Resistance Enzymes Research Articles

Phenotypic Detection of Β-Lactam Antibiotics, Methicillin and Inducible Clindamycin Resistance Among Bacterial Isolates in Patients with Otitis Externa

Background: Otitis is a general terminology used for inflammation or infection of the ear; Staphylococcus aureus and Pseudomonas spp. are the most common causes of otitis externa. The resistance mechanism against the beta-lactams group is due to the production of β-lactamase enzymes by the bacteria; the enzymes in staphylococci are encoded by erm genes that confer inducible Clindamycin resistance. Objectives: This study aimed at investigating bacterial resistance by evaluating samples collected from Otitis Externa patients admitted to Ayatollah Roohani Hospital of Babol, Iran. Methods: Ear samples were collected from 72 patients with Otitis Externa referred to Ayatollah Roohani hospital during May 2012 to 2013. At first, the isolated bacteria were identified using appropriate differential and selective media, and then were tested for antimicrobial susceptibility testing following the disk diffusion method. Special diagnostic tests were also performed for the identification of ESBL, iAmpC, pAmpC, metallo beta lactamase producers and inducible resistance to clindamycin and methicillin resistant strains. Data were analyzed by the SPSS 22 statistical software. Results: Among the 65 isolated bacteria, 24 (36.9%) cases were found to be gram negative and 41 (63.1%) were gram positive; pAmpC beta-lactamase producers were found to have the highest frequency in gram negative bacteria. From 36 (87.8%) isolated CoNS, 18 (50%) bacteria were found to be resistant to the methicillin group and 4 (11.1%) cases had inducible resistance to clindamycin; All isolated S. aureus were sensitive to methicillin and clindamycin. Conclusions: Considering that some bacteria are concurrently able to produce different types of resistance enzymes, and also the fact that high prevalence rate of resistance belongs to CoNS, it is important and necessary to perform antimicrobial susceptibility testing as per clinical and laboratory standards institute (CLSI) methods in clinical laboratories.

Isothermal Self-Assembly of Spermidine-DNA Nanostructure Complex as a Functional Platform for Cancer Therapy.

Programmable DNA nanostructure self-assembly offers great potentials in nanomedicine, drug delivery, biosensing, and bioimaging. However, due to the intrinsically negatively charged DNA backbones, the instability of DNA nanostructures in physiological settings poses serious challenges to their practical applications. To overcome this challenge, a strategy that combines the magnesium-free DNA self-assembly and functionalization is proposed in this study. We hypothesize that naturally abundant spermidine may not only mediate the self-assembly of DNA nanostructures, but also shield them from harsh physiological environments. As a proof of concept, a DNA nanoprism is designed and synthesized successfully through spermidine. It is found that spermidine can mediate the isothermal self-assembly of DNA nanoprisms. Compared to conventional Mg2+-assembled DNA nanostructures, the spermidine-DNA nanoprism complex shows higher thermal stability and better enzymatic resistance than Mg2+-assembled DNA nanoprisms, and more importantly, it has a much higher cellular uptake efficacy in multiple cancerous cell lines. The internalization mechanism is identified as clathrin-mediated endocytosis. To demonstrate the suitability of this new nanomaterial for biomedical applications, an mTOR siRNA, after being conjugated into the complex, is efficiently delivered into cancer cells and shows excellent gene knockdown efficacy and anticancer capability. These findings indicate that the spermidine-DNA complex nanomaterials might be a promising platform for biomedical applications in the future.

Plazomicin Retains Antibiotic Activity against Most Aminoglycoside Modifying Enzymes.

Plazomicin is a next-generation, semisynthetic aminoglycoside antibiotic currently under development for the treatment of infections due to multidrug-resistant Enterobacteriaceae. The compound was designed by chemical modification of the natural product sisomicin to provide protection from common aminoglycoside modifying enzymes that chemically alter these drugs via N-acetylation, O-adenylylation, or O-phosphorylation. In this study, plazomicin was profiled against a panel of isogenic strains of Escherichia coli individually expressing twenty-one aminoglycoside resistance enzymes. Plazomicin retained antibacterial activity against 15 of the 17 modifying enzyme-expressing strains tested. Expression of only two of the modifying enzymes, aac(2')-Ia and aph(2″)-IVa, decreased plazomicin potency. On the other hand, expression of 16S rRNA ribosomal methyltransferases results in a complete lack of plazomicin potency. In vitro enzymatic assessment confirmed that AAC(2')-Ia and APH(2'')-IVa (aminoglycoside acetyltransferase, AAC; aminoglycoside phosphotransferase, APH) were able to utilize plazomicin as a substrate. AAC(2')-Ia and APH(2'')-IVa are limited in their distribution to Providencia stuartii and Enterococci, respectively. These data demonstrate that plazomicin is not modified by a broad spectrum of common aminoglycoside modifying enzymes including those commonly found in Enterobacteriaceae. However, plazomicin is inactive in the presence of 16S rRNA ribosomal methyltransferases, which should be monitored in future surveillance programs.

IMPROVED QUALITY OF VIRAL INFECTED GRAPE PLANTS CULTIVATED IN SOIL INOCULATED WITH RHIZOPHERIC MICROORGANISMS

A considerable rhizopheric bacteria and mycorrhizae collectively known as plant growth promoting microorganisms (PGPM) have ability to induce acquire resistance in plant against pathogens and to provide benefits to their hosts. Grapevine viruses cause reducing yield and shortening the life span of infected plants in the vineyard. The current study aims to improve quality of Grape fan leaf virus (GFLV) infected grape plants via the soil inoculation with PGPM. Pot experiments were conducted under greenhouse during two seasons 2014/15 and 2015/16 in a Virology Greenhouse, Microbial. Dep. Fac. Agric., Ain Shams Univ. Cairo, Egypt. Grape cv. Flam grafted with GFLV infected stick and cultivated in inoculated soil with rhizopheric PGPB and mycorrhiza (VAM). GFLV was detected in infected leaves by DAS-ELISA. Plant growth parameters and chemical immune acquired resistance were assessment in GFLV infected grape cv. flam. The results were clearly indicated that PGPM inoculation in soil improved of plant growth in the second season (2015/16) compared with first season (2014/2015(. PGPM (Bacillus sp., Pseudomonas sp. and Serratia sp. isolates and VAM) improved quality of GFLV infected grape plants via increased plant growth parameters (leave number, stem diameter, plant length, and phosphorus and potassium components in leaves). PGPM induced acquire resistance in plant against GFLV; it was found that, significant increase of proline and SA contents in GFLV infected grape leaves compared as healthy ones. The results revealed that chlorophyll a; b and carotenoids were significant decreased while inoculated PGPM in soil showed significant increase compared with healthy control ones. Expressed proteins and resistance enzymes (POD and PPO) of antiviral proteins were significant increase in PGPM application of GFLV infected grape growth related no inoculated PGPM ones. So that the current study recommended that the combination among VAM and PGPB soil inoculation improved quality of GFLV infected grape plant under greenhouse conditions.

Morpho-anatomical, physiological and biochemical changes in rubber tree seeds

The physical, physiological and biochemical changes during the development until the dispersal of rubber tree seeds were evaluated with the purpose of estimating the point at physiological maturity. A total of 30 plants were selected at different points in a commercial planting area and had their flowers marked during the anthesis and every 15 days after marking. Fruits and seeds were collected for analysis of moisture content, dry matter, diameter and length. Details of the anatomy ultra-structure of the seeds were evaluated. The seed emergence, emergency speed index, heat resistant proteins and oxidative stress enzymes were examined. It was observed that fruits reached maximum size at 120 days after anthesis and seeds at 150 days. The seeds acquired germination capacity after 150 days. At 175 days, they presented the highest percentage of dry matter and lowest moisture, in addition to a higher percentage of germination and vigor. Therefore, it was possible to conclude that the physiological maturity of the rubber tree seeds occurs at 175 days after anthesis, and coincides with its maximum physiological quality. At 175 and 180 days post-anthesis, there is a greater expression of heat resistant proteins as well as low molecular weight and greater oxidative stress enzyme activity.

Unique Fluorescent Imaging Probe for Bacterial Surface Localization and Resistant Enzyme Imaging.

Emergence of antibiotic bacterial resistance has caused serious clinical issues worldwide due to increasingly difficult treatment. Development of a specific approach for selective visualization of resistant bacteria will be highly significant for clinical investigations to promote timely diagnosis and treatment of bacterial infections. In this article, we present an effective method that not only is able to selectively recognize drug resistant AmpC β-lactamases enzyme but, more importantly, is able to interact with bacterial cell wall components, resulting in a desired localization effect on the bacterial surface. A unique and specific enzyme-responsive cephalosporin probe (DFD-1) has been developed for the selective recognition of resistance bacteria AmpC β-lactamase, by employing fluorescence resonance energy transfer with an "off-on" bioimaging. To achieve the desired localization, a lipid-azide conjugate (LA-12) was utilized to facilitate its penetration into the bacterial surface, followed by copper-free click chemistry. This enables the probe DFD-1 to be anchored onto the cell surface. In the presence of AmpC enzymes, the cephalosporin β-lactam ring on DFD-1 will be hydrolyzed, leading to the quencher release, thus generating fluorescence for real-time resistant bacterial screening. More importantly, the bulky dibenzocyclooctyne group in DFD-1 allowed selective recognition toward the AmpC bacterial enzyme instead of its counterpart ( e.g., TEM-1 β-lactamase). Both live cell imaging and cell cytometry assays showed the great selectivity of DFD-1 to drug resistant bacterial pathogens containing the AmpC enzyme with significant fluorescence enhancement (∼67-fold). This probe presented promising capability to selectively localize and screen for AmpC resistance bacteria, providing great promise for clinical microbiological applications.

Production, Characterization and Biocompatibility Evaluation of Collagen Membranes Derived from Marine Sponge Chondrosia reniformis Nardo, 1847.

Collagen is involved in the formation of complex fibrillar networks, providing the structural integrity of tissues. Its low immunogenicity and mechanical properties make this molecule a biomaterial that is extremely suitable for tissue engineering and regenerative medicine (TERM) strategies in human health issues. Here, for the first time, we performed a thorough screening of four different methods to obtain sponge collagenous fibrillar suspensions (FSs) from C. reniformis demosponge, which were then chemically, physically, and biologically characterized, in terms of protein, collagen, and glycosaminoglycans content, viscous properties, biocompatibility, and antioxidant activity. These four FSs were then tested for their capability to generate crosslinked or not thin sponge collagenous membranes (SCMs) that are suitable for TERM purposes. Two types of FSs, of the four tested, were able to generate SCMs, either from crosslinking or not, and showed good mechanical properties, enzymatic degradation resistance, water binding capacity, antioxidant activity, and biocompatibility on both fibroblast and keratinocyte cell cultures. Finally, our results demonstrate that it is possible to adapt the extraction procedure in order to alternatively improve the mechanical properties or the antioxidant performances of the derived biomaterial, depending on the application requirements, thanks to the versatility of C. reniformis extracellular matrix extracts.

Molecular Detection of TEM, SHV and CTX-M Genes Among Gram-negative Klebsiella Isolates.

In the past few years, the use of antimicrobial drugs against a wide range of pathogens has increased significantly. This extensive use of drugs has increased the resistance rate in microbial community widely. Molecular techniques for the detection of resistance are more reliable as compared to the conventional phenotypic method. The family Enterobacteriaceae is considered to be an important cause of nosocomial infections due to its predominantly active species such as E. coli, Klebsiella and Pseudomonas. These organisms are mainly involved in causing pneumonia, sepsis, post-surgical and urinary tract infections. Resistance against antimicrobial drugs among these isolates is increasing more rapidly all over the world. This study primarily focuses on the resistant isolates of Klebsiella species. The drug resistance in Klebsiella isolates is found to be associated with the production of resistance enzymes such as beta lactamase and extended spectrum beta lactamase (ESBLs) which confer resistance, most specifically against cephalosporins and extended spectrum cephalosporins. Since these enzymes are plasmid mediated, they can also produce resistance against several other antimicrobials. It has been found that among the genus Klebsiella, ESBLs are more prevalent in K. pneumoniae followed by K. oxytoca. In this study, we estimated the distribution of ESBL producers among Klebsiella species and performed their genetic characterization. A total of 236 gram-negative isolates were collected from different microbiological laboratories, during the period January 2010 till January 2012. Among these gram-negative isolates, 125 were identified as Klebsiella species. After species identification, Kirby Bauer disk diffusion method was used for antimicrobial susceptibility profiling. Furthermore, the phenotypic detection of ESBL producers was performed by double disc synergy and combination disc methods. Resistance genes responsible for the production of beta lactamase and extended spectrum beta lactamase enzymes were detected by Polymerase chain reaction. DNA sequencing was performed by selecting an ESBL producing Klebsiella pneumoniae strain with a positive blaTEM gene. In this study, we found 48%, 43.2% and 2.4% of SHV, TEM, and CTX-M resistance genes respectively in Klebsiella isolates. The DNA sequence ESBL and blaTEM positive Klebsiella strain showed 94% similarity with Klebsiella pneumoniae KUN5033 blaTEM gene for class A beta-lactamase TEM-198 analyzed by the Basic Local Alignment Search Tool (BLAST). Since there is an intense need of research in the field of drug resistance for implementing strict antibiotic control policies in hospitals, health care centers, laboratories, etc., the present study is dedicatedly conducted to estimate the drug resistant Klebsiella isolates, specifically the Beta lactamase and Extended Spectrum Beta Lactamase producers, at molecular level.

Investigating insecticide resistance and knock-down resistance (kdr) mutation in Dielmo, Senegal, an area under long lasting insecticidal-treated nets universal coverage for 10\xa0years

BackgroundThe use of insecticides, through indoor residual spraying and long-lasting insecticide-treated nets (LLINs), is essential to control malaria vectors. However, the sustainability of these tools is challenged by the spread of insecticide resistance in Anopheles mosquitoes. This study was conducted to assess the susceptibility to insecticides and to determine the resistance mechanisms in malaria vectors in Dielmo, a rural area of western Senegal where LLINs were introduced a decade ago.MethodsCDC bottle bioassays were used to determine the susceptibility of 2–5 day-old unfed Anopheles gambiae s.l. females to alphacypermethrin (12.5 µg/bottle), deltamethrin (12.5 µg/bottle), etofenprox (12.5 µg/bottle), lambdacyhalothrin (12.5 µg/bottle), permethrin (21.5 µg/bottle), DDT (100 µg/bottle), bendiocarb (12.5 µg/bottle), pirimiphos-methyl (20 µg/bottle) and fenitrothion (50 µg/bottle). The involvement of glutathione-S-transferases (GSTs) in insecticide resistance was assessed using a synergist, etacrynic acid (EA, 80 µg/bottle). Polymerase chain reaction (PCR) was used to investigate the presence of ‘knock-down resistance (kdr)’ mutation and to identify sibling species within the An. gambiae complex.ResultsCDC bottle bioassays showed that mosquitoes were fully susceptible to lambdacyhalothrin, bendiocarb and fenitrothion. Overall, mortality rates of 97, 94.6, 93.5, 92.1, and 90.1% were, respectively, observed for permethrin, deltamethrin, pirimiphos-methyl, etofenprox and alphacypermethrin. Resistance to DDT was observed, with a mortality rate of 62%. The use of EA significantly improved the susceptibility of An. gambiae s.l. to DDT by inhibiting GSTs (p = 0.03). PCR revealed that Anopheles arabiensis was the predominant species (91.3%; IC 95 86.6–94%) within An. gambiae complex from Dielmo, followed by Anopheles coluzzii (5.4%; IC 95 2.7–8.1%) and Anopheles gambiae s.s. (3.3%; IC 95 0.6–5.9%). Both 1014F and 1014S alleles were found in An. arabiensis population with frequencies of 0.08 and 0.361, respectively, and 0.233 and 0.133, respectively in An. coluzzii. In An. gambiae s.s. population, only kdr L1014F mutation was detected, with a frequency of 0.167. It was observed that some individual mosquitoes carried both alleles, with 19 specimens recorded for An. arabiensis and 2 for An. coluzzii. The presence of L1014F and L1014S alleles were not associated with resistance to pyrethroids and DDT in An. arabiensis.ConclusionsThe co-occurrence of 1014F and 1014S alleles and the probable involvement of GSTs enzymes in insecticide resistance in An. gambiae s.l. should prompt the local vector programme to implement non-pyrethroid/DDT insecticides alternatives.

Starch film-coated microparticles for oral colon-specific drug delivery

The aim of this study was to prepare and characterize a novel type of starch-coated microparticles (MPs) allowing site-specific delivery of bioactives to the colon. An oral colon-specific controlled-release system was developed in the form of MPs coated with a resistant starch (RS2/RS3) film (RS@MPs) through an aqueous suspension coating process. The RS2 was chosen from a high-amylose cornstarch with 88.5% digestion resistibility. The RS3 was prepared by a high-temperature/pressure (HTP) treatment, with the following of enzymatic debranching, and retrogradation, resulting in a dramatic increase in enzymatic resistance (RS3 content: 76.6%). RS@MPs showed 40.7% of 5-aminosalicylic acid release within 8 h. The in vivo study of fluorescein-loaded RS@MPs indicated the high acidic and enzymatic resistibility of RS@MPs and a restrained release in the upper GIT. Therefore, RS@MPs has revealed to be a high potential system for accurately targeting bioactive compound delivery to the colon.

The current insecticide resistance status of Anopheles gambiae (s.l.) (Culicidae) in rural and urban areas of Bouak\xe9, C\xf4te d\u2019Ivoire

BackgroundSeveral studies were carried out in experimental hut station in areas surrounding the city of Bouaké, after the crisis in Côte d’Ivoire. They reported increasing resistance levels to insecticide for malaria transmiting mosquitoes. The present work aims to evaluate the current resistance level of An. gambiae(s.l.) in rural and urban areas in the city of Bouaké.MethodsLarvae of Anopheles gambiae (s.l.) were collected from five different study sites and reared to adult stages. The resistance status was assessed using the WHO bioassay test kits for adult mosquitoes, with eight insecticides belonging to pyrethroids, organochlorines, carbamates and organophosphates classes. Molecular assays were performed to identify the molecular forms of An. gambiae (s.l.), the L1014F kdr and the ace-1R alleles in individual mosquitoes. The synergist PBO was used to investigate the role of enzymes in resistance. Biochemical assays were performed to detect potential increased activities in mixed function oxidase (MFO) levels, non-specific esterases (NSE) and glutathione S-transferases (GST).ResultsHigh resistance levels to pyrethroids, organochlorines, and carbamates were observed in Anopheles gambiae (s.l.) from Bouaké. Mortalities ranged between 0 and 73% for the eight tested insecticides. The pre-exposure to PBO restored full or partial susceptibility to pyrethroids in the different sites. The same trend was observed with the carbamates in five sites, but to a lesser extent. With DDT, pre-exposure to PBO did not increase the mortality rate of An. gambiae (s.l.) from the same sites. Tolerance to organophosphates was observed. An increased activity of NSE and higher level of MFO were found compared to the Kisumu susceptible reference strain. Two molecular forms, S form [(An. gambiae (s.s)] and M form (An. coluzzi) were identified. The kdr allele frequencies vary from 85.9 to 99.8% for An. gambiae (s.s.) and from 81.7 to 99.6% for An. coluzzii. The ace-1R frequencies vary between 25.6 and 38.8% for An. gambiae (s.s.) and from 28.6 to 36.7% for An. coluzzii.ConclusionResistance to insecticides is widespread within both An. gambiae (s.s.) and An. coluzzii. Two mechanisms of resistance, i.e. metabolic and target-site mutation seemed to largely explain the high resistance level of mosquitoes in Bouaké. Pyrethroid resistance was found exclusively due to the metabolic mechanism.

Covalently crosslinked mussel byssus protein‐based materials with tunable properties

AbstractMussels’ anchoring threads, named byssus, are collagen‐rich fibers with outstanding mechanical properties. Our previous work has shown the possibility of producing a byssus protein hydrolyzate with good film‐forming ability, providing a promising new avenue for the preparation of biomaterials. Materials prepared from regenerated fibrous proteins often need additional treatments to reach the performance required for targeted applications. Here, we studied the effect of covalent crosslinking, using a carbodiimide or glutaraldehyde, on the mechanical properties and enzymatic resistance of byssus‐based materials. The results show that the mechanical properties of the films can be tuned, and that a higher crosslinking degree leads to increases in modulus and strength accompanied by a loss of extensibility. Structural analysis performed by infrared spectroscopy revealed that crosslinking induces an unexpected transition from aggregated strands to hydrated collagen/PPII‐related helical structures. The materials were nevertheless more resistant to collagenase degradation as a result of higher crosslinking density. This new set of materials prepared in aqueous environment could find a niche in tissue engineering.

α-l-Threose Nucleic Acids as Biocompatible Antisense Oligonucleotides for Suppressing Gene Expression in Living Cells.

Because of the chemical simplicity of α-l-threose nucleic acid (TNA) and its ability to exchange genetic information between itself and RNA, it has attracted significant interest as the RNA ancestor. We herein explore the biological properties and evaluate the potency of sequence-designed TNA polymers to suppress the gene expression in living environments. We found that sequence-specific TNA macromolecules exhibit strong affinity and specificity toward the complementary RNA targets, are highly biocompatible and nontoxic in a living cell system, and readily enter a number of cell lines without using transfecting agents. Particularly, TNA exhibited much stronger enzymatic resistance toward fetal bovine serum or human serum as compared to traditional antisense oligonucleotides, which means that the intrinsic structure of TNA is thoroughly resistant to biological degradation. Importantly, the efficacy of the TNA molecule with green fluorescent protein (GFP) target sequence (anti-GFP TNAs) as antisense agents was first demonstrated in living cells in which these polymers revealed high antisense activity in terms of the degree of inhibition of GFP gene expression. The GFP gene inhibition studies in HeLa and HEK293 cells characterize sequence-controlled TNA as a functional biomaterial and a valuable alternative to traditional antisense oligonucleotides such as peptide nucleic acids, phosphorodiamidate morpholino oligomers, and locked nucleic acids for a wide range of applications in drug discovery and life science research. Additionally, we also first reported the cost-efficient approach to synthesize the four TNA phosphoramidite monomers using 2-cyanoethyl N, N, N', N'-tetraisopropylphosphoramidite as a key reagent. Furthermore, by increasing the frequency of the deblocking and coupling reactions together with extending their reaction time in each synthesis cycle, sequence-controlled TNAs can be easily synthesized in a quantitative yield and high purity.

Elevated difenacoum metabolism is involved in the difenacoum-resistant phenotype observed in Berkshire rats homozygous for the L120Q mutation in the vitamin K epoxide reductase complex subunit 1 (Vkorc1) gene.

Soon after difenacoum began to be used, resistance to this rodenticide was detected in rats in northeast Hampshire and northwest Berkshire in England. Resistance to difenacoum has been reported to be stronger in rats from Berkshire than in rats from Hampshire. Surprisingly, after the discovery of the vitamin K epoxide reductase complex subunit 1 (Vkorc1) gene, rats from Berkshire and Hampshire were all shown to be homozygous for the L120Q mutation in Vkorc1. This study aimed to evaluate the resistance of Berkshire rats to confirm their extreme resistance and determine mechanisms supporting this resistance. For this purpose, we created a quasicongenic rat F7 strain by using a Berkshire rat as a donor to introduce the L120Q mutation in Vkorc1 into the genetic background of an anticoagulant-susceptible recipient strain. The use of F7 rats enabled demonstration of (i) the level of resistance to difenacoum conferred by the L120Q mutation, (ii) co-dominance of the L120 and Q120 alleles, (iii) the extreme resistance of Berkshire rats compared with Q120/Q120 rats as a consequence of additional resistance mechanisms, and (iv) the involvement of cytochrome P 450 (CYP450) enzymes in this extreme resistance. This study demonstrated that elevated CYP450 oxidative metabolism leading to accelerated difenacoum detoxification is involved in the Berkshire phenotype. © 2017 Society of Chemical Industry.

Strategies to Improve the Clinical Utility of Saporin-Based Targeted Toxins

Plant Ribosome-inactivating proteins (RIPs) including the type I RIP Saporin have been used for the construction of Immunotoxins (ITxs) obtained via chemical conjugation of the toxic domain to whole antibodies or by generating genetic fusions to antibody fragments/targeting domains able to direct the chimeric toxin against a desired sub-population of cancer cells. The high enzymatic activity, stability and resistance to conjugation procedures and especially the possibility to express recombinant fusions in yeast, make Saporin a well-suited tool for anti-cancer therapy approaches. Previous clinical work on RIPs-based Immunotoxins (including Saporin) has shown that several critical issues must be taken into deeper consideration to fully exploit their therapeutic potential. This review focuses on possible combinatorial strategies (chemical and genetic) to augment Saporin-targeted toxin efficacy. Combinatorial approaches may facilitate RIP escape into the cytosolic compartment (where target ribosomes are), while genetic manipulations may minimize potential adverse effects such as vascular-leak syndrome or may identify T/B cell epitopes in order to decrease the immunogenicity following similar strategies as those used in the case of bacterial toxins such as Pseudomonas Exotoxin A or as for Type I RIP Bouganin. This review will further focus on strategies to improve recombinant production of Saporin-based chimeric toxins.

An Investigation of the Effects of Riboflavin Concentration on the Efficacy of Corneal Cross-Linking Using an Enzymatic Resistance Model in Porcine Corneas.

PurposeTo investigate riboflavin concentration on enzymatic resistance following corneal cross-linking (CXL).MethodsNinety-six porcine eyes were divided into five groups in two treatment runs. Group 1 remained untreated. Group 2 received riboflavin 0.05%, group 3 riboflavin 0.1%, group 4 riboflavin 0.2%, and group 5 riboflavin 0.3%. Treated eyes underwent CXL with ultraviolet A at 9 mW/cm2 for 10 minutes. Eight-millimeter discs from each cornea were submerged in pepsin digest solution. In the first run, disc diameters were measured daily. After 10 days, dry weights were recorded from five samples in each group. In the second run, dry weights were recorded in five samples in each group at 10 and 20 days.ResultsCXL-treated corneas took longer to digest than untreated (P < 0.001). Although eyes treated with higher riboflavin concentrations generally took longer to digest, there were no significant differences between groups (P = 0.3). Dry weights at 10 days demonstrated, with each increase in concentration, an increase in weight of residual undigested tissue (P < 0.001). In the second run, with each increase in riboflavin concentration there was an increase in weight of residual tissue (P < 0.001) at 10 days. At 20 days, the dry weight was lower with 0.05% riboflavin compared to 0.3% (P < 0.001) and 0.2% and 0.1% solutions (P < 0.05), with no other difference between groups.ConclusionsThere is a consistent dose-response curve with higher concentrations of riboflavin achieving greater CXL efficacy, suggesting that manipulation of riboflavin dosage as well as the UVA protocol can be used to optimize CXL.

Rox, a Rifamycin Resistance Enzyme with an Unprecedented Mechanism of Action

Rifamycin monooxygenases (Rox) are present in a variety of environmental bacteria and are associated with decomposition of the clinically utilized antibiotic rifampin. Here we report the structure and function of a drug-inducible rox gene from Streptomyces venezuelae, which encodes a class A flavoprotein monooxygenase that inactivates a broad range of rifamycin antibiotics. Our findings describe a mechanism of rifamycin inactivation initiated by monooxygenation of the 2-position of the naphthyl group, which subsequently results in ring opening and linearization of the antibiotic. The result is an antibiotic that no longer adopts the basket-like structure essential for binding to the RNA exit tunnel of the target RpoB, thereby providing the molecular logic of resistance. This unique mechanism of enzymatic inactivation underpins the broad spectrum of rifamycin resistance mediated by Rox enzymes and presents a new antibiotic resistance mechanism not yet seen in microbial antibiotic detoxification.

Microbiological quality of refrigerated raw milk in the dairy farm and after transport to the processing dairy plant

ABSTRACT: Transport of cooled raw milk in bulk has greatly improved the quality of the raw material collected by dairy plants as it reduces the proliferation of mesophilic microorganisms that cause milk acidity and hinder its processing. However, refrigeration has favored the growth of psychrotrophic microorganisms which are able to grow at low temperatures (below 10ºC) and that produce heat resistant enzymes which degrade some milk components, reducing milk shelf life and causing organoleptic changes. The aim of this paper was to evaluate the microbiological quality of raw milk in dairy farms and after its transport to the processing dairy plant, through plate counting of mesophilic and psychrotrophic microorganisms. Fourteen milk samples were collected from tanks of the dairy-farming properties, and one sample was collected from their milk transport tanker at the entrance of the processing plant. Our results showed that the mean number of mesophilic microorganisms was higher in samples collected straight from the dairy farm tanks than in the samples collected from the transportation tankers at the entrance of the plant. Of the 14 sampled tanks, 64.3% were non-compliant with legislation. The sample collected from the milk transportation tanker containing milk from all properties showed a higher mean number of psychrotrophic microorganisms than the dairy farm samples. We conclude that the milk from dairy properties showed a higher amount of mesophilic microorganisms, and after transportation, at the entrance of the processing plant, there is a higher amount of psychrotrophic microorganisms.

Clonal relation and antimicrobial resistance pattern of extended-spectrum β-lactamase- and AmpC β-lactamase-producing Enterobacter spp. isolated from different clinical samples in Tehran, Iran.

Here, we determined the genes encoding antibiotic resistance enzymes and virulence factors and evaluated the genetic relationship between Enterobacter spp. isolated from different clinical samples. A total of 57 clinical isolates of Enterobacter spp. were tested for the production of extended-spectrum β-lactamases (ESBLs), carbapenemase, and AmpC using phenotypic and genotypic methods. The most common ESBLs and AmpC β-lactamases were bla TEM (63.3%) and bla EBC (57.7%), respectively. The most prevalent virulence gene was rpos (87.7%). The random amplified polymorphic DNA (RAPD) patterns of strains were genetically unrelated. RAPD polymerase chain reaction analysis revealed high genetic diversity among isolates.

Overcoming Aminoglycoside Enzymatic Resistance: Design of Novel Antibiotics and Inhibitors.

Resistance to aminoglycoside antibiotics has had a profound impact on clinical practice. Despite their powerful bactericidal activity, aminoglycosides were one of the first groups of antibiotics to meet the challenge of resistance. The most prevalent source of clinically relevant resistance against these therapeutics is conferred by the enzymatic modification of the antibiotic. Therefore, a deeper knowledge of the aminoglycoside-modifying enzymes and their interactions with the antibiotics and solvent is of paramount importance in order to facilitate the design of more effective and potent inhibitors and/or novel semisynthetic aminoglycosides that are not susceptible to modifying enzymes.