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This study examines the fermentation of autohydrolyzed and 1-n-butyl-3-methylimidazolium acetate (C4mimOAc) pretreated sugarcane bagasse, using four different yeast strains to determine the efficiency of bioethanol production. Three strains of Saccharomyces cerevisiae (S. cerevisiae) and one of Scheffersomyces stipitis (S. stipitis) were employed in this study. It was observed that the sugarcane bagasse autohydrolyzed at 205 °C for 6 min with subsequent enzymatic hydrolysis exhibited the maximum ethanol yield of 70.92 ± 0.09 mg/g-substrate when S. cerevisiae MZ-4 was used. However, a slightly higher ethanol yield of 78.78 ± 0.94 mg/g-substrate was obtained from C4mimOAc pretreated bagasse employing S. cerevisiae MZ-4. The study showed that the newly isolated MZ-4 strain exhibited better ethanol yield as compared to commercially available yeast strains S. cerevisiae Uvaferm-43, S. cerevisiae Lalvin EC-1118, and S. stipitis.

Cultureable bacterial diversity of previously unexplored Siachen glacier, Pakistan, was studied. Out of 50 isolates 33 (66%) were Gram negative and 17 (34%) Gram positive. About half of the isolates were pigment producers and were able to grow at 4-37°C. 16S rRNA gene sequences revealed Gram negative bacteria dominated by Proteobacteria (especially γ-proteobacteria and β-proteobacteria) and Flavobacteria. The genus Pseudomonas (51.51%, 17) was dominant among γ- proteobacteria. β-proteobacteria constituted 4 (12.12%) Alcaligenes and 4 (12.12%) Janthinobacterium strains. Among Gram positive bacteria, phylum Actinobacteria, Rhodococcus (23.52%, 4) and Arthrobacter (23.52%, 4) were the dominating genra. Other bacteria belonged to Phylum Firmicutes with representative genus Carnobacterium (11.76%, 2) and 4 isolates represented 4 genera Bacillus, Lysinibacillus, Staphylococcus and Planomicrobium. Most of the Gram negative bacteria were moderate halophiles, while most of the Gram positives were extreme halophiles and were able to grow up to 6.12 M of NaCl. More than 2/3 of the isolates showed antimicrobial activity against multidrug resistant S. aureus, E. coli, Klebsiella pneumonia, Enterococcus faecium, Candida albicans, Aspergillus flavus and Aspergillus fumigatus and ATCC strains. Gram positive bacteria (94.11%) were more resistant to heavy metals as compared to Gram negative (78.79%) and showed maximum tolerance against iron and least tolerance against mercury.

The degradation activities of bacterium, Roseateles depolymerans TB-87 and its depolymerases Est-H and Est-L against aliphatic as well as aliphatic–aromatic co-polyesters, were investigated. Strain TB-87 and its enzymes exhibited an ability to degrade aliphatic and aliphatic–aromatic co-polyesters. Monomers produced as a result of degradation of aliphatic polyesters [poly(butylene succinate) (PBS), poly(butylene succinate-co-adipate) (PBSA)] as well as aliphatic–aromatic co-polyester [poly(butylene succinate/terephthalate/isophthalate)-co-(lactate) (PBSTIL) by depolymerases Est-H and Est-L were investigated by liquid chromatography mass-spectrometry (LC-MS). Some common monomers like succinic acid and 1,4-butanediol were detected besides adipic acid and terephthalic/isophthalic acids as degradation products from PBSA and PBSTIL, respectively, whereas lactic acid was not detected. The succinic acid monomer was detected prior to adipic acid as a result of degradation of PBSA. The enzymes depolymerized PBS also into respective monomers. The analysis of PBSTIL degradation products revealed that enzymes easily degraded aliphatic segments as compared to aromatic segments and resulted in production of succinic acid prior to terephthalic and isophthalic acid. On the basis of these results, we speculate that both the enzymes Est-H and Est-L, attacked succinic acid segments (BS) first instead of adipic acid (BA) and terephthalic/isophthalic acid (BT or BI) segments of PBSA and PBSTIL, respectively. It is concluded from the results that R. depolymerans strain TB-87 can depolymerize aliphatic as well as aliphatic–aromatic co-polyesters; therefore, its enzymes can be applied in the process of biochemical monomer recycling.

Since polymeric materials do not decompose easily, disposal of waste polymers is a serious environmental concern. Widespread studies on the biodegradation of rubbers have been carried out in order to overcome the environmental problems associated with rubber waste. This report provides an overview on the microbial degradation of natural and synthetic rubbers. Rubber degrading microbes, bacteria and fungi, are ubiquitous in the environment especially soil. The qualitative data like plate assay, scanning electron microscopy (SEM), attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) and Sturm test indicated that both natural and synthetic rubbers can be degraded by microorganisms. It has confirmed that the enzymes latex clearing protein (Lcp) and rubber oxygenase A (RoxA) are responsible for the degradation of natural and synthetic rubbers. Lcp was obtained from Gram-positive bacterium Streptomyces sp. strain K30 and RoxA from Gram-negative bacterium Xanthomonas sp. strain 35Y. Analysis of degradation products of natural and synthetic rubbers indicated the oxidative cleavage of double bonds in polymer backbone. Aldehydes, ketones and other carbonyl groups were detected as degradation products from cultures of various rubber degrading strains. This review emphasizes the importance of biodegradation in environmental biotechnology for waste rubber disposal.

A poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) degrading bacterial strain designated as AF-111 was isolated from sewage sludge sample. The bacterium was identified by 16S rRNA gene sequencing. The results revealed that strain AF-111 showed 99 % similarity with Streptomyces althioticus strain NRRL B-3981 and designated as Streptomyces sp. strain AF-111. An extracellular PHBV depolymerase enzyme was produced under optimized conditions and purified through ammonium sulphate fractionation and column chromatography. The enzyme was purified to homogeneity, indicated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and molecular weight was found to be approximately 51 kDa. Effect of temperature, pH, metal ions and inhibitors on the PHBV depolymerase activity was determined. The enzyme was stable at wide range of temperature (35–55 °C) and pH (6–8). PHBV depolymerase was stable in the presence of different metal ions except iron and zinc which had inhibitory effect on depolymerase activity. Both ethylenediamine teteracetic acid and phenylmethyl sulphonyl fluoride strongly inhibited enzyme activity which indicates that this enzyme belongs to the serine hydrolase family like other polyhydroxyalkanoate depolymerases. The results show that a depolymerase from strain AF-111 can effectively degrade PHBV, therefore, it can be applied in the process of biochemical monomer recycling.

A novel aliphatic-aromatic co-polyester degrading bacterium designated as strain TB-87 was isolated from freshwater. Strain TB-87 has been characterized as aliphatic-aromatic co-polyester, poly[(butylene succinate/terephthalate/isophthalate)- co - (lactate)] (PBSTIL)-degrading microorganism since it grows efficiently and forms clear zones on PBSTIL emulsified NB agar plates. The bacterium was identified through 16S rRNA gene sequencing; it was completely matched with Roseateles depolymerans type strain. PBSTIL depolymerase was purified to homogeneity from R. depolymerans strain TB-87, by column chromatography. This strain produced both high and low molecular weight PBSTIL depolymerases with sizes of approximately 31 and 27 kDa, respectively, as determined by SDS-PAGE. The enzymes utilized p -nitrophenyl caproate (C 6 ) as preferred substrate among various p -NP acyl esters, indicating that these are a type of esterases, therefore, designated as Est-H (high) and Est-L (low). The peptide mapping as a result of limited proteolysis, indicated that the enzymes Est-H and Est-L have different pattern of peptide fragments from each other. The enzymes showed activity against PBSTIL, both in emulsified as well as in film form, however, the activity of Est-H was found higher than Est-L. These enzymes were found stable at 20–40 °C and pH 6.0–11.0. Both Est-H and Est-L were capable of degrading other aliphatic as well as aliphatic-aromatic co-polyesters like poly(butylene succinate) (PBS), poly(butylene succinate)- co -(butylene adipate) (PBSA), poly(e-caprolactone)(PCL), and poly(butylene succinate)- co -(butylene terephthalate) (PBST). It is concluded from the results that enzymes from strain TB-87 can be applied in the process of biochemical monomer recycling.

In this report, a polyester polyurethane (PU) degrading bacterium, designated as strain MZA-85, was isolated from soil through enrichment. The bacterium was identified through 16S rRNA gene sequencing; it was completely matched with Pseudomonas aeruginosa type strain. The degradation of PU film pieces by P. aeruginosa strain MZA-85 was investigated by scanning electron microscopy (SEM), Fourier transformed infra-red spectroscopy (FT-IR) and gel permeation chromatography (GPC). SEM micrographs of PU film pieces, treated with strain MZA-85, revealed changes in the surface morphology. FTIR spectrum showed increase in organic acid functionality and corresponding decrease in ester functional group. GPC results revealed increase in polydispersity, which shows that long chains of polyurethane polymer are cleaved into shorter chains by microbial action. The bacterium was found to produce cell associated esterases based on p -Nitrophenyl acetate ( p NPA) hydrolysis assay. 1,4-Butanediol and adipic acid monomers were detected by gas chromatography–mass spectrometry (GC–MS), which were produced as a result of hydrolysis of ester linkages in PU by cell bound esterases. Strain MZA-85 not only depolymerized PU but also mineralized it into CO 2 and H 2 O, as indicated by increase in cells growth in the presence of degradation products as well as detection of CO 2 evolution through Sturm test. From the results presented above, it is finally concluded that P. aeruginosa strain MZA-85, as well as its enzymes, can be applied in the process of biochemical monomerization for the pure monomers recycling.

The xenobiotic nature and lack of degradability of polymeric materials has resulted in vast levels of environmental pollution and numerous health hazards. Different strategies have been developed and still more research is being in progress to reduce the impact of these polymeric materials. This work aimed to isolate and characterize polyester polyurethane (PU) degrading fungi from the soil of a general city waste disposal site in Islamabad, Pakistan. A novel PU degrading fungus was isolated from soil and identified as Aspergillus tubingensis on the basis of colony morphology, macro- and micro-morphology, molecular and phylogenetic analyses. The PU degrading ability of the fungus was tested in three different ways in the presence of 2% glucose: (a) on SDA agar plate, (b) in liquid MSM, and (c) after burial in soil. Our results indicated that this strain of A. tubingensis was capable of degrading PU. Using scanning electron microscopy (SEM), we were able to visually confirm that the mycelium of A. tubingensis colonized the PU material, causing surface degradation and scarring. The formation or breakage of chemical bonds during the biodegradation process of PU was confirmed using Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) spectroscopy. The biodegradation of PU was higher when plate culture method was employed, followed by the liquid culture method and soil burial technique. Notably, after two months in liquid medium, the PU film was totally degraded into smaller pieces. Based on a comprehensive literature search, it can be stated that this is the first report showing A. tubingensis capable of degrading PU. This work provides insight into the role of A. tubingensis towards solving the dilemma of PU wastes through biodegradation.

Background & objectives Acinetobacter baumannii can cause a wide range of infections, including bacteremia, pneumonia, urinary tract infection, peritonitis, etc. This organism is becoming resistant to a large group of antibiotics, especially β-lactam antibiotics. The reason for multi-drug resistance may be the production of extended- spectrum β-lactamses (ESBLs), carbapenemases/metallo β-lactamases or AmpC β-lactamases. The aim of the present study was to determine the prevalence of multi-drug resistant Acinetobacter baumannii isolated from the patients in Surgical Intensive Care Units (SICUs) of Pakistan Institute of Medical Sciences (PIMS), Islamabad, Pakistan. Methods A total of 91 A. baumanni isolates were collected from PIMS during the period from February 2011 to December 2011. The antibiotic susceptibility testing was performed by standard disc diffusion method as recommended by CLSI. Combination disc method, Modified Hodge test, EDTA disc synergy test and AmpC disc test were performed for detection of ESBLs, carbapenemases, metallo β-lactamases, and AmpC β-lactamases, respectively. Results The prevalence of MDRs was reported 100% among A. baumannii. The antibiotic susceptibility profile showed that minocycline and tigecycline were the most effective drugs against A. baumannii. Almost all of A. baumannii isolates were carbapenemase and metallo β-lactamase producers. AmpC prevalence was observed in 41.76%, while none of the isolates was ESBL producer. Antibiogram and minimal inhibitory concentrations (MICs) indicated tetracycline is relatively effective against A. baumanii. Conclusions Increased frequency of multi-drug resistance supports the need for continuous surveillance to determine prevalence and evolution of these enzymes in Pakistan.

To determine the prevalence of cytomegalovirus in pregnant women and types of overt congenital infection in neonates.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.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 avidity50%. The remaining 12(23%) sera had avidity assay value50%. 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.Human cytomegalovirus infection in pregnant women was the main reason of congenital defects among neonates.

Efficient utilization of hemicellulose entails high catalytic capacity containing xylanases. In this study, proline rich sequence was fused together with a C-terminal of xylanase gene from Geobacillus thermodenitrificans C5 and designated as GthC5ProXyl. Both GthC5Xyl and GthC5ProXyl were expressed in Escherichia coli BL21 host in order to determine effect of this modification. The C-terminal oligopeptide had noteworthy effects and instantaneously extended the optimal temperature and pH ranges and progressed the specific activity of GthC5Xyl. Compared with GthC5Xyl, GthC5ProXyl revealed improved specific activity, a higher temperature (70 degrees C versus 60 degrees C) and pH (8 versus 6) optimum, with broad ranges of temperature and pH (60-80 degrees C and 6.0-9.0 versus 40-60 degrees C and 5.0-8.0, respectively). The modified enzyme retained more than 80% activity after incubating in xylan for 3 h at 80 degrees C as compared to wild type with only 45% residual activity. Our study demonstrated that proper introduction of proline residues on C-terminal surface of xylanase family might be very effective in improvement of enzyme thermostability. Moreover, this study reveals an engineering strategy to improve the catalytic performance of enzymes. (C) 2016 Elsevier Inc. All rights reserved.

The purpose of this study was to isolate the poly (3-hydroxybutyrate- co -3-hydroxyvalerate) (PHBV) degrading microorganisms from soil, and the analysis of degradation by scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) spectroscopy. After soil burial for 120 days at 37 °C, a new actinobacterial strain, Actinomadura sp. AF-555, was isolated from the surface of PHBV film, through an enrichment technique. The increase in biomass on the surface of PHBV film pieces and in the medium after culturing in basal salt medium indicated that it utilizes PHBV as a source of carbon and energy. Scanning electron microscopy of PHBV film at the end of incubation showed many changes in surface morphology, such as erosion and extensive roughening of the surface with pit formation, as compared to the untreated plastic pieces. The FTIR spectra of PHBV film showed a decrease in the peak from 1725 cm −1 (untreated plastic film) to 1721 cm −1 , and disappearance of a peak present in controls, at 2745 cm −1 , indicating the breakdown of ester (>C O) or O–R groups and –C–H bonds, respectively. The intensity of the band at 1185 cm −1 , the amorphous state-sensitive band, increased, indicating an increase in 3HV content. It can be concluded that soil contains microorganisms that can potentially be used for biodegradation of plastic wastes.

Microbial lipases have a great potential due to the rapid development of enzyme technology and their industrial applications. Lipase was produced by Bacillus sp. FH5 and partially purified by acetone precipitation. It was immobilized on silica gel. Lipolytic activity of the enzyme in free as well as immobilized state was compared. Lipolytic activity was determined by using p-nitrophenyl laurate as substrate and measuring the absorbance at 420 nm. Lipolytic activity was higher in immobilized crude enzyme as compared to that of free-state. A sharp decrease in lipolytic activity was observed in case of free and immobilized crude enzyme at 50 and 55°C, respectively. The lipase activity of both free and immobilized crude extracts was inhibited when incubated with serine reagent PMSF and SDS. Both the free and immobilized crude lipase forms were found to have a stimulatory effect in the presence of Mg+2, Fe+3, and Ca+2, whereas K+, Cu+, Na+2, Zn+2, and Ni(NO3)2 showed an inhibitory effect on the activity of free as well as immobilized crude lipase. Maximum lipolytic activity was found in the presence of 20% ethanol both in the case of free and immobilized crude lipase.

The present study describes the isolation of bacteria from soil with the ability to degrade plastic polyurethane (PU). Bacterial strains attached on the polyurethane film, after soil burial for 6 months, were isolated and identified asBacillus sp. AF8,Pseudomonas sp AF9,Micrococcus sp. 10,Arthrobacter sp. AF11, andCorynebacterium sp. AF12. In plate assay, zones of hydrolysis were visualised around the bacterial colonies on mineral salt agar plates containing polyurethane as a sole carbon source. The results of the Sturm test for degradability showed more CO2 production in the test than in control. The production of esterase was detected in the presence of polyurethane as a substrate. The Scanning Electron Microscopy and Fourier Transform Infrared Spectroscopy showed certain changes on the surface of PU film and formation of some new intermediate products after polymer breakdown.

Lack of degradability and the closing of landfill sites as well as growing water and land pollution problems have led to concern about plastics. With the excessive use of plastics and increasing pressure being placed on capacities available for plastic waste disposal, the need for biodegradable plastics and biodegradation of plastic wastes has assumed increasing importance in the last few years. Awareness of the waste problem and its impact on the environment has awakened new interest in the area of degradable polymers. The interest in environmental issues is growing and there are increasing demands to develop material which do not burden the environment significantly. Biodegradation is necessary for water-soluble or water-immiscible polymers because they eventually enter streams which can neither be recycled nor incinerated. It is important to consider the microbial degradation of natural and synthetic polymers in order to understand what is necessary for biodegradation and the mechanisms involved. This requires understanding of the interactions between materials and microorganisms and the biochemical changes involved. Widespread studies on the biodegradation of plastics have been carried out in order to overcome the environmental problems associated with synthetic plastic waste. This paper reviews the current research on the biodegradation of biodegradable and also the conventional synthetic plastics and also use of various techniques for the analysis of degradation in vitro.

High levels of extracellular xylanase activity (994.50 IU/ml) produced by Geobacillus thermodenitrificans C5 originated gene was detected when it was expressed in E. coli BL21 host. Thermostable xylanase (GthC5Xyl) was purified to homogeneity and showed a molecular mass of approximately 44 kDa according to SDS-PAGE. The specific activity of the purified GthC5Xyl was up to 1243.125IU/mg with a 9.89-fold purification. The activity of GthC5Xyl was stimulated by CoCl2, MnSO4, CuSO4, MnCl2 but was inhibited by FeSO4, Hg, CaSO4. GthC5Xyl showed resistant to SDS, Tween 20, Triton X-100, beta-Mercaptoethanol, PMSF, DTT, NEM and DEPC, SDS, and EDTA. A greater affinity for oat spelt xylan was exhibited by GthC5Xyl with maximum enzymatic activity at 60 degrees C and 6.0 pH. The activity portrayed by GthC5Xyl was found to be acellulytic with stability at high temperature (70 degrees C-80 degrees C) and low pH (4.0 to 8.0). Xylobiose and xylopentose were the end products of proficient oat spelt xylanase hydrolysis by GthC5Xyl. High SDS resistance and broader stability of GthC5Xyl proves it to be worthy of impending application in numerous industrial processes especially textile, detergents and animal feed industry.

Background Drug resistant tuberculosis (DR-TB) is a major public health problem in developing countries such as Pakistan. Objective The current study was conducted to assess the frequency of drug resistant tuberculosis including multi drug resistance (MDR- TB) as well as risk factors for development of DR-TB, in Punjab, Pakistan. Methodology Drug susceptibility testing (DST) was performed, using proportion method, for 2367 culture positive Mycobacterium tuberculosis (MTB) cases that were enrolled from January 2012 to December 2013 in the province of Punjab, Pakistan, against first-line anti-tuberculosis drugs. The data was analyzed using statistical software; SPSS version 18. Results Out of 2367 isolates, 273 (11.5%) were resistant to at least one anti-TB drug, while 221 (9.3%) showed MDR- TB. Risk factors for development of MDR-TB were early age (ranges between 10–25 years) and previously treated TB patients. Conclusion DR-TB is a considerable problem in Pakistan. Major risk factors are previous history of TB treatment and younger age group. It emphasizes the need for effective TB control Program in the country.

Streptoverticillium kashmirense AF1 with the ability to degrade a natural polymer, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) was isolated from municipal sewage sludge by soil burial technique. The PHBV film was degraded by the action of extracellular enzymes secreted by the microorganisms. Degradation of PHBV was evident by the formation of clear zones of hydrolysis on the polymer containing mineral salt agar plates. The extent of PHBV degradation increased up to 30 days of incubation. Maximum production of PHBV depolymerase was observed both at pH 8 and pH 7, 45 °C, 1% substrate concentration and in the presence of lactose as an additional carbon source. Two types of extracellular PHBV depolymerases were purified fromS. kashmirense AF1 by gel permeation chromatography using Sephadex G-75. The molecular weights of the two proteins were found to be 35 and 45 kDa approximately, as determined by SDS-PAGE. The results of the Sturm test also showed more CO2 production as a result of PHBV degradation, in the test as compared to control. The present findings indicated the degradation capabilities ofS. kashmirense AF1.

Lipases are a class of enzymes which catalyze the hydrolysis of long-chain triglycerides. Microbial lipases are currently receiving much attention with the rapid development of enzyme technology. Bacillus subtilis FH5, isolated from tannery wastes, produced a thermostable alkalophilic lipase and was purified to homogeneity as judged by SDS-PAGE. The purification steps included acetone fractionation and sequential column chromatography on DEAE-cellulose, Sephadex G-75 and adsorption chromatography on Hydroxylapatite. The results of chromatographies showed that two types of lipases were present having molecular weights approximately 62 kDa and 24 kDa, respectively. The purified enzyme was found to be 100% stable at pH 10 and about 80% residual activity was present at 60 degrees C. The enzyme was found to be stable in the presence of Mg2+, Mn2+ and Ca2+ ions. Km value was calculated as 5.05 mM and Vmax as 0.416 micromol/ml/min. Bacillus subtilis FH5 was isolated from tannery waste, therefore, enzyme is environmentally compatible for application in leather degreasing process.

This work was focused on the possibility of accelerating the biodegradation process of low density polyethylene (LDPE). Comparative studies, between the properties of untreated LDPE pieces and those exposed to UV irradiation and nitric acid plus microbial culture treatment, were performed. The LDPE pieces were irradiated by UV light (254 nm wavelength) for 250 h and incubated with nitric acid (99.0%) at 80°C for 6 days. These pretreated LDPE pieces were used as sole carbon source in mineral salt medium and inoculated with the Fusarium sp. AF4, isolated from soil. The efficiency of the LDPE degradation depended on the growth phase in pure cultivation of the fungus. The changes in the structural properties of LDPE film because of UV-nitric acid and microbial treatment were determined by Fourier transform infrared spectroscopy (FTIR). It was observed that in case of UV and nitric acid treated LDPE, peaks appeared at 1710 cm−1 and 831, which were then reduced to 1708 cm−1 and 830 after microbial treatment, indicating breakdown of polymer chain. In this study, it was observed that a synergistic effect of UV-nitric acid and microbial treatment induced oxidation reaction that enhanced and accelerated the biodegradability rate of LDPE pieces. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci 2007

The contribution of bacteria, isolated from sewage sludge, towards the microbial degradation of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) was studied. A microbial consortium was inoculated on mineral salt agar plates containing different concentrations of PHBV as a substrate. The PHBV degraders showed optimum depolymerase production at pH 7.0, 37 °C, and 0.4% substrate concentration during 2–5 weeks of incubation. Additional carbon sources like glucose, fructose, sucrose, lactose and nitrogen sources including yeast extract were used as co-metabolites. The highest degree of degradation of PHBV was observed in the presence of glucose. Among the surfactants, Tween 80, was found to be a good inducer for the release of extracellular depolymerase enzyme as compared to Tween 20. When the PHBV depolymerase activity of isolates present in the consortium was determined separately, it was found that the enzyme activity was higher in case of Bacillus sp. AF3. The purification steps included ammonium sulfate fractionation and gel permeation chromatography on Sephadex G-75. The enzyme has been purified to homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and molecular weight was found to be approximately 37 kDa.

Background Congenital cytomegalovirus (cCMV) infection contributes to considerable long-term sequelae in neonates and children all over the world. The association between viral genotypes and severity of clinical cytomegalovirus (CMV) infection is yet to be defined. The objective of this study was to find the impact of active CMV infection during pregnancy and the clinical significance of genotypes in neonates with congenital cytomegalovirus infections in Pakistan. Methods A total of 409 blood samples from pregnant women seeking health care services at the two antenatal hospitals of Islamabad during January to December 2012 were tested by ELISA and nested-PCR. Pregnant women with active infection (detected as IgM positive, PCR positive or positive on both assays) were followed until delivery, to detect the outcome of overt cCMV infection in neonates. Genetic characterization of CMV strains was performed by sequence analysis of envelope glycoproteins: gB, gN and gH to detect the contributing CMV genotypes. Results The seroprevalence of anti-CMV IgG and IgM was 97.5% (399 out of 409) and 12.7% (52 out of 409), respectively, while 20% (82/409) pregnant women were found positive for CMV DNA by PCR. Logistic regression analysis showed a significant association of active infection with parity [OR = 2.56, 95% CI = 1.82–2.62, p = 0.04], febrile illness [OR = 1.84, 95% CI = 1.76–3.65, p = 0.01] and jaundice [OR = 22.5, 95% CI = 4.53–85.02, p = 0.002]. We were able to isolate virus in 41 out of 70 neonates; 36.6% (15 out of 41) of them were symptomatic at birth while 63.4% (26 out of 41) were asymptomatic. The most prominent clinical feature observed in symptomatic neonates was hepatosplenomegaly (26.6%; 4 out of 15). All three genotypes gB, gN and gH were found with the highest frequency of gB1 genotype, found in 75% infants with hepatic damage. Phylogenetic analysis of Pakistani strains showed 96%-100% homology to their prototype strains. Conclusions Active CMV infection during pregnancy is a major cause of congenital CMV infection with comparable distribution of all three genotypes: gB, gN and gH in symptomatic and asymptomatic neonates. Our findings emphasize to conduct a comprehensive large scale survey and introduction of country wide routine screening at maternity clinics for early diagnosis of CMV to reduce its associated devastating outcomes.

Lipases are a class of enzymes, which catalyse the hydrolysis of long chain triglycerides. Microbial lipases are currently receiving much attention with the rapid development of enzyme technology. Lipases have industrial potential in the chemical, pharmaceutical, medical, cosmetic, leather and paper manufacturing industries, biosurfactant synthesis, and agrochemicals. ABacillus strain isolated from soil was tested for the production of extracellular lipase, by batch culturing in shake flask. The growth conditions were optimised for the maximum production of enzyme. Various parameters for the production of lipase, such as temperature, incubation period, pH, carbon source, nitrogen source and lipids were studied. Maximum lipase production was found in 48-h-old culture filtrate at 37 °C, pH 8.0. Among all the carbon sources, salicin gave the maximum activity and among all the nitrogen sources yeast extract gave maximum production/activity. Tween (20 and 80) does not stimulate the growth much but assisted in enzyme production.

Lipases are a class of enzymes which catalyse the hydrolysis of long chain triglycerides. Microbial lipases are currently receiving much attention with the rapid development of enzyme technology. Lipases constitute the most important group of biocatalysts for biotechnological applications. This review describes various industrial applications of microbial lipases in the detergent, food, flavour industry, biocatalytic resolution of pharmaceuticals, esters and amino acid derivatives, making of fine chemicals, agrochemicals, use as biosensor, bioremediation and cosmetics and perfumery.

Beta-lactam antimicrobial agents represent the most common treatment for bacterial infections and continue to be the leading cause of resistance to beta-lactam antibiotics among Gram-negative bacteria worldwide. The persistent exposure of bacterial strains to a multitude of beta-lactams has induced dynamic and continuous production and mutation of beta-lactamases in these bacteria, expanding their activity even against the newly developed beta-lactam antibiotics. These enzymes are known as extended-spectrum beta-lactamases (ESBLs). The majority of ESBLs are derived from the widespread broad-spectrum beta-lactamases TEM-1 and SHV-1. There are also new families of ESBLs, including the CTX-M and OXA-type enzymes as well as novel unrelated beta-lactamases. In recent years, there has been an increased incidence and prevalence of ESBLs. ESBLs are mainly found in strains of Escherichia coli and Klebsiella pneumoniae but have also been reported in other Enterobacteriaceae strains and Pseudomonas aeruginosa. Infections with ESBL-producing bacterial strains are encountered singly or in outbreaks, especially in critical care units in hospitals, resulting in increasing cost of treatment and prolonged hospital stays. Not only may nursing home patients be an important reservoir of ESBL-containing multiple antibiotic-resistant organisms, but ambulatory patients with chronic conditions may also harbor ESBL-producing organisms.

Beta-lactamases of Enterobacteriaceae are the most important mechanism of resistance against beta-lactam drugs. Two types of beta-lactamases can confer resistance against 3rd generation cephalosporins. Chromosomally mediated beta-lactamases are inducible and are not inhibited by clavulanic acid. Resistance due to these enzymes is non-transferable. The 2nd type of enzyme is plasmid-mediated beta-lactamases, which are inhibited by clavulanic acid. These enzymes are more important clinically as these can be transferred between various species of Enterobacteriaceae. These enzymes are called extended-spectrum beta-lactamases (ESBLs). ESBL-producing Enterobacteriaceae have been responsible for numerous outbreaks of infection throughout the world and pose challenging infection control issues. Antibacterial choice is often complicated by multi-resistance. ESBLs can confer resistance against all beta-lactam drugs except carbapenems and cephamycins. Nursing home patients may be an important reservoir of ESBL-containing multiple antibiotic-resistant organisms. Use of broad-spectrum oral antibiotics and probably poor infection control practices may facilitate spread of this plasmid-mediated resistance. In addition to known populations at risk, ambulatory patients with chronic conditions represent another patient population that may harbor ESBL-producing organisms. Various methods can be used for detection of ESBLs in the laboratory. These tests include double disc diffusion test, Vitek ESBL test, E Tests, MIC Determination, genetic method, and isoelectric focusing (IEF).

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(e-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.

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.

Three psychrotrophic bacteria, morpho-physiologically, identified as Bacillus subtilis MRLBA7, Bacillus licheniformis MRLBA8 and Bacillus megaterium MRLBA9 were isolated from -20°C freezer of the Microbiology Research Laboratory (MRL), Quaid-i-Azam University, Islamabad, Pakistan. These strains were able to grow aerobically at 6°C but not at 40°C except MRLBA8 that could grow at 48°C. None of the isolates showed inhibition of growth in the presence of glycerol. Isolate MRLBA7, bearing central spore, grew in the presence of 30% glycerol at 0°C after 48 h of incubation and showed maximum growth without glycerol at 25°C after 24 h. Isolate MRLBA8 showed growth in the presence of 50% glycerol at 4°C after 72 h of incubation and maximum growth was observed at 20°C in the absence ofglycerol. Isolate MRLBA9 showed growth at 6°C in the presence of 40% glycerol after 48 h of incubation and maximum growth was observed at 25°C in the absence of glycerol. Isolates were susceptible to antibiotics except Bacillus subtilis MRLBA7 that exhibited antibiotic resistance against penicillin and fosphomycin, Bacillus licheniformis MRLBA8 against aztreonam and fosphomycin, and Bacillus megaterium MRLBA9 against vancomycin and penicillin. The growth profile and biochemical characteristics of all the isolates were rather similar to that of mesophilic counterparts except adaptation to low temperature. These strains could be used as model microbial strains for characterization of food contaminants in freezers, to understand the mechanism of antibiotic resistanceinduced at low temperature and as a source of psychrotrophic enzymes.

Endophytic fungi are known for the production of valuable metabolites, but information on the gibberellin production capacity of this group is limited. We isolated 9 endophytic fungi from the roots of salt-stressed soybean plants and screened them on waito-c rice, in order to identify plant growth promoting fungal strains. The fungal isolate TK- 2-4 gave maximum plant length (20.35 cm) promotion in comparison with wild-type Gibberella fujikuroi (19.5 cm). In a separate experiment, bioassay of TK-2-4 promoted plant length and biomass of soybean cultivar Taegwangkong. The TK-2-4 culture filtrate was analyzed for the presence of gibberellins, and it was found that all physiologically active gibberellins, especially GA(4) and GA(7), were present in higher amounts (GA(1), 0.11 ng/ml; GA(3), 2.91 ng/ml; GA(4), 3.21 ng/ml; and GA(7), 1.4 ng/ml) in conjunction with physiologically inactive GA(9) (0.05 ng/ml), GA(12) (0.23 ng/ ml), GA(15) (0.42 ng/ml), GA(19) (0.53 ng/ml), and GA(20) (0.06 ng/ml). The fungal isolate TK-2-4 was later identified as a new strain of Phoma herbarum, through the phylogenetic analysis of 28S rDNA sequence.

Microbial lipases are very prominent biocatalysts because of their ability to catalyze a wide variety of reactions in aqueous and non-aqueous media. The chemo-, regio- and enantio-specific behaviour of these enzymes has caused tremendous interest among scientists and industrialists. Lipases from a large number of bacterial, fungal and a few plant and animal sources have been purified to homogeneity. This article presents a critical review of different strategies which have been employed for the detection, purification and characterization of microbial lipases.

A new bacterial strain, capable of degrading poly(3-hydroxybutyrate) (PHB) was isolated from soil. This organism, identified as Streptoverticillium kashmirense AF1, secreted PHB depolymerases both on solid as well as in liquid mineral salt medium containing poly(3-hydroxybutyrate) as sole carbon source. The optimum production of PHB depolymerase was observed at pH 8 and 7, at 45 degrees C, 1% substrate concentration and in the presence of lactose as an additional carbon source. The extracellular PHB depolymerase was purified by gel permeation chromatography using Sephadex G-75. The Streptoverticillium kashmirense AF1 produced two types of PHB depolymerases having molecular weights of about 37 and 45 kDa as determined by SDS-PAGE. The difference in dry cell mass and amount of CO2 evolved in the test and control calculated gravimetrically through Sturm test indicated the degradative capabilities of Streptoverticillium kashmirense AF1.

To determine the prevalence and routes of transmission of hepatitis C virus (HCV) infection in Hafizabad, Pakistan, we collected sera in 1993 from a geographically based random sample of residents, and in 1994 identified 15 HCV-infected individuals (cases) and 67 age and sex matched uninfected individuals (controls). Initially we approached 504 households, and collected serum from a randomly selected household member in 309 (64%). Twenty persons (6.5%) had anti-HCV antibody; 31% percent had hepatitis B core antibodies, and 4.3% had hepatitis B surface antigen. In the case-control study, persons who received more therapeutic injections (categorized as averaging 1, 2-4, 5-9 or10 injections per year in the previous 10 years) were more likely to be infected with HCV (odds ratio 0, 1.5, 2.5 and 6.9 respectively, P = 0.008) compared to persons averaging 0 injections per year. Efforts to limit therapeutic injections to only those that are medically indicated and that use sterile equipment are essential in order to prevent transmission of HCV.To determine the prevalence of hepatitis C virus (HCV) infection in Hafizabad, Pakistan, sera were collected in 1993 from a geographically representative random sample of 309 adults and children. 20 serum samples (6.5%) had anti-HCV antibodies, 89 (31%) had hepatitis B core antibodies, and 12 (4.3%) had hepatitis B surface antigens. The prevalence of HCV increased markedly with age; no cases were detected among children under 10 years old. A follow-up study of 15 of these HCV cases and 67 age- and sex-matched healthy controls was conducted in 1994. Compared to noninfected controls, HCV cases were significantly more likely to have received 5 or more injections in the previous 10 years (odds ratio, 5.4; 95% confidence interval, 1.2-28.0). Only 5 HCV cases had received blood transfusions, and none reported intravenous drug use. Inspection of 3 private health clinics in Hafizabad exposed disposable syringes and needles--used primarily for vitamin B complex, chloroquine, and penicillin--soaking in a bowl of tepid water. Extrapolation of this study's findings suggests 800 new cases of HCV and 109 HCV-related deaths in Hafizabad each year. HCV infection could be reduced substantially by efforts to limit injections to those that are medically essential and to ensure the use of sterile equipment.

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) degrading thermophilic fungus was isolated from soil sample collected from waste disposal site, Islamabad, Pakistan. It was able to grow efficiently on a medium containing PHBV as a sole source of carbon and has been identified as Aspergillus sp. NA-25 by 18S rRNA. Using 9% of inoculum maximum production of PHBV depolymerase was observed at 45 degrees C, pH 7.0 in the presence of 0.2% lactose as an additional carbon source. PHBV depolymerase was purified by precipitation with 80% ammonium sulfate and gel filtration chromatography on Sephadex G-75. The four enzyme forms obtained after gel filtration were analyzed on SDS-PAGE and their molecular weights (36, 68, 72 and 90 kDa) were determined. They were characterized on the basis of effect of different temperatures, pH, metal ions and different reagents on the PHBV activity and stability. It is obvious that the fungal strain Aspergillus sp. NA-25 is capable of degrading PHBV with the help of different types of depolymerases.

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 %).

Various microorganisms were screened for their ability to degrade polyisoprene rubber (natural rubber latex gloves). Strain AF-666, newly isolated from a soil sample, was selected as the best strain having the ability to grow on polyisoprene containing plates. The strain identified as Bacillus sp. AF-666, was found to degrade polyisoprene rubber, both on basal agar plates (latex overlay) as well as in liquid medium. Qualitative analysis of degradation was done through scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) spectroscopy SEM showed changes in surface morphology, like appearance of pits and cracks, and marked difference in transmittance spectra of test and control due to changes in the functional groups, was detected through FTIR. CO2 evolution as a result of rubber degradation, was calculated gravimetrically by Sturm Test. About 4.43 g/1 of CO2 was produced in case of test, whereas, 1.57 g/1 in case of control. The viable number of cells (CFU/ml) was also higher in test than in control. Present study may provide an opportunity for further studies on the applications of biotechnological processes as a tool for rubber waste management.