Resistance Enzymes Research Articles

Inositol hexakisphosphate biosynthesis underpins PAMP-triggered immunity to Pseudomonas syringae pv. tomato in Arabidopsis thaliana but is dispensable for establishment of systemic acquired resistance.

Phytic acid (inositol hexakisphosphate, InsP 6) is an important phosphate store and signal molecule necessary for maintenance of basal resistance to plant pathogens. Arabidopsis thaliana (‘arabidopsis’) has three genes encoding myo‐inositol phosphate synthases (IPS1–3), the enzymes that catalyse conversion of glucose‐6‐phosphate to InsP, the first step in InsP 6 biosynthesis. There is one gene for inositol‐(1,3,4,5,6)‐pentakisphosphate 2‐kinase (IPK1), which catalyses the final step. Previously, we showed that mutation of IPS2 and IPK1 but not IPS1 increased susceptibility to pathogens. Our aim was to better understand the InsP 6 biosynthesis pathway in plant defence. Here we found that the susceptibility of arabidopsis (Col‐0) to virulent and avirulent Pseudomonas syringae pv. tomato was also increased in ips3 and ips2/3 double mutants. Also, ipk1 plants had compromised expression of local acquired resistance induced by treatment with the pathogen‐derived molecular pattern (PAMP) molecule flg22, but were unaffected in other responses to flg22, including Ca2+ influx and the oxidative burst, seedling root growth inhibition, and transcriptional up‐regulation of the PAMP‐triggered genes MITOGEN‐ACTIVATED PROTEIN KINASE (MPK) 3, MPK11, CINNAMYL ALCOHOL DEHYDROGENASE 5, and FLG22‐INDUCED RECEPTOR‐LIKE KINASE 1. IPK1 mutation did not prevent the induction of systemic acquired resistance by avirulent P. syringae. Also, ips2 and ips2/3 double mutant plants, like ipk1, were hypersusceptible to P. syringae but were not compromised in flg22‐induced local acquired resistance. The results support the role of InsP 6 biosynthesis enzymes in effective basal resistance and indicate that there is more than one basal resistance mechanism dependent upon InsP 6 biosynthesis.

Structural and functional characterization of three Type B and C chloramphenicol acetyltransferases from Vibrio species.

Chloramphenicol acetyltransferases (CATs) were among the first antibiotic resistance enzymes identified and have long been studied as model enzymes for examining plasmid-mediated antibiotic resistance. These enzymes acetylate the antibiotic chloramphenicol, which renders it incapable of inhibiting bacterial protein synthesis. CATs can be classified into different types: Type A CATs are known to be important for antibiotic resistance to chloramphenicol and fusidic acid. Type B CATs are often called xenobiotic acetyltransferases and adopt a similar structural fold to streptogramin acetyltransferases, which are known to be critical for streptogramin antibiotic resistance. Type C CATs have recently been identified and can also acetylate chloramphenicol, but their roles in antibiotic resistance are largely unknown. Here, we structurally and kinetically characterized three Vibrio CAT proteins from a nonpathogenic species (Aliivibrio fisheri) and two important human pathogens (Vibrio cholerae and Vibrio vulnificus). We found all three proteins, including one in a superintegron (V. cholerae), acetylated chloramphenicol, but did not acetylate aminoglycosides or dalfopristin. We also determined the 3D crystal structures of these CATs alone and in complex with crystal violet and taurocholate. These compounds are known inhibitors of Type A CATs, but have not been explored in Type B and Type C CATs. Based on sequence, structure, and kinetic analysis, we concluded that the V. cholerae and V. vulnificus CATs belong to the Type B class and the A. fisheri CAT belongs to the Type C class. Ultimately, our results provide a framework for studying the evolution of antibiotic resistance gene acquisition and chloramphenicol acetylation in Vibrio and other species.

Relationship between Serum Glypican-4 Level and Metabolic Parameters in Experimentally-Induced Non-Alcoholic Fatty Liver Disease in Adult Male Albino Rats

AbstractBackground: Glypican-4 (GPC4) is a novel adipokine thataffects insulin signaling, and adipocyte differentiation. Serum GPC4 level was associatedwith obesity-related param-eters such as hyperglycaemia, Insulin Resistance (IR), and elevated liver enzymes aspartate Amino-Transferase (AST), and alanine Amino-Transferase (ALT).Non-Alcoholic Fatty Liver Disease (NAFLD) has emerged as one of the manifestations of metabolic syndrome. It is usually associated with IR, dyslipidemia, and elevated AST & ALT levels.Few studies were performed to elucidate the role of GPC4 in the development of NAFLD in human, and its relation to other metabolic parameters. But, its exact role hasn't been clarified yet.Aim of Study: To estimate serum level of GPC4 in NAFLD induced by high fat diet in adult male albino rats, and to examine its relationship to other metabolic parameters.Material and Methods: A total number of 60 healthy adult male local strain albino rats were divided into 2 main groups: Control group (I) (n=30) were fed on standard chow, and was further subdivided according to the period of their nourishment on standard chowinto 3 equal subgroups (n=10), subgroup (IA), for 4 weeks, subgroup (IB), for 12 weeks, andsubgroup (IC), for 24 weeks High Fat Diet (HFD) fed group (II) (n=30) were fed on high fat chow, and was further subdivided accord-ing to the period of their nourishment on HFDinto 3 equal subgroups (n=10), subgroup (IIA): For 4 weeks, subgroup (IIB), for 12 weeks, andsubgroup (IIC), for 24 weeks. Body Mass Index (BMI), Abdominal Circumference (AC) were measured. Serum GPC4, glucose, insulin, ofhomeostasis model assessment (HOMA-IR)-index, Total Cholesterol (TC), Triglycerides (TG), High Density Lipoprotein (HDL), Low Density Lipoprotein (LDL), AST, ALT, and C-Reactive Protein (CRP) were estimated. Liver histopathology was studied.Results: GPC4 was positively correlated with BMI, AC, TC, TG, LDL, AST, ALT, and CRP in all HFD-fed rats groups. Concerning, IR and HOMA-IR, it was found that, GPC4 was positively correlated with both of them in HFD-fed rats for 4 weeks and 12 weeks groups, however, no correlations were reported between GPC4 and IR & HOMA-IR in HFD-fed rats for 24 weeks group.Conclusion: Serum GPC4 level was increased in HFD-fed rats for 4 weeks and 12 weeks groups as a compensatory mechanism to overcome IR, and was reduced in HFD-fed rats for 24 weeks group due to failure of compensation as a result of marked progress of IR.

Antifungal resistance profile and enzymatic activity of Candida species recovered from human and animal samples

Candida is currently the most implicated pathogenic fungal species recognized as the major cause of a variety of human infections all over the world. This study investigated species distribution, enzymatic activities, and antifungal resistance profiles of human and animal Candida species. Clinical Candida species (n=220) were isolated from urine, high vaginal swab (HVS) and blood while Candida species (n=128) were isolated from rectal swab, ear swab, blood, feces, and milk in animals: goat, sheep, cattle, pig and chicken. The identification of the species was performed using standard methods. Enzymatic activity was screened using plate methods. Susceptibility testing was carried out using disk diffusion and broth microdilution methods. A statistically significant difference (P=0.031) was observed in the distribution of Candida spp. recovered from humans and animals. The Pz values of human Candida species for proteinase, hemolysin, lipase and phospholipase were 0.65±0.97, 0.61±0.81, 0.59±0.47 and 0.76±0.74 respectively while that of Candida species recovered from animal were 0.67±0.13, 0.61±0.95, 0.62±0.67 and 0.69±0.70 respectively. No statistically significant difference (P>0.05) in the in vitro enzymatic activity was observed between the two groups. High azole-resistance rate was observed. Resistance was higher among human Candida isolates compared to animal isolates although the difference was not considered statistically significant (p = 0.519). Our findings suggest that the enzymatic activity (virulence potential) and resistance patterns are similar in the two groups investigated. This study underscores the importance of animals especially pets and their products as potential sources/reservoirs of pathogenic and multi-azole resistant Candida species in Nigeria.

Differential physiological and biochemical responses of Quercus infectoria and Q. libani to drought and charcoal disease.

The vast oak-dominated forests of the Zagros Mountains in southwestern Iran currently undergo large-scale dieback driven by a combination of drought and increasing incidence of charcoal disease caused by the fungal pathogens Biscogniauxia mediterranea and Obolarina persica. Here, we explore the interactive effects between drought and charcoal disease agents on the physiology and biochemistry of Quercus infectoria and Quercus libani seedlings. The combination of pathogen attack and water limitation hampered plant development, especially in Q. libani seedlings, negatively affecting growth, biomass production, photosynthetic efficiency, and leaf water potential. An increase in markers of oxidative damage together with the upregulation of the antioxidant defense revealed that drought stress and pathogen infection led to pro-oxidative conditions in both oak species, especially in Q. libani, where larger changes in malondialdehyde and hydrogen peroxide occurred. The upregulation of the antioxidant system was more prominent in Q. infectoria than in Q. libani, resulting in enhanced enzyme activity and accumulation of non-enzymatic antioxidants. Fungal infection stimulated the activity of chitinase, phenylalanine ammonia lyase and β-1,3-glucanase in Q. infectoria leaves and this response became more pronounced under water shortage. Our study highlights that drought stress greatly intensifies the effects of the charcoal disease. Moreover, our findings imply superior stress resistance of Q. infectoria conferred by a highly efficient antioxidant system, strong osmotic adjustment (through proline), and increases in resistance enzymes and secondary metabolites (phenols and flavonoids). Future investigations should focus on adult trees in their natural habitat including interactions with soil factors and other pathogens like nematodes, bacteria and other fungi. Because the present research was conducted on oak seedlings, the findings can be considered by forest nursery managers.

Fine-tuning the properties of the thrombin binding aptamer through cyclization: Effect of the 5'-3' connecting linker on the aptamer stability and anticoagulant activity.

A small library of cyclic TBA analogues (named cycTBA I-IV), obtained by covalently connecting its 5'- and 3'-ends with flexible linkers, has been synthesized with the aim of improving its chemical and enzymatic stability, as well as its anticoagulant properties. Two chemical procedures have been exploited to achieve the desired cyclization, based on the oxime ligation method (providing cycTBA I and II) or on Cu(I)-assisted azide-alkyne cycloaddition (CuAAC) protocols (for cycTBA III and IV), leading to analogues containing circularizing linkers with different chemical nature and length, overall spanning from 22 to 48 atoms. The resulting cyclic TBAs have been characterized using a variety of biophysical methods (UV, CD, gel electrophoresis, SE-HPLC analyses) and then tested for their serum resistance and anticoagulant activity under in vitro experiments. A fine-tuning of the length and flexibility of the linker allowed identifying a cyclic analogue, cycTBA II, with improved anticoagulant activity, associated with a dramatically stabilized G-quadruplex structure (ΔTm = +17 °C) and a 6.6-fold higher enzymatic resistance in serum compared to unmodified TBA.

Characterisation of the β-lactam resistance enzyme in Acanthamoeba castellanii

β-Lactams are well known as the best antibiotics for inhibiting the cross-linking between adjacent polysaccharide chains and peptides in the peptidoglycan layer of bacterial cell walls, causing bacterial cell lysis. There are no reports on the action of and resistance mechanisms to β-lactams in protozoa. Acanthamoeba castellanii is a free-living protozoan pathogen capable of causing blinding keratitis and fatal granulomatous encephalitis. When Acanthamoeba is exposed to harsh conditions, it differentiates into the cyst stage to avoid environmental stresses, such as drug treatment. In this study, it was shown that the mature encystation rate of A. castellanii is decreased by treatment with cefotaxime (CTX) and clavulanic acid (CLA); however, the drugs do not kill the amoeba. We hypothesise that β-lactam antibiotics may disturb synthesis of the double cell wall during the encystation process of Acanthamoeba. Interestingly, CTX is considered a powerful β-lactam, whereas CLA is considered a weak β-lactam but an efficient β-lactamase inhibitor. It was demonstrated that Acanthamoeba expresses β-lactamases to prevent inhibition of the encystation process by β-lactams. To reveal the functions of Acanthamoeba β-lactamases, a recombinant Acanthamoeba β-lactamase was produced in Escherichia coli that conferred resistance to β-lactams such as CTX, cefuroxime, penicillin and meropenem. Consequently, we suggest that Acanthamoeba produces enzymes similar to β-lactamases to avoid interference from the environment. Here we provide a new point of view on an important gene responsible for drug resistance and advocate for the development of more efficient treatment against Acanthamoeba infection.

Distinguish metabolic profiles and defense enzymes in Alternaria leaf blight resistant and susceptible genotypes of groundnut.

Alternaria leaf blight is major fungal disease of summer groundnut, causes significant loss of haulm and pod yield. Aims of this study were to understand the role of metabolites and phenylpropanoid related enzymes in Alternaria leaf blight resistance and to find out metabolic marker for disease resistance. Alternaria leaf blight resistant (GPBD4 and CS186) and susceptible genotypes (GG2 and TPG41) of groundnut were grown in pots during rabi-summer 2015. Groundnut plants were infected with Alternaria alternata (Fr.) Keissler at 40days after sowing. 5days after infection, upper second leaves were collected from both control and infected plants for analysis. A total of 67 metabolites comprising sugars, sugar alcohols, amino acids, organic acids, fatty acids, sterols and phenolic were identified using gas chromatography-mass spectrometry (non-targeted metabolomics). Constitutive levels of alpha-d-galactoside, d-mannitol, d-erythropentitol, glycine, and hexadecanoic acid were observed higher in resistant genotypes compared to susceptible genotypes. Moreover, arabinofuranose, cinnamic acid, 2-butendioic acid, and linoleic acid were observed only in resistant genotypes at both control and infected stage. In susceptible genotypes myo-inositol, glucose and fructose content was increased after infection with pathogen while decreased in resistant genotypes. Resistant genotypes had higher constitutive level of cinnamic and salicylic acid compared to susceptible genotypes. Non-infected leaves of resistant genotypes also had higher activities of phenylalanine ammonia lyase and tyrosine ammonia lyase activities. Our results suggest that metabolites specifically present in resistant genotypes impart defense mechanism against Alternaria pathogen and can be used as bio-marker for screening of germplasm.

Toward Broad Spectrum Dihydrofolate Reductase Inhibitors Targeting Trimethoprim Resistant Enzymes Identified in Clinical Isolates of Methicillin Resistant Staphylococcus aureus

The spread of plasmid borne resistance enzymes in clinical Staphylococcus aureus isolates is rendering trimethoprim and iclaprim, both inhibitors of dihydrofolate reductase (DHFR), ineffective. Continued exploitation of these targets will require compounds that can broadly inhibit these resistance-conferring isoforms. Using a structure-based approach, we have developed a novel class of ionized nonclassical antifolates (INCAs) that capture the molecular interactions that have been exclusive to classical antifolates. These modifications allow for a greatly expanded spectrum of activity across these pathogenic DHFR isoforms, while maintaining the ability to penetrate the bacterial cell wall. Using biochemical, structural, and computational methods, we are able to optimize these inhibitors to the conserved active sites of the endogenous and trimethoprim resistant DHFR enzymes. Here, we report a series of INCA compounds that exhibit low nanomolar enzymatic activity and potent cellular activity with human selectivity against a panel of clinically relevant TMP resistant (TMPR) and methicillin resistant Staphylococcus aureus (MRSA) isolates.

Biocompatible green synthesized silver nanoparticles impact on insecticides resistant developing enzymes of dengue transmitted mosquito vector

The growing interest in nanomaterial-based products in various fields urged to study the influence of different nano-products on the ecosystem and on various insect pests. In this study, we evaluated insecticides resistant influenced important biochemical constituents of A. aegypti upon the exposure of green synthesized A. reticulata silver nanoparticle (Ar–AgNPs) for 24 h at their lethal threshold concentration. The AgNPs exposed and control mosquito larval homogenates of the protein content, acetylcholinesterase, specific detoxification enzymes α- and β-carboxylesterase and other anti-oxidant or effective insecticides resistant developed enzyme named glutathione S-transferase (GST) were studied. The larvicidal activity of AgNPs at their lethal threshold concentration (4.5 µg/ml) were monitored for 12 and 24 h and prompted time dependent mortality rate were 23.3 ± 0.57 and 46.6 ± 0.57% respectively. Further, the biochemical constituents of protein content of AgNPs exposed larval homogenates were quantitatively higher compared to the control, but, in the qualitative analysis of separated protein in the electrophoresed gel revealed, the expression protein was down regulated. The important esterase enzymes of acetylcholinesterase, α-and β-carboxylesterase and GST enzymes were significantly reduced upon the exposure of AgNPs compared to the control larval body homogenates respectively. In the qualitative analysis, isoenzyme of β-carboxylesterase expression level was also down regulated. Followed by these investigations, it is clearly showed that A. reticulata mediated synthesized AgNPs have the potent deleterious impact on fourth instar larvae of A. aegypti physiological system by inhibiting important esterase enzyme groups.

Biochemical mechanisms and molecular analysis of fenvalerate resistant population of Spodoptera litura (Fabricius)

Spodoptera litura (Fabricius) is an important polyphagous insect pest of several host plants worldwide and has developed resistance to many insecticides. The present study aimed at analysing the effect of selection with fenvalerate on a resistant field population from Sangrur, Punjab, India (SGR) through synergist and biochemical assays. Following selection with fenvalerate for 14 generations, the selected population (SGR-Sel) was analysed through bioassays to assess changes in levels of resistance, synergism and detoxification enzyme activities. Resistance ratio increased from 12.61 to 52.81 fold when compared to the laboratory susceptible strain (H-Sus). Synergism of piperonyl butoxide (6.19 fold) and triphenyl phosphate (5.88) was observed with SGR-Sel, which suggested the involvement of mainly monooxygenases (mixed function oxidases or MFOs) and esterases in resistance to fenvalerate. A strong positive correlation (r = 0.98) was observed between MFO activity and fenvalerate resistance in SGR-Sel, the activity was 4.26- and 1.84- fold that of H-Sus and SGR. Similarly, esterase activity was 3.70- and 1.73- fold that of H-Sus and SGR populations, with a strong positive correlation between esterases and fenvalerate resistance (r = 0.97). Alignment between COI sequences of SGR-Sel and SGR identified the existence of a single base/nucleotide polymorphism at position 421 represented by replacement of C in SGR with T in SGR-Sel. In the translated protein sequence, serine in SGR was replaced by phenylalanine in SGR-Sel population at position 139.

Stochastic Gene Expression Influences the Selection of Antibiotic Resistance Mutations

Bacteria can resist antibiotics by expressing enzymes that remove or deactivate drug molecules. Here, we study the effects of gene expression stochasticity on efflux and enzymatic resistance. We construct an agent-based model that stochastically simulates multiple biochemical processes in the cell and we observe the growth and survival dynamics of the cell population. Resistance-enhancing mutations are introduced by varying parameters that control the enzyme expression or efficacy. We find that stochastic gene expression can cause complex dynamics in terms of survival and extinction for these mutants. Regulatory mutations, which augment the frequency and duration of resistance gene transcription, can provide limited resistance by increasing mean expression. Structural mutations, which modify the enzyme or efflux efficacy, provide most resistance by improving the binding affinity of the resistance protein to the antibiotic; increasing the enzyme’s catalytic rate alone may contribute to resistance if drug binding is not rate limiting. Overall, we identify conditions where regulatory mutations are selected over structural mutations, and vice versa. Our findings show that stochastic gene expression is a key factor underlying efflux and enzymatic resistances and should be taken into consideration in future antibiotic research.

Population-dependent reproducible deviation from natural bread wheat genome in synthetic hexaploid wheat.

Sequence elimination is one of the main mechanisms that increases the divergence among homoeologous chromosomes after allopolyploidization to enhance the stability of recently established lineages, but it can cause a loss of some economically important genes. Synthetic hexaploid wheat (SHW) is an important source of genetic variation to the natural hexaploid wheat (NHW) genepool that has low genetic diversity. Here, we investigated the change between SHW and NHW genomes by utilizing a large germplasm set of primary synthetics and synthetic derivatives. Reproducible segment elimination (RSE) was declared if a large chromosomal chunk (>5cM) produced no aligned reads in more than five SHWs. RSE in five genomic regions was the major source of variation between SHW and NHW. One RSE eliminated almost the complete short arm of chromosome 1B, which contains major genes for flour quality, disease resistance and different enzymes. The occurrence of RSE was highly dependent on the choice of diploid and tetraploid parental lines, their ancestral subpopulation and admixture, e.g. SHWs derived from Triticum dicoccon or from one of two Aegilops tauschii subpopulations were almost free of RSE, while highly admixed parents had higher RSE rates. The rate of RSE in synthetic derivatives was almost double that in primary synthetics. Genome-wide association analysis detected four loci with minor effects on the occurrence of RSE, indicating that both parental lines and genetic factors were affecting the occurrence of RSE. Therefore, pre-pre-breeding strategies should be applied before introducing SHW into pre-breeding programs to ensure genomic stability and avoid undesirable gene loss.

New Trimodal Phenotypic Reporter of Extended-Spectrum β-Lactamase Activity.

Bacterial resistance to β-lactam antibiotics continues to grow as misadministration presents evolutionary pressure that drives bacteria to develop improved resistance enzymes. Known as extended-spectrum β-lactamases (ESBLs), these enzymes are capable of hydrolyzing advanced β-lactam antibiotics such as third-generation (and higher) cephalosporins. Phenotypic detection substrates can be used to rapidly identify a cultured patient sample prior to confirmation by more exhaustive but slower means, critically aiding in the antibiotic stewardship essential in maintaining the effectiveness of not only the cephalosporins but also indirectly the carbapenems, our last-resort β-lactams. To enhance the phenotypic detection arsenal, we have designed an ESBL detection substrate that releases a glucose molecule upon β-lactamase hydrolysis. Because many forms of detection for glucose exist, the substrate enables ESBL quantification via three modalities commonly found in the clinical laboratory: optical absorbance, for use with the most common microbiology platforms; fluorescence, for enhanced sensitivity; and electrochemistry, which offers the potential for integration into a hand-held platform similar to a personal glucometer. Moreover, we demonstrate that, as opposed to currently available phenotypic detection substrates, our new substrate is engineered to be resistant to older and narrower β-lactamases, thus enabling specific identification of newer and more dangerous ESBLs.

Cohnella sp. A01 laccase: thermostable, detergent resistant, anti-environmental and industrial pollutants enzyme

Laccase (EC 1.10.3.2; benzenediol; oxygen oxidoreductases) is a multi-copper oxidase that catalyzes the oxidation of phenols, polyphenols, aromatic amines, and different non-phenolic substrates with concomitant reduction of O2 to H2O. Enzymatic oxidation techniques have the potential of implementation in different areas of industrial fields. In this study, the Cohnella sp. A01 laccase gene was cloned into pET-26 (b+) vector and was transformed to E. coli BL21. Then it was purified using His tag affinity (Ni sepharose resin) chromatography. The estimated molecular weight was approximately 60 kDa using SDS-PAGE. The highest enzyme activity and best pH for 2,6-dimethoxyphenol (DMP) oxidation were recorded as 8 at 90 °C respectively. The calculated half-life and kinetic values including Km, Vmax, turn over number (kcat), and catalytic efficiency (kcat/Km) of the enzyme were 106 min at 90 °C and 686 μM, 10.69 U/ml, 20.3 S−, and 0.029 s−1 μM−1, respectively. The DMP was available as the substrate in all the calculations. Enzyme activity enhanced in the presence of Cu2+, NaCl, SDS, n-hexane, Triton X-100, tween 20, and tween 80, significantly. The binding residues were predicted and mapped upon the modeled tertiary structure of identified laccase. The remaining activity and structural properties of Cohnella sp. A01 laccase in extreme conditions such as high temperatures and presence of metals, detergents, and organic solvents suggest the potential of this enzyme in biotechnological and industrial applications. This process has been patented in Iranian Intellectual Property Centre under License No: 91325.

Investigation of Relative Development and Reproductivity Fitness Cost in Three Insecticide-Resistant Strains of Aedes aegypti from Thailand.

Knockdown resistance (kdr) and detoxification enzymes are major resistance mechanisms in insecticide-resistant Aedes aegypti throughout the world. Persistence of the resistance phenotype is associated with high fitness of resistance alleles in the absence of insecticide pressure. This study determined the relative fitness cost of three insecticide-resistant strains of Aedes aegypti—PMD, PMD-R, and UPK-R—and a hybrid under similar laboratory conditions in the absence of insecticide. The PMD strain is resistant to DDT with no kdr alleles; the PMD-R is resistant to DDT and permethrin with 1534C homozygous kdr alleles; and UPK-R is resistant to DDT, permethrin, and deltamethrin with 989P + 1016G homozygous alleles. The DDT-resistant PMD strain had the highest fitness compared with the two DDT/pyrethroid-resistant strains (PMD-R and UPK-R) and hybrid. Consistent fitness costs were observed in the DDT/pyrethroid-resistant strains and hybrid, including shorter wing length, reduced egg hatchability, shorter female lifespan, and shorter viability of eggs after storage, whereas no effect was observed on blood feeding rate. In addition, reduced egg production was observed in the PMD-R strain and prolonged developmental time was seen in the UPK-R strain. The corresponding hybrid that is heterozygous for kdr alleles was fitter than either of the homozygous mutant genotypes. This is in accordance with the high frequency of heterozygous genotypes observed in natural populations of Ae. aegypti in Chiang Mai city.

Limited distribution and mechanism of the TetX4 tetracycline resistance enzyme

Limited distribution and mechanism of the TetX4 tetracycline resistance enzyme

Probing the Robustness of Inhibitors of Tuberculosis Aminoglycoside Resistance Enzyme Eis by Mutagenesis.

Each year, millions of people worldwide contract tuberculosis (TB), the deadliest infection. The spread of infections with drug-resistant strains of Mycobacterium tuberculosis (Mtb) that are refractory to treatment poses a major global challenge. A major cause of resistance to antitubercular drugs of last resort, aminoglycosides, is overexpression of the Eis (enhanced intracellular survival) enzyme of Mtb, which inactivates aminoglycosides by acetylating them. We showed previously that this inactivation of aminoglycosides could be overcome by our recently reported Eis inhibitors that are currently in development as potential aminoglycoside adjunctive therapeutics against drug-resistant TB. To interrogate the robustness of the Eis inhibitors, we investigated the enzymatic activity of Eis and its inhibition by Eis inhibitors from three different structural families for nine single-residue mutants of Eis, including those found in the clinic. Three engineered mutations of the substrate binding site, D26A, W36A, and F84A, abolished inhibitor binding while compromising Eis enzymatic activity 2- to 3-fold. All other Eis mutants, including clinically observed ones, were potently inhibited by at least one inhibitor. This study helps position us one step ahead of Mtb resistance to Eis inhibitors as they are being developed for TB therapy.

Chrysin ameliorates nonalcoholic fatty liver disease in rats.

Nonalcoholic fatty liver disease (NAFLD) is regarded as the hepatic manifestation of the metabolic syndrome. It begins with the accumulation of fat in the liver (simple steatosis), which if untreated can progress to nonalcoholic steatohepatitis, cirrhosis, and hepatocellular carcinoma. Chrysin is a flavonoid present in bee propolis and many other plants. The objective of this study was to determine if chrysin can ameliorate NAFLD induced by feeding a high fructose diet (HFD) in rats. The rats were divided into five groups: normal control, HFD control, chrysin (25, 50, and 100mg/kg p.o. body weight). Biochemical estimations were carried out in the serum and liver of rats. The gene expressions of SREBP-1c and PPAR α were determined in the liver. The histopathology of the liver was also studied. Chrysin caused a significant decrease in the serum fasting glucose and improved the insulin resistance, dyslipidemia, and liver enzymes. It caused a significant decrease in the liver weight and hepatic free fatty acids, triglyceride, and cholesterol content. Chrysin exerted antioxidant effects, reduced carbonyl content, advanced glycation end products, collagen, TNF-α, and IL-6 concentrations in the liver. Chrysin significantly reduced the hepatic gene expression of SREBP-1c and increased that of PPAR-α. The histopathology of liver of rats treated with chrysin showed significant decrease in the steatosis, ballooning, and lobular inflammation when compared to the HFD control group. Thus, chrysin demonstrated anti-steatotic, antiglycating, antioxidant, anti-inflammatory, and antifibrotic effects and seems to be a promising molecule for the management of NAFLD.

Cooccurrence of NDM-1, ESBL, RmtC, AAC(6')-Ib, and QnrB in Clonally Related Klebsiella pneumoniae Isolates Together with Coexistence of CMY-4 and AAC(6')-Ib in Enterobacter cloacae Isolates from Saudi Arabia.

The aim of this study was to investigate the mechanisms responsible for resistance to antimicrobials in a collection of enterobacterial isolates recovered from two hospitals in Saudi Arabia. A total of six strains isolated from different patients showing high resistance to carbapenems was recovered in 2015 from two different hospitals, with four being Klebsiella pneumoniae and two Enterobacter cloacae. All isolates except one K. pneumoniae were resistant to tigecycline, but only one K. pneumoniae was resistant to colistin. All produced a carbapenemase according to the Carba NP test, and all were positive for the EDTA-disk synergy test for detection of MBL. Using PCR followed by sequencing, the four K. pneumoniae isolates produced the carbapenemase NDM-1, while the two E. cloacae isolates produced the carbapenemase VIM-1. Genotyping analysis by Multilocus Sequence Typing (MLST) showed that three out of the four K. pneumoniae isolates were clonally related. They had been recovered from the same hospital and belonged to Sequence Type (ST) ST152. In contrast, the fourth K. pneumoniae isolate belonged to ST572. Noticeably, the NDM-1-producing K. pneumoniae additionally produced an extended-spectrum ß-lactamase (ESBL) of the CTX-M type, together with OXA-1 and TEM-1. Surprisingly, the three clonally related isolates produced different CTX-M variants, namely, CTX-M-3, CTX-M-57, and CTX-M-82, and coproduced QnrB, which confers quinolone resistance, and the 16S rRNA methylase RmtC, which confers high resistance to all aminoglycosides. The AAC(6′)-Ib acetyltransferase was detected in both K. pneumoniae and E. cloacae. Mating-out assays using Escherichia coli as recipient were successful for all isolates. The blaNDM-1 gene was always identified on a 70-kb plasmid, whereas the blaVIM-1 gene was located on either a 60-kb or a 150-kb plasmid the two E. cloacae isolates, respectively. To the best of our knowledge, this is the first report of the coexistence of an MBL (NDM-1), an ESBL (CTX-M), a 16S rRNA methylase (RmtC), an acetyltransferase (AAC[6′]-Ib), and a quinolone resistance enzyme (QnrB) in K. pneumoniae isolates recovered from different patients during an outbreak in a Saudi Arabian hospital.