Laboratory Isolates Research Articles

Identifying Recrudescent Plasmodium falciparum in Treated Malaria Patients by Real-time PCR and High Resolution Melt Analysis of Genetic Diversity

Recurrent parasitaemia during follow up of clinical trials of antimalarial drug efficacy results from either recrudescence of parasites surviving treatment or from parasites newly emerging from the hepatic stage of infection. Nested PCR is used to distinguish these two possibilities and the technique is difficult to standardise. There is risk of both false positive and false negative results, leading to misclassification errors. The high-resolution melt (HRM) assay was developed with pairs of conserved primers targeting blocks of merozoite surface protein 1 and 2 (msp1 and msp2) genes, and polymorphisms were compared using sequence-confirmed Plasmodium falciparum DNA samples from laboratory isolates. In this study, the HRM dissociation profiles of msp1 and msp2 amplicons were determined and validated against parasite isolates from malaria patients. The msp1 and msp2 profiles of both laboratory and clinical isolates were reproducibly differentiated by HRM. These rapid assays are performed in a closed-tube system, and so avoid cross-contamination while increasing throughput, which are two major advantages. The HRM assays offer significant gains in simplicity, speed and interpretation of results, and reduced analysis cost, for studies that require discrimination of parasite clones. Assay performance in large-scale studies utilizing DNA samples derived from filter-paper bloodspots should now be evaluated.

In vitro activity of fluralaner and commonly used acaricides against Dermanyssus gallinae isolates from Europe and Brazil

BackgroundThe poultry red mite Dermanyssus gallinae negatively impacts bird welfare and health, and interferes with egg production and quality, while emerging acaricide resistance limits control options. Fluralaner, a novel miticide for administration in drinking water, is approved for control of D. gallinae infestations. Mite sensitivity testing is relevant to gauge field isolate susceptibility to available treatments.MethodsThirteen D. gallinae isolates collected during 2014 through 2016 from farms in Germany, France, Spain and Brazil, and a 2001 laboratory-maintained isolate were used for acaricide contact sensitivity testing. Tested compounds were cypermethrin, deltamethrin, phoxim, propoxur, and the recently available acaricides, spinosad and fluralaner. In each study, at least one isolate was exposed to increasing concentrations of at least one acaricide. In one study, additional testing determined the sensitivity of the 2001 isolate to fluralaner using a mite-feeding test, and of fluralaner, phoxim and spinosad using an immersion test. At least two replicates were used for each dilution. Vehicle and untreated controls were also included.ResultsBased on 90% mortality (LC90) values, the laboratory isolate was susceptible to fluralaner (15.6–62.5 parts per million, ppm), phoxim (< 500 ppm), propoxur (< 125 ppm), and deltamethrin (500–1000 ppm). All field isolates remained sensitive to fluralaner concentrations ≤ 125 ppm. Spinosad LC90 values for laboratory and field isolates ranged between 2000–4000 ppm. For phoxim, relative to the laboratory isolate, there was reduced sensitivity of two German isolates (LC90 up to 4000 ppm) and two French isolates (> 4000 ppm). An isolate from Spain demonstrated reduced sensitivity to phoxim, propoxur and deltamethrin; an isolate from Brazil showed reduced sensitivity to propoxur and cypermethrin. Mite LC90 when exposed to fluralaner by blood feeding was < 0.1 ppm.ConclusionsContact sensitivity testing indicated apparent resistance to at least one of phoxim, deltamethrin, cypermethrin and propoxur in 13 field isolates from Europe and Brazil. All isolates were highly susceptible to fluralaner. Fluralaner was approximately 1000 times more active by feeding than by contact. Fluralaner’s distinct mode of action and efficacy against isolates largely refractory to those acaricides, makes it a promising option for the control of D. gallinae infestations of poultry.

The effect of &lt;i&gt;Piper&lt;/i&gt; &lt;i&gt;betle&lt;/i&gt; against &lt;i&gt;Candida&lt;/i&gt; &lt;i&gt;albicans&lt;/i&gt; adherence to denture acrylic surfaces

Antimicrobial activity of medicinal plants has been identified since ancient times, and has been used to cure bacterial and fungal diseases. Piper betle is an important medicinal plant with its medicinal properties extending to antimicrobial activity against several pathogenic fungi and bacteria. The objective of this study was to evaluate the effect of P. betle leaf extract on Candida albicans adhesion on denture acrylics. Young betel leaves (1 st - 3 rd leaf) were subjected to vacuum infiltration with ethanol. Anticandidal activity of a range of concentrations of the leaf extract (5,000 – 10,000 ppm) was assessed against C. albicans (ATCC 90028) and seven laboratory isolates using the agar well bioassay. Further, the same concentrations were used to test their ability to suppress the adhesion of C. albicans on denture acrylics, as compared to a commercial denture cleanser (positive control), and sterile distilled water (negative control) using an adhesion assay. The results indicated that sterile distilled water had the highest adhesion (23.75 ±5.3 cells per unit area) while both, the leaf extract of concentrations of 8,000 – 10,000 ppm and the commercial denture cleanser showed significant (p C. albicans adhesion on denture acrylics. There was no significant difference between adhesion in an 8000 ppm concentration extract of P. betle and the commercial denture cleanser of equal concentration, indicating that betel leaf extract is as efficient as the commercial denture cleanser, in suppressing the adhesion of C. albicans on denture acrylic surfaces

Malaria infection in mosquitoes decreases the personal protection offered by permethrin-treated bednets

BackgroundInsecticides targeting adult mosquitoes are the main way of controlling malaria. They work not only by killing mosquitoes, but also by repelling and irritating them. Indeed their repellent action gives valuable personal protection against biting mosquitoes. In the context of malaria control this personal protection is especially relevant when mosquitoes are infectious, whereas to protect the community we would prefer that the mosquitoes that are not yet infectious are killed (so, not repelled) by the insecticide. As the infectious stage of malaria parasites increases the motivation of mosquitoes to bite, we predicted that it would also change their behavioural response to insecticides.ResultsWith two systems, a laboratory isolate of the rodent malaria Plasmodium berghei infecting Anopheles gambiae and several isolates of P. falciparum obtained from schoolchildren in Tanzania that infected Anopheles arabiensis, we found that mosquitoes harbouring the infectious stage (the sporozoites) of the parasite were less repelled by permethrin-treated nets than uninfected ones.ConclusionsOur results suggest that, at least in the laboratory, malaria infection decreases the personal protection offered by insecticide-treated nets at the stage where the personal protection is most valuable. Further studies must investigate whether these results hold true in the field and whether the less effective personal protection can be balanced by increased community protection.

PH-responsive delivery of Griffithsin from electrospun fibers

Human immunodeficiency virus (HIV-1) affects over 36 million people globally. Current prevention strategies utilize antiretrovirals that have demonstrated protection, but result in antiviral resistance, adverse toxicity, and require frequent administration. A novel biologic, griffithsin (GRFT), has demonstrated outstanding safety and efficacy against laboratory and primary HIV isolates and against intravaginal murine herpes simplex virus 2 (HSV-2) challenge, making it a promising microbicide candidate. However, transient activity and instability remain concerns surrounding biologic delivery, particularly in the harsh environment of the female reproductive tract (FRT). Recently, electrospun fibers (EFs) have demonstrated promise for intravaginal delivery, with the potential to conserve active agent until release is needed. The goal of this study was to fabricate and characterize pH-responsive fibers comprised of poly(lactic-co-glycolic acid) (PLGA) or methoxypolyethylene glycol-b-PLGA (mPEG-PLGA) with varying ratios of poly(n-butyl acrylate-co-acrylic acid) (PBA-co-PAA), to selectively release GRFT under pH-conditions that mimic semen introduction. Fibers comprised of mPEG-PLGA:PBA-co-PAA (90:10 w/w) demonstrated high GRFT loading that was maintained within simulated vaginal fluid (SVF), and pH-dependent release upon exposure to buffered and SVF:simulated semen solutions. Moreover, GRFT fibers demonstrated potent in vitro efficacy against HIV-1 and safety in vaginal epithelial cells, suggesting their future potential for efficacious biologic delivery to the FRT.

Leishmania naiffi and Leishmania guyanensis reference genomes highlight genome structure and gene evolution in the Viannia subgenus.

The unicellular protozoan parasite Leishmania causes the neglected tropical disease leishmaniasis, affecting 12 million people in 98 countries. In South America, where the Viannia subgenus predominates, so far only L. (Viannia) braziliensis and L. (V.) panamensis have been sequenced, assembled and annotated as reference genomes. Addressing this deficit in molecular information can inform species typing, epidemiological monitoring and clinical treatment. Here, L. (V.) naiffi and L. (V.) guyanensis genomic DNA was sequenced to assemble these two genomes as draft references from short sequence reads. The methods used were tested using short sequence reads for L. braziliensis M2904 against its published reference as a comparison. This assembly and annotation pipeline identified 70 additional genes not annotated on the original M2904 reference. Phylogenetic and evolutionary comparisons of L. guyanensis and L. naiffi with 10 other Viannia genomes revealed four traits common to all Viannia: aneuploidy, 22 orthologous groups of genes absent in other Leishmania subgenera, elevated TATE transposon copies and a high NADH-dependent fumarate reductase gene copy number. Within the Viannia, there were limited structural changes in genome architecture specific to individual species: a 45 Kb amplification on chromosome 34 was present in all bar L. lainsoni, L. naiffi had a higher copy number of the virulence factor leishmanolysin, and laboratory isolate L. shawi M8408 had a possible minichromosome derived from the 3’ end of chromosome 34. This combination of genome assembly, phylogenetics and comparative analysis across an extended panel of diverse Viannia has uncovered new insights into the origin and evolution of this subgenus and can help improve diagnostics for leishmaniasis surveillance.

Finished Genome Sequence of a Polyurethane-Degrading Pseudomonas Isolate.

ABSTRACTPseudomonas sp. strain WP001 is a laboratory isolate capable of polyurethane polymer degradation and harbors a predicted lipase precursor gene. The genome of strain WP001 is 6.15 Mb in size and is composed of seven scaffolds with a G+C content of 60.54%. Strain WP001 is closely related to Pseudomonas fluorescens based on ribosomal DNA comparisons.

Effect of atmospheric carbon dioxide concentration on the cultivation of bovine Mycoplasma species

Recommendations for bovine mycoplasma culture CO2 concentrations are varied and were not empirically derived. The objective of this study was to determine whether the growth measures of bovine mycoplasma isolates differed when incubated in CO2 concentrations of 10 or 5% or in candle jars (2.7 ± 0.2% CO2). Growth of Mycoplasma bovis (n = 22), Mycoplasma californicum (n = 18), and other Mycoplasma spp. (n = 10) laboratory isolates was evaluated. Isolate suspensions were standardized to approximately 108 cfu/mL and serially diluted in pasteurized whole milk to achieve test suspensions of 102 and 106 cfu/mL. One hundred microliters of each test dilution was spread in duplicate onto the surface of a modified Hayflick's agar plate. Colony growth was enumerated on d 3, 5, and 7 of incubation. A mixed linear model included the fixed effects of CO2 treatment (2.7, 5, or 10%), species, day (3, 5, or 7), and their interactions, with total colony counts as the dependent variable. Carbon dioxide concentration did not significantly affect overall mycoplasma growth differences, but differences between species and day were present. Colony counts (log10 cfu/mL) of M. bovis were 2.6- and 1.6-fold greater than M. californicum and other Mycoplasma spp., respectively. Growth at 7 d of incubation was greater than d 3 and 5 for all species. These findings were confirmed using field isolates (n = 98) from a commercial veterinary diagnostic laboratory. Binary growth responses (yes/no) of the field isolates were not different between CO2 treatments but did differ between species and day of incubation. On average, 57% of all field isolates were detected by 3 d of incubation compared with 93% on d 7. These results suggest that the range of suitable CO2 culture conditions and incubation times for the common mastitis-causing Mycoplasma spp. may be broader than currently recommended.

Variability in Disinfection Resistance between Currently Circulating Enterovirus B Serotypes and Strains.

The susceptibility of waterborne viruses to disinfection is known to vary between viruses and even between closely related strains, yet the extent of this variation is not known. Here, different enteroviruses (six strains of coxsackievirus B5, two strains of coxsackievirus B4 and one strain of coxackievirus B1) were isolated from wastewater and inactivated by UV254, sunlight, free chlorine (FC), chlorine dioxide (ClO2), and heat. Inactivation kinetics of these isolates were compared with those of laboratory enterovirus strains (CVB5 Faulkner and echovirus 11 Gregory) and MS2 bacteriophage. FC exhibited the greatest (10-fold) variability in inactivation kinetics between different strains, whereas inactivation by UV254 differed only subtly. The variability in inactivation kinetics was greater between serotypes than it was among the seven strains of the CVB5 serotype. MS2 was a conservative surrogate of enterovirus inactivation by UV254, sunlight, or heat but frequently underestimated the disinfection requirements for FC and ClO2. Similarly, laboratory strains did not always reflect the inactivation behavior of the environmental isolates. Overall, there was considerable variability in inactivation kinetics among and within enteroviruses serotypes, as well as between laboratory and environmental isolates. We therefore recommend that future disinfection studies include a variety of serotypes and environmental isolates.

Meropenem potentiation of aminoglycoside activity against Pseudomonas aeruginosa: involvement of the MexXY-OprM multidrug efflux system.

To assess the ability of meropenem to potentiate aminoglycoside (AG) activity against laboratory and AG-resistant cystic fibrosis (CF) isolates of Pseudomonas aeruginosa and to elucidate its mechanism of action. AG resistance gene deletions were engineered into P. aeruginosa laboratory and CF isolates using standard gene replacement technology. Susceptibility to AGs ± meropenem (at ½ MIC) was assessed using a serial 2-fold dilution assay. mexXY expression and MexXY-OprM efflux activity were quantified using quantitative PCR and an ethidium bromide accumulation assay, respectively. A screen for agents that rendered WT P. aeruginosa susceptible to a sub-MIC concentration of the AG paromomycin identified the carbapenem meropenem, which potentiated several additional AGs. Meropenem potentiation of AG activity was largely lost in a mutant lacking the MexXY-OprM multidrug efflux system, an indication that it was targeting this efflux system in enhancing P. aeruginosa susceptibility to AGs. Meropenem failed to block AG induction of mexXY expression or MexXY-OprM efflux activity, suggesting that it may be interfering with some MexXY-dependent process linked to AG susceptibility. Meropenem potentiated AG activity versus AG-resistant CF isolates, enhancing susceptibility to at least one AG in all isolates and susceptibility to all tested AGs in 50% of the isolates. Notably, meropenem potentiation of AG activity was linked to MexXY in some but not all CF isolates in which this was examined. Meropenem potentiates AG activity against laboratory and CF strains of P. aeruginosa, both dependent on and independent of MexXY, highlighting the complexity of AG resistance in this organism.

Proteomic analysis of extracellular vesicles from a Plasmodium falciparum Kenyan clinical isolate defines a core parasite secretome.

Many pathogens secrete effector molecules to subvert host immune responses, to acquire nutrients, and/or to prepare host cells for invasion. One of the ways that effector molecules are secreted is through extracellular vesicles (EVs) such as exosomes. Recently, the malaria parasite P. falciparum has been shown to produce EVs that can mediate transfer of genetic material between parasites and induce sexual commitment. Characterizing the content of these vesicles may improve our understanding of P. falciparum pathogenesis and virulence. Previous studies of P. falciparum EVs have been limited to long-term adapted laboratory isolates. In this study, we isolated EVs from a Kenyan P. falciparum clinical isolate adapted to in vitro culture for a short period and characterized their protein content by mass spectrometry (data are available via ProteomeXchange, with identifier PXD006925). We show that P. falciparum extracellular vesicles ( PfEVs) are enriched in proteins found within the exomembrane compartments of infected erythrocytes such as Maurer's clefts (MCs), as well as the secretory endomembrane compartments in the apical end of the merozoites, suggesting that these proteins play a role in parasite-host interactions. Comparison of this novel clinically relevant dataset with previously published datasets helps to define a core secretome present in Plasmodium EVs. P. falciparum extracellular vesicles contain virulence-associated parasite proteins. Therefore, analysis of PfEVs contents from a range of clinical isolates, and their functional validation may improve our understanding of the virulence mechanisms of the parasite, and potentially identify targets for interventions or diagnostics.

CRISPR/Cas9 System as a Valuable Genome Editing Tool for Wine Yeasts with Application to Decrease Urea Production.

An extensive repertoire of molecular tools is available for genetic analysis in laboratory strains of S. cerevisiae. Although this has widely contributed to the interpretation of gene functionality within haploid laboratory isolates, the genetics of metabolism in commercially-relevant polyploid yeast strains is still poorly understood. Genetic engineering in industrial yeasts is undergoing major changes due to Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas) engineering approaches. Here we apply the CRISPR/Cas9 system to two commercial “starter” strains of S. cerevisiae (EC1118, AWRI796), eliminating the CAN1 arginine permease pathway to generate strains with reduced urea production (18.5 and 35.5% for EC1118 and AWRI796, respectively). In a wine-model environment based on two grape musts obtained from Chardonnay and Cabernet Sauvignon cultivars, both S. cerevisiae starter strains and CAN1 mutants completed the must fermentation in 8–12 days. However, recombinant strains carrying the can1 mutation failed to produce urea, suggesting that the genetic modification successfully impaired the arginine metabolism. In conclusion, the reduction of urea production in a wine-model environment confirms that the CRISPR/Cas9 system has been successfully established in S. cerevisiae wine yeasts.

Dirofilaria immitis JYD-34 isolate: whole genome analysis

BackgroundMacrocyclic lactone (ML) anthelmintics are used for chemoprophylaxis for heartworm infection in dogs and cats. Cases of dogs becoming infected with heartworms, despite apparent compliance to recommended chemoprophylaxis with approved preventives, has led to such cases being considered as suspected lack of efficacy (LOE). Recently, microfilariae collected from a small number of LOE isolates were used as a source of infection of new host dogs and confirmed to have reduced susceptibility to ML in controlled efficacy studies using L3 challenge in dogs. A specific Dirofilaria immitis laboratory isolate named JYD-34 has also been confirmed to have less than 100% susceptibility to ML-based preventives. For preventive claims against heartworm disease, evidence of 100% efficacy is required by FDA-CVM. It was therefore of interest to determine whether JYD-34 has a genetic profile similar to other documented LOE and confirmed reduced susceptibility isolates or has a genetic profile similar to known ML-susceptible isolates.MethodsIn this study, the 90Mbp whole genome of the JYD-34 strain was sequenced. This genome was compared using bioinformatics tools to pooled whole genomes of four well-characterized susceptible D. immitis populations, one susceptible Missouri laboratory isolate, as well as the pooled whole genomes of four LOE D. immitis populations. Fixation indexes (FST), which allow the genetic structure of each population (isolate) to be compared at the level of single nucleotide polymorphisms (SNP) across the genome, have been calculated. Forty-one previously reported SNP, that appeared to differentiate between susceptible and LOE and confirmed reduced susceptibility isolates, were also investigated in the JYD-34 isolate.ResultsThe FST analysis, and the analysis of the 41 SNP that appeared to differentiate reduced susceptibility from fully susceptible isolates, confirmed that the JYD-34 isolate has a genome similar to previously investigated LOE isolates, and isolates confirmed to have reduced susceptibility, and to be dissimilar to the susceptible isolates.ConclusionsThese results provide additional evidence for the link between genotype and the reduced susceptibility phenotype observed in such isolates as JYD-34. Further work on other isolates showing reduced susceptibility to ML is required to demonstrate the value of genetic analysis in predicting the response to ML chemoprophylaxis. The authors suggest that genetic analysis may be useful in helping to interpret the results of in vivo efficacy testing of ML heartworm preventives against D. immitis isolates.

A re-assessment of gene-tag classification approaches for describing var gene expression patterns during human Plasmodium falciparum malaria parasite infections

PfEMP1 are variant parasite antigens that are inserted on the surface of Plasmodium falciparum infected erythrocytes (IE). Through interactions with various host molecules, PfEMP1 mediate IE sequestration in tissues and play a key role in the pathology of severe malaria. PfEMP1 is encoded by a diverse multi-gene family called var. Previous studies have shown that that expression of specific subsets of var genes are associated with low levels of host immunity and severe malaria. However, in most clinical studies to date, full-length var gene sequences were unavailable and various approaches have been used to make comparisons between var gene expression profiles in different parasite isolates using limited information. Several studies have relied on the classification of a 300 – 500 base-pair “DBLα tag” region in the DBLα domain located at the 5’ end of most var genes. We assessed the relationship between various DBLα tag classification methods, and sequence features that are only fully assessable through full-length var gene sequences. We compared these different sequence features in full-length var gene from six fully sequenced laboratory isolates. These comparisons show that despite a long history of recombination, DBLα sequence tag classification can provide functional information on important features of full-length var genes. Notably, a specific subset of DBLα tags previously defined as “group A-like” is associated with CIDRα1 domains proposed to bind to endothelial protein C receptor. This analysis helps to bring together different sources of data that have been used to assess var gene expression in clinical parasite isolates.

The response of human bacteria to static magnetic field and radiofrequency electromagnetic field.

Cell phones and electronic appliances and devices are inseparable from most people in modern society and the electromagnetic field (EMF) from the devices is a potential health threat. Although the direct health effect of a cell phone and its radiofrequency (RF) EMF to human is still elusive, the effect to unicellular organisms is rather apparent. Human microbiota, including skin microbiota, has been linked to a very significant role in the health of a host human body. It is important to understand the response of human skin microbiota to the RF-EMF from cell phones and personal electronic devices, since this may be one of the potential mechanisms of a human health threat brought about by the disruption of the intimate and balanced host-microbiota relationship. Here, we investigated the response of both laboratory culture strains and isolates of skin bacteria under static magnetic field (SMF) and RF-EMF. The growth patterns of laboratory cultures of Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus epidermidis under SMF were variable per different species. The bacterial isolates of skin microbiota from 4 subjects with different cell phone usage history also showed inconsistent growth responses. These findings led us to hypothesize that cell phone level RF-EMF disrupts human skin microbiota. Thus, the results from the current study lay ground for more comprehensive research on the effect of RF-EMF on human health through the human-microbiota relationship.

GRL-09510, a Unique P2-Crown-Tetrahydrofuranylurethane -Containing HIV-1 Protease Inhibitor, Maintains Its Favorable Antiviral Activity against Highly-Drug-Resistant HIV-1 Variants in vitro

We report that GRL-09510, a novel HIV-1 protease inhibitor (PI) containing a newly-generated P2-crown-tetrahydrofuranylurethane (Crwn-THF), a P2′-methoxybenzene, and a sulfonamide isostere, is highly active against laboratory and primary clinical HIV-1 isolates (EC50: 0.0014–0.0028 μM) with minimal cytotoxicity (CC50: 39.0 μM). Similarly, GRL-09510 efficiently blocked the replication of HIV-1NL4-3 variants, which were capable of propagating at high-concentrations of atazanavir, lopinavir, and amprenavir (APV). GRL-09510 was also potent against multi-drug-resistant clinical HIV-1 variants and HIV-2ROD. Under the selection condition, where HIV-1NL4-3 rapidly acquired significant resistance to APV, an integrase inhibitor raltegravir, and a GRL-09510 congener (GRL-09610), no variants highly resistant against GRL-09510 emerged over long-term in vitro passage of the virus. Crystallographic analysis demonstrated that the Crwn-THF moiety of GRL-09510 forms strong hydrogen-bond-interactions with HIV-1 protease (PR) active-site amino acids and is bulkier with a larger contact surface, making greater van der Waals contacts with PR than the bis-THF moiety of darunavir. The present data demonstrate that GRL-09510 has favorable features for treating patients infected with wild-type and/or multi-drug-resistant HIV-1 variants, that the newly generated P2-Crwn-THF moiety confers highly desirable anti-HIV-1 potency. The use of the novel Crwn-THF moiety sheds lights in the design of novel PIs.

Biodegradation of thermally treated high-density polyethylene (HDPE) by Klebsiella pneumoniae CH001.

Biodegradation of plastics, which are the potential source of environmental pollution, has received a great deal of attention in the recent years. We aim to screen, identify, and characterize a bacterial strain capable of degrading high-density polyethylene (HDPE). In the present study, we studied HDPE biodegradation using a laboratory isolate, which was identified as Klebsiella pneumoniae CH001 (Accession No MF399051). The HDPE film was characterized by Universal Tensile Machine (UTM), Fourier Transform Infrared Spectroscopy (FTIR), Scanning Electron Microscope (SEM), and Atomic Force Microscope (AFM) before and after microbial incubation. We observed that this strain was capable of adhering strongly on HDPE surface and form a thick biofilm, when incubated in nutrient broth at 30°C on 120rpm for 60days. UTM analysis showed a significant decrease in weight (18.4%) and reduction in tensile strength (60%) of HDPE film. Furthermore, SEM analysis showed the cracks on the HDPE surface, whereas AFM results showed an increase in surface roughness after bacterial incubation. Overall, these results indicate that K. pneumoniae CH001 can be used as potential candidate for HDPE degradation in eco-friendly and sustainable manner in the environment.

A re-assessment of gene-tag classification approaches for describing var gene expression patterns during human Plasmodium falciparum malaria parasite infections.

PfEMP1 are variant parasite antigens that are inserted on the surface of Plasmodium falciparum infected erythrocytes (IE). Through interactions with various host molecules, PfEMP1 mediate IE sequestration in tissues and play a key role in the pathology of severe malaria. PfEMP1 is encoded by a diverse multi-gene family called var. Previous studies have shown that that expression of specific subsets of var genes are associated with low levels of host immunity and severe malaria. However, in most clinical studies to date, full-length var gene sequences were unavailable and various approaches have been used to make comparisons between var gene expression profiles in different parasite isolates using limited information. Several studies have relied on the classification of a 300 - 500 base-pair "DBLα tag" region in the DBLα domain located at the 5' end of most var genes. We assessed the relationship between various DBLα tag classification methods, and sequence features that are only fully assessable through full-length var gene sequences. We compared these different sequence features in full-length var gene from six fully sequenced laboratory isolates. These comparisons show that despite a long history of recombination, DBLα sequence tag classification can provide functional information on important features of full-length var genes. Notably, a specific subset of DBLα tags previously defined as "group A-like" is associated with CIDRα1 domains proposed to bind to endothelial protein C receptor. This analysis helps to bring together different sources of data that have been used to assess var gene expression in clinical parasite isolates.

Reversion of High-level Mecillinam Resistance to Susceptibility in Escherichia coli During Growth in Urine

Mecillinam (amdinocillin) is a β-lactam antibiotic used to treat uncomplicated urinary tract infections (UTIs). We have previously shown that inactivation of the Escherichia coli cysB gene is the major cause of mecillinam resistance (MecR) in clinical isolates. In this study, we used different E. coli strains (laboratory and clinical isolates) that were MecR due to cysB mutations to determine how mecillinam susceptibility was affected during growth in urine compared to growth in the commonly used growth medium Mueller Hinton (MHB). We also examined mecillinam susceptibility when bacteria were grown in urine obtained from 48 different healthy volunteers. Metabolome analysis was done on the urine samples and the association between the mecillinam susceptibility patterns of the bacteria and urine metabolite levels was studied.Two major findings with clinical significance are reported. First, MecRE. coli cysB mutant strains (both laboratory and clinical isolates) were always more susceptible to mecillinam when grown in urine as compared to laboratory medium, with many strains showing complete phenotypic susceptibility in urine. Second, the degree of reversion to susceptibility varied between urine samples obtained from different individuals. This difference was correlated with osmolality such that in urine with low osmolality the MecR mutants were more susceptible to mecillinam than in urine with high osmolality.This is the first example describing conditional resistance where a genetically stable antibiotic resistance can be phenotypically reverted to susceptibility by metabolites present in urine. These findings have several important clinical implications regarding the use of mecillinam to treat UTIs. First, they suggest that mecillinam can be used to treat also those clinical strains that are identified as MecR in standard laboratory tests. Second, the results suggest that testing of mecillinam susceptibility in the laboratory ought to be performed in media that mimics urine to obtain clinically relevant susceptibility testing results. Third, these findings imply that changes in patient behavior, such as increased water intake or use of diuretics to reduce urine osmolality and increased intake of cysteine, might induce antibiotic susceptibility in an infecting MecRE. coli strain and thereby increase treatment efficiency.

Excretory/secretory products from two Fasciola hepatica isolates induce different transcriptional changes and IL-10 release in LPS-activated bovine "BOMA" macrophages.

Fasciola hepatica are trematodes that reside in the bile ducts of mammals. Infection causes US$3 billion in losses annually in animal production and is considered a zoonosis of growing importance. An under-represented area in F. hepatica research has been the examination of the different immunomodulatory abilities of various parasite isolates on the host immune system. In this paper, this issue was explored, with the bovine macrophage cell line "BOMA". The cells were matured by LPS treatment and stimulated with excretory/secretory antigens (ES) from two Fasciola hepatica isolates: a laboratory isolate "Weybridge" (Fh-WeyES) and a wild isolate (Fh-WildES). As expected, stimulation with antigen mixtures with highly similar compositions resulted in mild transcriptomic differences. However, there were significant differences in cytokine levels. Compared to Fh-WeyES, exposure to Fh-WildES upregulated 27 and downregulated 30 genes. Fh-ES from both isolates diminished the release of TNF-α, whereas only Fh-WildES decreased IL-10 secretion. Neither Fh-WeyES nor Fh-WildES had an impact on IL-12 release. Our results indicate that various isolates can have different immunomodulatory abilities and impacts on the bovine immune system.