Pakistan, between October 2011 and December 2014, were analyzed by Xpert MTB/RIF test. MTB positive with rifampicin resistance were further analyzed to first line anti-mycobacterial drug susceptibility testing (DST) using middle brook 7H10 medium. The data was analyzed using statistical software; SPSS version 18. RESULTS: Out of 2391 specimens, 1408 (59 %) were found positive for MTB and among them, 408 (29 %) showed rifampicin-resistance with four different rpoB gene mutations within 81 bp RRDR. The frequency of various probes among RIF-resistant isolates was observed as: probe E, in 314 out of 408 isolates; B, 44 out of 408; A, 5 out of 408; D, 34 out of 408; and probe C was observed among 6 out of 408 RIF-resistant isolates. The probe A&B and E&D mutation combination was found in only 1 isolate in each case, while B&D mutation combination was detected among 3 out of 408 RIF-resistant isolates. CONCLUSIONS: Hence, it is concluded from our study on a selected population, 29 % of patients had MDR-TB. Probe E related mutations (also known as codon 531and 533) were the most common rpoB genetic mutation [314 (77 %)], acknowledged by Xpert MTB/RIF assay. Least mutation was detected within the sequence 511 (1.2 %).

Seroprevalence of Human Cytomegalovirus (HCMV) infection in pregnant women and outcomes of pregnancies with active infection.

OBJECTIVE: To determine the prevalence of cytomegalovirus in pregnant women and types of overt congenital infection in neonates. METHODS: This cross-sectional study was conducted at the Pakistan Institute of Medical Sciences and Federal Government Services Hospital in Islamabad, Pakistan, from March 2010 to June 2011, and comprised blood samples of pregnant women. Seroprevalence of human cytomegalovirus, immunoglobulin G and immunoglobulin M was determined by enzyme-linked immunosorbent assay while its deoxyribonucleic acid was detected by nested polymerase chain reaction.The congenital human cytomegalovirus infection was also identified in newborn babies from actively infected pregnant women. SPSS 18 was used for data analysis. RESULTS: Of the 409 pregnant women enrolled, 399(97.55%) were seropositive for cytomegalovirus immunoglobulinG and 52(12.71%) for immunoglobulinM, while cytomegalovirus deoxyribonucleic acid was detected in 82(20%). Of the cytomegalovirus immunoglobulinM-positive women, sera of 40(80%) had immunoglobulinG avidity >50%. The remaining 12(23%) sera had avidity assay value <50%. Among the 82(20%) infected pregnant women, 70(85.4%) were successfully followed up. Among them, the virus was isolated from 41(58.5%) newborns babies, of which 15(21%) were symptomatic while 26(47.2%) were asymptomatic. Of the former, 4(26.6%) had hepatosplenomegaly. CONCLUSIONS: Human cytomegalovirus infection in pregnant women was the main reason of congenital defects among neonates.

Microbial degradation of aliphatic and aliphatic-aromatic co-polyesters.

Biodegradable plastics (BPs) have attracted much attention since more than a decade because they can easily be degraded by microorganisms in the environment. The development of aliphatic-aromatic co-polyesters has combined excellent mechanical properties with biodegradability and an ideal replacement for the conventional nondegradable thermoplastics. The microorganisms degrading these polyesters are widely distributed in various environments. Although various aliphatic, aromatic, and aliphatic-aromatic co-polyester-degrading microorganisms and their enzymes have been studied and characterized, there are still many groups of microorganisms and enzymes with varying properties awaiting various applications. In this review, we have reported some new microorganisms and their enzymes which could degrade various aliphatic, aromatic, as well as aliphatic-aromatic co-polyesters like poly(butylene succinate) (PBS), poly(butylene succinate)-co-(butylene adipate) (PBSA), poly(ε-caprolactone) (PCL), poly(ethylene succinate) (PES), poly(L-lactic acid) (PLA), poly(3-hydroxybutyrate) and poly(3-hydoxybutyrate-co-3-hydroxyvalterate) (PHB/PHBV), poly(ethylene terephthalate) (PET), poly(butylene terephthalate) (PBT), poly(butylene adipate-co-terephthalate (PBAT), poly(butylene succinate-co-terephthalate) (PBST), and poly(butylene succinate/terephthalate/isophthalate)-co-(lactate) (PBSTIL). The mechanism of degradation of aliphatic as well as aliphatic-aromatic co-polyesters has also been discussed. The degradation ability of microorganisms against various polyesters might be useful for the treatment and recycling of biodegradable wastes or bioremediation of the polyester-contaminated environments.

Characterization of ES10 lytic bacteriophage isolated from hospital waste against multidrug-resistant uropathogenic E. coli.

Escherichia coli is the major causative agent of urinary tract infections worldwide and the emergence of multi-drug resistant determinants among clinical isolates necessitates the development of novel therapeutic agents. Lytic bacteriophages efficiently kill specific bacteria and seems promising approach in controlling infections caused by multi-drug resistant pathogens. This study aimed the isolation and detailed characterization of lytic bacteriophage designated as ES10 capable of lysing multidrug-resistant uropathogenic E. coli. ES10 had icosahedral head and non-contractile tail and genome size was 48,315 base pairs long encoding 74 proteins. Antibiotics resistance, virulence and lysogenic cycle associated genes were not found in ES10 phage genome. Morphological and whole genome analysis of ES10 phage showed that ES10 is the member of Drexlerviridae. Latent time of ES10 was 30 min, burst size was 90, and optimal multiplicity of infection was 1. ES10 was stable in human blood and subsequently caused 99.34% reduction of host bacteria. Calcium chloride shortened the adsorption time and latency period of ES10 and significantly inhibited biofilm formation of host bacteria. ES10 caused 99.84% reduction of host bacteria from contaminated fomites. ES10 phage possesses potential to be utilized in standard phage therapy.

Degradation of polyester polyurethane by a newly isolated soil bacterium, Bacillus subtilis strain MZA-75.

A polyurethane (PU) degrading bacterial strain MZA-75 was isolated from soil through enrichment technique. The bacterium was identified through 16S rRNA gene sequencing, the phylogenetic analysis indicated the strain MZA-75 belonged to genus Bacillus having maximum similarity with Bacillus subtilis strain JBE0016. The degradation of PU films by strain MZA-75 in mineral salt medium (MSM) was analyzed by scanning electron microscopy (SEM), fourier transform infra-red spectroscopy (FT-IR) and gel permeation chromatography (GPC). SEM revealed the appearance of widespread cracks on the surface. FTIR spectrum showed decrease in ester functional group. Increase in polydispersity index was observed in GPC, which indicates chain scission as a result of microbial treatment. CO2 evolution and cell growth increased when PU was used as carbon source in MSM in Sturm test. Increase in both cell associated and extracellular esterases was observed in the presence of PU indicated by p-Nitrophenyl acetate (pNPA) hydrolysis assay. Analysis of cell free supernatant by gas chromatography-mass spectrometry (GC-MS) revealed that 1,4-butanediol and adipic acid monomers were produced. Bacillus subtilis strain MZA-75 can degrade the soft segment of polyester polyurethane, unfortunately no information about the fate of hard segment could be obtained. Growth of strain MZA-75 in the presence of these metabolites indicated mineralization of ester hydrolysis products into CO2 and H2O.

Genetic characterization of human echinococcosis in Southern Punjab, Pakistan.

Introduction: Echinococcosis is a neglected tropical zoonotic infection that affects both the human and livestock populations. In Pakistan, the infection is long-standing, but data on its molecular epidemiology and genotypic characterization in the southern Punjab region are limited. The aim of the current study was the molecular characterization of human echinococcosis in southern Punjab, Pakistan. Methods: Echinococcal cysts were obtained from a total of 28 surgically treated patients. Patients' demographic characteristics were also recorded. The cyst samples were subjected to further processing to isolate DNA in order to probe the Nad1 and Cyt-b genes, followed by DNA sequencing and phylogenetic analysis for genotypic identification. Results: The majority of the echinococcal cysts were from male patients (60.7%). The liver was the most commonly infected organ (60.71%), followed by the lungs (25%), spleen (7.14%), and the mesentery (7.14%). Molecular and genotypic identification through sequencing and phylogenetic tree analysis showed that most of the cysts (24/28, 85.7%) were caused by the species Echinococcus granulosus sensu stricto (E. granulosus s.s.) (G1 and G3), followed by Echinococcus multilocularis (E. multilocularis) and Echinococcus canadensis (E. canadensis) (G6/G7) (3/28, 10.8%, and 1/28, 3.5%, respectively). Conclusion: The current study concluded that the majority of human infections were caused by E. granulosus s.s., followed by the E. multilocularis and E. canadensis species (G6/G7). Genotypic characterization among both human and livestock populations is needed to explore the genetic diversity of echinococcosis.