Resistance Enzymes Research Articles

Structure-based design of haloperidol analogues as inhibitors of acetyltransferase Eis from Mycobacterium tuberculosis to overcome kanamycin resistance.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is a deadly bacterial disease. Drug-resistant strains of Mtb make eradication of TB a daunting task. Overexpression of the enhanced intracellular survival (Eis) protein by Mtb confers resistance to the second-line antibiotic kanamycin (KAN). Eis is an acetyltransferase that acetylates KAN, inactivating its antimicrobial function. Development of Eis inhibitors as KAN adjuvant therapeutics is an attractive path to forestall and overcome KAN resistance. We discovered that an antipsychotic drug, haloperidol (HPD, 1), was a potent Eis inhibitor with IC50 = 0.39 ± 0.08 μM. We determined the crystal structure of the Eis-haloperidol (1) complex, which guided synthesis of 34 analogues. The structure-activity relationship study showed that in addition to haloperidol (1), eight analogues, some of which were smaller than 1, potently inhibited Eis (IC50 ≤ 1 μM). Crystal structures of Eis in complexes with three potent analogues and droperidol (DPD), an antiemetic and antipsychotic, were determined. Three compounds partially restored KAN sensitivity of a KAN-resistant Mtb strain K204 overexpressing Eis. The Eis inhibitors generally did not exhibit cytotoxicity against mammalian cells. All tested compounds were modestly metabolically stable in human liver microsomes, exhibiting 30-60% metabolism over the course of the assay. While direct repurposing of haloperidol as an anti-TB agent is unlikely due to its neurotoxicity, this study reveals potential approaches to modifying this chemical scaffold to minimize toxicity and improve metabolic stability, while preserving potent Eis inhibition.

腐植酸浸种对低温胁迫下玉米幼苗抗氧化系统的影响

低温冷害是导致黑龙江省玉米产量不稳、品质不高的重要农业气象灾害，而关于低温胁迫下玉米抗氧化应激能力和程度的研究较少。采用清水为对照（CK），以腐植酸质量浓度为0.2%、0.4%、0.8%、1.2%、1.6%和2.0%浸种，研究了昼夜温度为15℃/8℃低温胁迫48 h下玉米幼苗的代表性抗氧化酶活性、抗坏血酸-谷胱甘肽（AsA-GSH）循环、系统抗性及碳氮代谢的变化，以明确腐植酸对玉米的耐低温胁迫响应机制及适宜的浸种浓度。结果表明：低温胁迫条件下腐植酸浸种主要受过氧化氢（H₂O₂）积累诱导，随着浸种浓度的增加，大多数抗氧化酶和系统抗性酶活性、AsA-GSH循环、碳氮代谢主要物质含量、根系活力和鲜重均呈单峰曲线分布，其中0.8%-2.0%浓度处理的叶片和根系可溶性蛋白含量分别显著增加了23.3%-34.9%和31.9%-89.6%；1.6%浓度处理的叶片和根系苯丙氨酸解氨酶活性分别显著增加了16.8%和38.8%，多酚氧化酶活性分别增加了27.7%和13.4%，抗坏血酸含量分别显著增加了62.6%和55.3%，脱氢抗坏血酸含量分别显著增加了31.9%和66.4%；0.8%浓度处理的叶片和根系过氧化氢酶活性分别显著增加了66.4%和108.1%。因此，腐植酸浸种处理的叶片H₂O₂含量较对照显著降低了18.7%-37.6%，根系H₂O₂含量显著降低了27.5%-49.7%。0.4%和0.8%浓度处理的根系鲜重较对照分别显著增加了85.2%和105.3%，单株鲜重分别显著增加了31.9%和40.8%。腐植酸0.4%-0.8%处理增强了AsA为核心的AsA-GSH循环系统的运转活性和系统抗性，提高了玉米幼苗的抗氧化应激能力，获得最大的根系活力和植株鲜重，是水培条件下较为适宜的浸种浓度。;Chilling damage in Heilongjiang Province is an important agricultural meteorological disaster that leads to unstable maize yield and low quality in this region. However, there are few studies on the ability and degree of antioxidant stress in maize under low-temperature stress. Mass concentration of humic acid were 0.2%, 0.4%, 0.8%, 1.2%, 1.6% and 2.0%, and water as the control, the changes of activities of representative antioxidant enzymes, ascorbic acid-glutathione (AsA-GSH) cycle, systemic resistance and carbon-nitrogen metabolism of maize seedlings under low-temperature stress of day and night for 48 h at 15℃/8℃ were studied to illustrate the tolerance mechanism of humic acid for low-temperature stress and the appropriate concentration of soaking seed. The results showed that soaking seed with humic acid was mainly induced by H₂O₂ accumulation under low temperature stress. With the increase of seed soaking concentration, most of the activity of antioxidant enzymes and system resistance enzymes, the AsA-GSH cycle and main substance content of carbon and nitrogen metabolism, root activity and fresh weight showed unimodal curve distribution. The soluble protein content of leaves and roots increased by 23.3%-34.9% and 31.9%-89.6% at the concentration of 0.8%-2.0%, respectively. The activity of phenylalanine ammonia-lyase increased by 16.8% and 38.8%, the activity of polyphenol oxidase increased by 27.7% and 13.4%, the content of ascorbic acid increased by 62.6% and 55.3%, and the content of dehydroascorbic acid increased by 31.9% and 66.4% in leaves and roots at the concentration of 1.6%, respectively. The activity of hydrogen peroxide in leaves and roots increased by 66.4% and 108.1% at the concentration of 0.8%. Therefore, H₂O₂ content in leaves and roots after soaking seed significantly reduced by 18.7%-37.6% and 27.5%-49.7% compared with the CK, respectively. The fresh root weight increased significantly by 85.2% and 105.3%, and the fresh weight of single plant increased significantly by 31.9% and 40.8% at the concentration of 0.4% and 0.8% compared with the control, respectively. In this study, the treatment at the concentration of 0.4%-0.8% enhanced the operation activity and systemic resistance of the AsA-GSH circle system with AsA as the core, improved the antioxidant stress ability of maize seedlings, and obtained the maximum root activity and fresh weight of plant, which were a relatively appropriate seed soaking concentration under hydroponic condition.

1446. Dynamics of Enterococcus faecalis Cardiolipin Synthase Gene Expression Reveal Compensatory Roles in Daptomycin Resistance

BackgroundDaptomycin (DAP) is a lipopeptide antibiotic targeting membrane anionic phospholipids (APLs) at the division septum, and resistance (DAP-R) has been linked to mutations in genes encoding i) the LiaFSR stress response system or its effector LiaX, and ii) cardiolipin synthase (Cls). Activation of the E. faecalis (Efs) LiaFSR response is associated with DAP-R and redistribution of APL microdomains away from the septum, and cardiolipin is predicted to be a major component of these APL microdomains. Efs harbors two putative cls genes, cls1 and cls2. While changes in Cls1 have been implicated in DAP-R, the exact roles of each enzyme in resistance are unknown. We aim to characterize the contributions of Cls1 and Cls2 in the development of DAP-R.Methods cls1 and cls2 were deleted individually and in tandem from DAP-S Efs OG117 and DAP-R Efs OG117∆liaX (a DAP-R derivative strain with an activated LiaFSR response). Mutants were characterized by DAP minimum inhibitory concentration (MIC) using E-test on Mueller-Hinton II agar and localization of APL microdomains with 10-N-nonyl-acridine orange staining. Quantitative PCR (qRT-PCR) was used to study gene expression profiles of cls1 and cls2 in Efs OG117∆liaX relative to Efs OG117 across the cell growth cycle.ResultsqRT-PCR revealed differential expression profiles of cls1 and cls2 associated with DAP-R. cls1 was highly upregulated in stationary phase concurrent with a decrease in cls2 expression. However, independent deletion of cls1 or cls2 in the DAP-R background resulted in no significant changes in DAP MICs or localization of APL microdomains (remaining non-septal). Further studies revealed that cls2 expression is upregulated upon deletion of cls1 in both the DAP-S and DAP-R background, suggesting a potential compensatory role for Cls2. Double deletion of both cls genes in the DAP-R strain decreased DAP MIC and restored the septal localization of APL microdomains.ConclusionCls1 is the major and predominant enzyme involved in cell membrane adaptation associated with the development of DAP-R in E. faecalis. However, we describe a novel compensatory and overlapping role for cardiolipin synthases to ensure bacterial survival upon attack from antimicrobial peptides and related antibiotics.Disclosures Cesar A. Arias, MD, MSc, PhD, FIDSA, Entasis Therapeutics (Scientific Research Study Investigator)MeMed (Scientific Research Study Investigator)Merck (Grant/Research Support)

Aminoglycoside 6'-N-acetyltransferase Type Ib [AAC(6')-Ib]-Mediated Aminoglycoside Resistance: Phenotypic Conversion to Susceptibility by Silver Ions.

Clinical resistance to amikacin and other aminoglycosides is usually due to the enzymatic acetylation of the antimicrobial molecule. A ubiquitous resistance enzyme among Gram-negatives is the aminoglycoside 6′-N-acetyltransferase type Ib [AAC(6′)-Ib], which catalyzes acetylation using acetyl-CoA as a donor substrate. Therapies that combine the antibiotic and an inhibitor of the inactivation reaction could be an alternative to treat infections caused by resistant bacteria. We previously observed that metal ions such as Zn2+ or Cu2+ in complex with ionophores interfere with the AAC(6′)-Ib-mediated inactivation of aminoglycosides and reduced resistance to susceptibility levels. Ag1+ recently attracted attention as a potentiator of aminoglycosides′ action by mechanisms still in discussion. We found that silver acetate is also a robust inhibitor of the enzymatic acetylation mediated by AAC(6′)-Ib in vitro. This action seems to be independent of other mechanisms, like increased production of reactive oxygen species and enhanced membrane permeability, proposed to explain the potentiation of the antibiotic effect by silver ions. The addition of this compound to aac(6′)-Ib harboring Acinetobacter baumannii and Escherichia coli cultures resulted in a dramatic reduction of the resistance levels. Time-kill assays showed that the combination of silver acetate and amikacin was bactericidal and exhibited low cytotoxicity to HEK293 cells.

In vitro assessment of protease production and stress tolerance of mutant isolates of Trichoderma sp.

Soil-borne plant pathogenic fungi cause serious losses in agricultural products. The antagonistic fungi for the control of plant diseases have increased efficiency and use of space has emerged as an alternative to other methods for the protection of agricultural products. One of the fungi used for this purpose is Trichoderma species. In this study mu-tant isolates of Trichoderma spp. were used. The resistance and pro-tease enzyme activities of mutant isolates against abiotic factors such as temperature, drought and salinity were investigated. Mutant iso-lates of Trichoderma sp. were showed differed in tolerance to differ-ent abiotic stress factors. Protease enzyme activity produced by iso-lates was influenced by the tested abiotic factors. In the medium con-taining 30% PEG, the highest protease activity was determined in Tm13 isolate. Indigenous Trichoderma strains produced proteases in high temperature, drought and saline conditions. This indicates that isolates may be promising candidates in agricultural production.

Transmission of Engineered Plastids in Sugarcane, a C4 Monocotyledonous Plant, Reveals that Sorting of Preprogrammed Progenitor Cells Produce Heteroplasmy.

We report here plastid transformation in sugarcane using biolistic transformation and embryogenesis-based regeneration approaches. Somatic embryos were developed from unfurled leaf sections, containing preprogrammed progenitor cells, to recover transformation events on antibiotic-containing regeneration medium. After developing a proficient regeneration system, the FLARE-S (fluorescent antibiotic resistance enzyme, spectinomycin and streptomycin) expression cassette that carries species-specific homologous sequence tails was used to transform plastids and track gene transmission and expression in sugarcane. Plants regenerated from streptomycin-resistant and genetically confirmed shoots were subjected to visual detection of the fluorescent enzyme using a fluorescent stereomicroscope, after genetic confirmation. The resultant heteroplasmic shoots remained to segregate on streptomycin-containing MS medium, referring to the unique pattern of division and sorting of cells in C4 monocotyledonous compared to C3 monocotyledonous and dicotyledonous plants since in sugarcane bundle sheath and mesophyll cells are distinct and sort independently after division. Hence, the transformation of either mesophyll or bundle sheath cells will develop heteroplasmic transgenic plants, suggesting the transformation of both types of cells. Whilst developed transgenic sugarcane plants are heteroplasmic, and selection-based regeneration protocol envisaging the role of division and sorting of cells in the purification of transplastomic demands further improvement, the study has established many parameters that may open up exciting possibilities to express genes of agricultural or pharmaceutical importance in sugarcane.

2',4'-BNA/LNA with 9-(2-Aminoethoxy)-1,3-diaza-2-oxophenoxazine Efficiently Forms Duplexes and Has Enhanced Enzymatic Resistance*.

Artificial nucleic acids are widely used in various technologies, such as nucleic acid therapeutics and DNA nanotechnologies requiring excellent duplex‐forming abilities and enhanced nuclease resistance. 2′‐O,4′‐C‐Methylene‐bridged nucleic acid/locked nucleic acid (2′,4′‐BNA/LNA) with 1,3‐diaza‐2‐oxophenoxazine (BNAP (BH)) was previously reported. Herein, a novel BH analogue, 2′,4′‐BNA/LNA with 9‐(2‐aminoethoxy)‐1,3‐diaza‐2‐oxophenoxazine (G‐clamp), named BNAP‐AEO (BAEO), was designed. The BAEO nucleoside was successfully synthesized and incorporated into oligodeoxynucleotides (ODNs). ODNs containing BAEO possessed up to 104‐, 152‐, and 11‐fold higher binding affinities for complementary (c) RNA than those of ODNs containing 2′‐deoxycytidine (C), 2′,4′‐BNA/LNA with 5‐methylcytosine (L), or 2′‐deoxyribonucleoside with G‐clamp (PAEO), respectively. Moreover, duplexes formed by ODN bearing BAEO with cDNA and cRNA were thermally stable, even under molecular crowding conditions induced by the addition of polyethylene glycol. Furthermore, ODN bearing BAEO was more resistant to 3′‐exonuclease than ODNs with phosphorothioate linkages.

Peptide-Based Polyion Complex Vesicles That Deliver Enzymes into Intact Plants To Provide Antibiotic Resistance without Genetic Modification.

Direct delivery of enzymes into intact plants using cell-penetrating peptides (CPPs) is an attractive approach for modifying plant functions without genetic modification. However, by conventional methods, it is difficult to maintain the enzyme activity for a long time because of proteolysis of the enzymes under physiological conditions. Here, we developed a novel enzyme delivery system using polyion complex vesicles (PICsomes) to protect the enzyme from proteases. We created PICsome-bearing reactive groups at the surface by mixing an anionic block copolymer, alkyne-TEG-P(Lys-COOH), and a cationic peptide, P(Lys). The PICsome encapsulated neomycin phosphotransferase II (NPTII), a kanamycin resistance enzyme, and protected NPTII from proteases in vitro. A CPP-modified PICsome delivered NPTII into the root hair cells of Arabidopsis thaliana seedlings and provided kanamycin resistance in the seedlings that lasted for 7 days. Thus, the PICsome-mediated enzyme delivery system is a promising method for imparting long-term transient traits to plants without genetic modification.

Modulation of antioxidant defence system in response to berberine in Candida albicans.

Emergence of multidrug resistant species of Candida is evolving, which advocates an urgent need for the development of new therapeutic strategies and antifungal drugs. Activation of antioxidant defence system in Candida albicans is known as forefront mechanism to escape drug toxicity. This study evaluated the role of antioxidant defence genes in the susceptibility to fluconazole in C. albicans and also determined the effect of berberine on growth, antioxidant enzymes and the expression of their genes in C. albicans isolates. Expression of major antioxidant genes was significantly increased in fluconazole-resistant isolates in comparison with the susceptible group. Antifungal susceptibility against berberine showed MIC values ranging from 125 to 500 μg/ml. Berberine treatment caused upregulation of mRNA expression and enzymatic activities of the targeted major antioxidants. Interestingly, C. albicans exhibited efficient antioxidant response at lower concentrations but could not sufficiently alleviate berberine-induced oxidative stress occurring at concentrations greater than 250 μg/ml. Therefore, berberine could serve as a potent Reactive Oxygen Species (ROS)-inducing agent, disrupting the antioxidant system especially in fluconazole-resistant C. albicans to overcome antifungal drug resistance. TAKE AWAYS: Evaluated the role of antioxidant enzymes in FLC resistance in C. albicans Studied the effect of berberine on growth of different C. albicans isolates Investigated the modulation of antioxidant enzymes by berberine in C. albicans Studied the effect of berberine on antioxidant gene expression in C. albicans.

Elevated production of reactive oxygen species is related to host plant resistance to sugarcane aphid in sorghum.

ABSTRACT Sugarcane aphid (Melanaphis sacchari) is a phloem-feeding insect that severely affects the growth and productivity of sorghum and other related crops. While a growing body of knowledge is accumulating regarding plant, and insect interactions, the role of reactive oxygen species (ROS) against aphid infestation in sorghum has not been established yet. Here, the involvement of H2O2 and ROS detoxification enzymes in host plant resistance to sugarcane aphid in sorghum was demonstrated. The H2O2 accumulation and expression patterns of selected ROS scavenging enzymes including ascorbate peroxidase (APX), glutathione S transferase (GST), superoxide dismutase (SOD), and catalase (CAT) in response to sugarcane aphid infestation at 3, 6, 9, and 12 days post infestation (dpi) in resistant (Tx2783) and susceptible (Tx7000) sorghum genotypes were assessed, respectively. A significant increase in H2O2 accumulation was observed in resistant genotypes at all time points studied as compared to susceptible plants. Furthermore, gene expression analysis revealed that in responding to attack by sugarcane aphid, antioxidant genes were induced in both genotypes, but much stronger in the resistant line. Furthermore, aphid survival and fecundity were significantly inhibited in resistant plants compared to susceptible plants. Taken together, our results suggest that the elevated accumulation of H2O2 and the strong upregulation of the antioxidant genes in sorghum may have contributed to host plant resistance in Tx2783 against sugarcane aphid but the weak expression of those antioxidant genes in Tx7000 resulted in the failure of attempting defense against sugarcane aphid. This report also provides the experimental evidence for the role of ROS involvement in the early defensive response to an attack by sugarcane aphid in sorghum.

Hepatitis E Virus Seroprevalence in Patients With Chronic Hepatitis C at a University Hospital in Brazil

Aim: We investigated the prevalence of anti-hepatitis E virus (HEV) antibodies in patients with chronic hepatitis C and the relationship with liver injury stage. Materials & methods: In total, 451 patients were included and the presence of anti-HEV antibodies was evaluated by ELISA. Results: Anti-HEV IgG antibodies were detected in 45 (10.0%) patients and anti-HEV IgM were detected in two IgG-positive patients (4.4%). The distributions of liver fibrosis, steatosis, inflammatory activity, homeostasis model assessment of insulin resistance and liver enzyme levels were similar between HEV-positive and HEV-negative patients. However, HEV-positive patients had a higher mean age (p = 0.030). The seroprevalence by age group increased from 2.2 (18–30 years) to 53.3% (>60 years). HEV infection was not related to advanced fibrosis. Conclusion: This investigation showed that the seroprevalence of HEV among patients with chronic hepatitis C is similar to that of blood donors in the same region.

The Effect of Aerobic Continuous Training on Myonectin, Insulin Resistance and Liver Enzymes in Rats with Nonalcoholic Fatty Liver Disease

The Effect of Aerobic Continuous Training on Myonectin, Insulin Resistance and Liver Enzymes in Rats with Nonalcoholic Fatty Liver Disease

Environmentally Friendly Quinolones Design for a Two-Way Choice between Biotoxicity and Genotoxicity through Double-Activity 3D-QSAR Model Coupled with the Variation Weighting Method.

Quinolone (QN) antibiotics are widely used, which lead to their accumulation in soil and toxic effects on ryegrass in pasture. In this study, we employed ryegrass as the research object and selected the total scores of 29 QN molecules docked with two resistant enzyme structures, superoxide dismutase (SOD, PDB ID: 1B06) and proline (Pro, PPEP-2, PDB ID: 6FPC), as dependent variables. The structural parameters of QNs were used as independent variables to construct a QN double-activity 3D-QSAR model for determining the biotoxicity on ryegrass by employing the variation weighting method. This model was constructed to determine modification sites and groups for designing QNs molecules. According to the 3D contour map of the model, by considering enrofloxacin (ENR) and sparfloxacin (SPA) as examples, 23 QN derivatives with low biotoxicity were designed, respectively. The functional properties and environmental friendliness of the QN derivatives were predicted through a two-way selection between biotoxicity and genotoxicity before and after modification; four environmentally friendly derivatives with low biotoxicity and high genotoxicity were screened out. Mixed toxicity index and molecular dynamics methods were used to verify the combined toxicity mechanism of QNs on ryegrass before and after modification. By simulating the combined pollution of ENR and its derivatives in different soils (farmland, garden, and woodland), the types of combined toxicity were determined as partial additive and synergistic. Binding energies were calculated using molecular dynamics. The designed QN derivatives with low biotoxicity, high genotoxicity, and environmental friendliness can highly reduce the combined toxicity on ryegrass and can be used as theoretic reserves to replace QN antibiotics.

Shared requirements for key residues in the antibiotic resistance enzymes ErmC and ErmE suggest a common mode of RNA recognition

Erythromycin-resistance methyltransferases are SAM dependent Rossmann fold methyltransferases that convert A2058 of 23S rRNA to m62A2058. This modification sterically blocks binding of several classes of antibiotics to 23S rRNA, resulting in a multidrug-resistant phenotype in bacteria expressing the enzyme. ErmC is an erythromycin resistance methyltransferase found in many Gram-positive pathogens, whereas ErmE is found in the soil bacterium that biosynthesizes erythromycin. Whether ErmC and ErmE, which possess only 24% sequence identity, use similar structural elements for rRNA substrate recognition and positioning is not known. To investigate this question, we used structural data from related proteins to guide site-saturation mutagenesis of key residues and characterized selected variants by antibiotic susceptibility testing, single turnover kinetics, and RNA affinity-binding assays. We demonstrate that residues in α4, α5, and the α5-α6 linker are essential for methyltransferase function, including an aromatic residue on α4 that likely forms stacking interactions with the substrate adenosine and basic residues in α5 and the α5-α6 linker that likely mediate conformational rearrangements in the protein and cognate rRNA upon interaction. The functional studies led us to a new structural model for the ErmC or ErmE-rRNA complex.

Acinetobacter baumannii as a causative agent of health care associated infections

Acinetobacter baumannii is an opportunistic pathogen that seriously affects sick patients, causing Health Care Associated Infections (HCAI) such as pneumonia associated with mechanical ventilation, urinary tract infections and bacteremia, in recent years this bacterium has become a health problem worldwide, its isolation from infections present in hospitalized patients has been increasing, and it also has various mechanisms of resistance to antibiotics. The present documentary research aims to describe the mechanisms of pathogenicity and resistance to antibiotics used by Acinetobacter baumannii as a causal agent of HCAI. To carry out this work, a literature search was carried out in databases such as: Scielo, PubMed, NCBI, and Elsevier. The pathogenicity mechanisms that allow it to colonize and develop infections in hospitalized patients are: porins, biofilms, pili, lipopolysaccharides, phospholipases, outer membrane vesicles and the production of siderophores. It has enzymatic resistance mechanisms such as beta-lactamases and non-enzymatic mechanisms such as porin modification, efflux pumps, and modifications in DNA gyrase. There are extrinsic factors that favor the development of HCAI, such as the immunological and health condition of the patient, as well as the association with medical equipment. Studies carried out between the years 2005 to 2018 reveal that A. baumannii is one of the main causative agents of pneumonia associated with mechanical ventilation in patients admitted to the Intensive care unit.

The Novel CarbaLux Test for Carbapenemases and Carbapenem Deactivating AmpC Beta-Lactamases.

ObjectivesTo evaluate the rapid phenotypic CarbaLux test for routine diagnostics in the medical laboratory in a proof of concept study.Methodsisolates of Gram-negative bacteria suspicious for carbapenem resistance including Enterobacterales (67), Pseudomonas (10), Acinetobacter (5), and Stenotrophomonas (1) species, collected between 2016 and 2018 from in-patients, were tested for carbapenemase activity using a novel fluorescent carbapenem. When subjected to extracted bacterial carbapenemases its fluorescence disappears. All bacteria to be tested were cultured on Columbia blood agar and few on other commercial media. MALDI TOF MS, molecular assays, automated MIC testing, and in part, agar diffusion tests served to characterize the isolates. For comparison, few selected bacteria were also investigated by prior phenotypic tests for carbapenemase detection.ResultsUnder UV light, the CarbaLux test allowed a rapid detection of 39/39 carbapenemase-producing bacteria, including 15 isolates with OXA carbapenemases (e.g., OXA-23, OXA-24/40-like OXA-48-like or OXA-181). Several isolates had low MICs but still expressed carbapenemases. Among Enterobacter spp., it detected six strains with hyper-produced AmpC beta-lactamases, which deactivated carbapenems but were not detectable by prior rapid phenotypic assays. An unexpected high carbapenemase activity appeared with these enzymes. They were identified as AmpC variants by inhibition with cloxacillin.ConclusionOther than prior rapid phenotypic assessments for carbapenemases, which use secondary effects such as a change of pH, the inactivation of the fluorescent carbapenem substrate can be visualized directly under UV light. The new test works at 100 to 200-fold lower, therapy-like substrate concentrations. It takes advantage of the high substrate affinity to carbapenemases allowing also the detection of less reactive resistance enzymes via a trapping mechanism, even from bacteria, which might appear unsuspicious from initial antibiograms. The novel fluorescence method allows simple and safe handling, reliable readings, and documentation and is suitable for primary testing in the clinical laboratory.

Genome Mining and Expression Analysis of Carboxylesterase and Glutathione S-Transferase Genes Involved in Insecticide Resistance in Eggplant Shoot and Fruit Borer, Leucinodes orbonalis (Lepidoptera: Crambidae).

The shoot and fruit borer, Leucinodes orbonalis (Lepidoptera: Crambidae) is the major cause of low productivity in eggplant and insecticides being the mainstay of management of L. orbonalis. However, field control failures are widespread due to the evolution of insecticide resistance. Taking advantage of the whole genome sequence information, the present study investigated the level of insecticide resistance and the expression pattern of individual carboxylesterase (CE) and glutathione S-transferases (GSTs) genes in various field collected populations of L. orbonalis. Dose-mortality bioassays revealed a very high level of resistance development against fenvalerate (48.2–160-fold), phosalone (94-534.6-fold), emamectin benzoate (7.2–55-fold), thiodicarb (9.64–22.7-fold), flubendiamide (187.4–303.0-fold), and chlorantraniliprole (1.6–8.6-fold) in field populations as compared to laboratory-reared susceptible iso-female colony (Lo-S). Over-production of detoxification enzymes viz., CE and GST were evident upon enzyme assays. Mining of the draft genome of L. orbonalis yielded large number of genes potentially belonging to the CE and GST gene families with known history of insecticide resistance in other insects. Subsequent RT-qPCR studies on relative contribution of individual genes revealed over-expression of numerous GSTs and few CEs in field populations, indicating their possible involvement of metabolic enzymes in insecticide resistance. The genomic information will facilitate the development of novel resistance management strategies against this pest.

Fabrication of Bio-Based Gelatin Sponge for Potential Use as A Functional Acellular Skin Substitute.

Gelatin possesses biological properties that resemble native skin and can potentially be fabricated as a skin substitute for full-thickness wound treatment. The native property of gelatin, whereby it is easily melted and degraded at body temperature, could prevent its biofunctionality for various applications. This study aimed to fabricate and characterise buffalo gelatin (Infanca halal certified) crosslinked with chemical type crosslinker (genipin and genipin fortified with EDC) and physicaly crosslink using the dihydrothermal (DHT) method. A porous gelatin sponge (GS) was fabricated by a freeze-drying process followed by a complete crosslinking via chemical—natural and synthetic—or physical intervention using genipin (GNP), 1-ethyl-3-(3-dimethylaminopropyl) (EDC) and dihydrothermal (DHT) methods, respectively. The physicochemical, biomechanical, cellular biocompatibility and cell-biomaterial interaction of GS towards human epidermal keratinocytes (HEK) and dermal fibroblasts (HDF) were evaluated. Results showed that GS had a uniform porous structure with pore size ranging between 60 and 200 µm with high porosity (>78.6 ± 4.1%), high wettability (<72.2 ± 7.0°), high tensile strain (>13.65 ± 1.10%) and 14 h of degradation rate. An increase in the concentration and double-crosslinking approach demonstrated an increment in the crosslinking degree, enzymatic hydrolysis resistance, thermal stability, porosity, wettability and mechanical strength. The GS can be tuned differently from the control by approaching the GS via a different crosslinking strategy. However, a decreasing trend was observed in the pore size, water retention and water absorption ability. Crosslinking with DHT resulted in large pore sizes (85–300 µm) and low water retention (236.9 ± 18.7 g/m2·day) and a comparable swelling ratio with the control (89.6 ± 7.1%). Moreover no changes in the chemical content and amorphous phase identification were observed. The HEK and HDF revealed slight toxicity with double crosslinking. HEK and HDF attachment and proliferation remain similar to each crosslinking approach. Immunogenicity was observed to be higher in the double-crosslinking compared to the single-crosslinking intervention. The fabricated GS demonstrated a dynamic potential to be tailored according to wound types by manipulating the crosslinking intervention.

Adaptation by copy number variation increases insecticide resistance in the fall armyworm

Understanding the genetic basis of insecticide resistance is a key topic in agricultural ecology. The adaptive evolution of multi-copy detoxification genes has been interpreted as a cause of insecticide resistance, yet the same pattern can also be generated by the adaptation to host-plant defense toxins. In this study, we tested in the fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae), if adaptation by copy number variation caused insecticide resistance in two geographically distinct populations with different levels of resistance and the two host-plant strains. We observed a significant allelic differentiation of genomic copy number variations between the two geographic populations, but not between host-plant strains. A locus with positively selected copy number variation included a CYP gene cluster. Toxicological tests supported a central role for CYP enzymes in deltamethrin resistance. Our results indicate that copy number variation of detoxification genes might be responsible for insecticide resistance in fall armyworm and that evolutionary forces causing insecticide resistance could be independent of host-plant adaptation.

Identification and characterization of striking multiple-insecticide resistance in a Tetranychus urticae field population from Greece.

Tetranychus urticae is a notorious crop pest with a worldwide distribution that has developed resistance to a wide range of acaricides. Here, we investigated the resistance levels of a T. urticae population collected from an ornamental greenhouse in Peloponnese, Greece, and analyzed its resistance mechanisms at the molecular level. Toxicological assays showed resistance against compounds with different modes of action, with resistance ratios of: 89-fold for abamectin; > 1000-fold for clofentezine; > 5000-fold for etoxazole; 27-fold for fenpyroximate and pyridaben; 20- and 36-fold for spirodiclofen and spirotetramat, respectively; and 116- and > 500-fold for cyenopyrafen and cyflumetofen, respectively. Bioassays with synergists indicated the involvement of detoxification enzymes in resistance to abamectin, but not to cyflumetofen and spirodiclofen. RNA sequencing (RNA-seq) analysis showed significant over-expression of several genes encoding detoxification enzymes such as cytochrome P450 monooxygenases and UDP-glycosyltransferases, which have been previously associated with acaricide resistance. Known target-site resistance mutations were identified in acetyl-choline esterase, chitin synthase 1 and NDUFS7/psst, but putative novel resistance mutations were also discovered in targets such as glutamate-gated chloride channel subunit 3. Interestingly, target-site resistance mutations against pyrethroids or bifenazate were not identified, possibly indicating a recent reduced selection pressure in Greece, as well as a possible opportunity to rotate these chemistries. We identified and characterized a striking case of multiple acaricide resistance in a field population of T. urticae. Exceptionally strong resistance phenotypes, with accumulation of multiple resistance mutations and over-expression of P450s and other detoxification genes in the same field population are reported.