Control of Multidrug-Resistant Gene Flow in the Environment Through Bacteriophage Intervention

The spread of multidrug-resistant (MDR) bacteria is an emerging threat to the environment and public wellness. Inappropriate use and indiscriminate release of antibiotics in the environment through un-metabolized form create a scenario for the emergence of virulent pathogens and MDR bugs in the surroundings. Mechanisms underlying the spread of resistance include horizontal and vertical gene transfers causing the transmittance of MDR genes packed in different host, which pass across different food webs. Several controlling agents have been used for combating pathogens; however, the use of lytic bacteriophages proves to be one of the most eco-friendly due to their specificity, killing only target bacteria without damaging the indigenous beneficial flora of the habitat. Phages are part of the natural microflora present in different environmental niches and are remarkably stable in the environment. Diverse range of phage products, such as phage enzymes, phage peptides having antimicrobial properties, and phage cocktails also have been used to eradicate pathogens along with whole phages. Recently, the ability of phages to control pathogens has extended from the different areas of medicine, agriculture, aquaculture, food industry, and into the environment. To avoid the arrival of pre-antibiotic epoch, phage intervention proves to be a potential option to eradicate harmful pathogens generated by the MDR gene flow which are uneasy to cure by conventional treatments.     

Potential of MALDI-TOF mass spectrometry as a rapid detection technique in plant pathology: identification of plant-associated microorganisms

Plant diseases caused by plant pathogens substantially reduce crop production every year, resulting in massive economic losses throughout the world. Accurate detection and identification of plant pathogens is fundamental to plant pathogen diagnostics and, thus, plant disease management. Diagnostics and disease-management strategies require techniques to enable simultaneous detection and quantification of a wide range of pathogenic and non-pathogenic microorganisms. Over the past decade, rapid development of matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) techniques for characterization of microorganisms has enabled substantially improved detection and identification of microorganisms. In the biological sciences, MALDI-TOF MS is used to analyze specific peptides or proteins directly desorbed from intact bacteria, fungal spores, nematodes, and other microorganisms. The ability to record biomarker ions, in a broad m/z range, which are unique to and representative of individual microorganisms, forms the basis of taxonomic identification of microorganisms by MALDI-TOF MS. Recent advances in mass spectrometry have initiated new research, i.e. analysis of more complex microbial communities. Such studies are just beginning but have great potential for elucidation not only of the interactions between microorganisms and their host plants but also those among different microbial taxa living in association with plants. There has been a recent effort by the mass spectrometry community to make data from large scale mass spectrometry experiments publicly available in the form of a centralized repository. Such a resource could enable the use of MALDI-TOF MS as a universal technique for detection of plant pathogens and non-pathogens. The effects of experimental conditions are sufficiently understood, reproducible spectra can be obtained from computational database search, and microorganisms can be rapidly characterized by genus, species, or strain.     

Bacteriophage reporter technology for sensing and detecting microbial targets

Bacteriophages (phages) are bacterial viruses evolutionarily tuned to very specifically recognize, infect, and propagate within only a unique pool of host cells. Knowledge of these phage host ranges permits one to devise diagnostic tests based on phage–host recognition profiles. For decades, fundamental phage typing assays have been used to identify bacterial pathogens on the basis of the ability of phages to kill, or lyse, the unique species, strain, or serovar to which they are naturally targeted. Over time, and with a better understanding of phage–host kinetics and the realization that there exists a phage specific for nearly any bacterial pathogen of clinical, foodborne, or waterborne consequence, a variety of improved, rapid, sensitive, and easy-to-use phage-mediated detection assays have been developed. These assays exploit every stage of the phage recognition and infection cycle to yield a wide variety of pathogen monitoring, detection, and enumeration formats that are steadily advancing toward new biosensor integrations and advanced sensing technologies.     

Chemically immobilized T4-bacteriophage for specific Escherichia coli detection using surface plasmon resonance

A bioassay platform using T4 bacteriophage (T4) as the specific receptor and surface plasmon resonance (SPR) as the transduction technique has been developed for the detection of Escherichia coli K12 bacteria. The T4 phages have been covalently immobilized onto gold surfaces using a self-assembled monolayer of dithiobis(succinimidyl propionate) (DTSP). Substrates of BSA/EA-T4/DTSP/Au prepared using different T4 phage concentrations have been characterized using scanning electron microscopy (SEM). The studies reveal that the use of DTSP results in a uniform binding of T4 phages onto the surface. The SPR analysis demonstrates that these BSA/EA-T4/DTSP/Au interfaces can detect the E. coli K12 with high specificity against non-host E. coli NP10 and NP30. Results of SEM and SPR studies indicate that the maximum host bacterial capture is obtained when 1.5 × 10(11) pfu ml(-1) concentration of T4 phages was used for immobilization. The surface of these chemically anchored phage substrates can be regenerated for repeated detection of E. coli K12 and can be used for detection in 7 × 10(2) to 7 × 10(8) cfu ml(-1) range. The results of these studies have implications for the development of online bioassays for the detection of various food and water borne pathogens using the inherent selectivity of bacteriophage recognition.     

Bacteria love our sugars: Interaction between soluble lectins and human fucosylated glycans,structures, thermodynamics and design of competing glycocompounds

The specific recognition of host epithelial surfaces by pathogen receptors is often the first step of infection. Bacterial carbohydrate-binding proteins, called adhesins or lectins, specifically recognize glycoconjugates on host tissues. Opportunistic pathogens responsible for lung infections, such as some Pseudomonas and Burkholderia species, produce soluble lectins that bind to human fucosylated oligosaccharides. Among these targets, the observed variations in ABO and Lewis histo-blood group phenotypes are proposed to be a result of co-evolution between pathogens and mammals. The analysis of the available crystal structures of bacterial lectins helps deciphering the structure/function relationship involving this important class of protein. The high affinity observed between bacterial lectins and fucosylated oligosaccharides can be reached by different strategies in term of structures and thermodynamics. Structure-based design of glycomimetics and glycodendrimers resulted in very high affinity ligands that open the route towards novel anti-infectious strategies.     

Dyes as guests in ordered systems: current understanding and future directions

ABSTRACT

The optical properties of dyes dissolved in liquid crystals have led to their proposed use in a diverse range of practical applications. Such guest–host systems are typically required to fulfil a range of criteria relating to their absorption properties, degree of alignment and stability, but concurrently satisfying these requirements has proven a barrier to their widespread use. In this article, many of the proposed applications and their requirements are discussed, and an outline of some of the most prevalent classes of dye proposed in the context of guest–host systems is given, along with a summary of recent reports of dyes that exhibit thermotropic mesophases. Theoretical approaches to describing the alignment within guest–host systems are outlined, and possible strategies for the future rational design of guest–host systems are discussed.     

Investigation of separation and identification possibilities of some metal-DEDTC complexes by sequential TLC-IR system

The separation and identification possibilities of some metal-diethyldithiocarbamate (DEDTC) complexes are investigated by thin-layer chromatography-IR sequential system. DEDTC complexes of metal cations are prepared in a predetermined optimum pH or pH range. Optimum separation conditions of these complexes and the reproducibilities of their retention factor (Rf) values are investigated on a series of precoated or laboratory-made TLC plates. Rf values are determined using the one-dimensional ascending technique. The precision of the procedures is determined for five replicates in terms of standard deviation. Detection limits are also determined for each of the metal complexes. The IR spectra are scanned and interpreted for their precise identifications after preconcentration with a wick-stick procedure for the complexes, giving the overlapped Rf values. The complexes could be identified by the aid of IR absorption bands. It has been determined that these IR absorption bands can also be used as reference spectral bands in possible quantitative studies. It is concluded that an efficient and successful qualitative analysis is possible for incompletely-separated complex compounds using an IR spectrophotometer as the TLC detector.     

A method for the analysis of intermediates of oxidative hair dyes in cosmetic products

A method has been developed and validated for the analysis of some commonly used intermediates of oxidative hair dyes: phenylenediamines, toluenediamines, aminophenols, 1-Naphthol, resorcinol, and hydroquinone. The target analytes are ion-paired prior to HPLC analysis with gradient elution employing phosphate buffer-acetonitrile as mobile phase and detection in the wavelength range 220–400 nm by a photodiode array detector. A spectral-library, consisting of 220–400 nm spectra of the target substances and their HPLC retention times, has been prepared for the identification. A method of sample preparation was established and applied to the analysis of a series of cosmetic formulations for hair dyeing. The method has been found to be suitable for routine analysis of the target intermediates of oxidative hair dyes, and it may also be suitable for the analysis of some non-target intermediates of these substances.     

Bacterial capture efficiency and antimicrobial activity of phage-functionalized model surfaces

The rise of antibiotic-resistant bacteria has directed substantial attention toward the use of bacteriophages as a means to control bacterial populations. It has been proposed that bacteriophages can be applied as a coating on surfaces in healthcare settings or on indwelling medical devices to create an antimicrobial surface. In this study, antimicrobial model surfaces functionalized with five different types of bacteriophage were prepared and characterized with X-ray photoelectron spectroscopy and atomic force microscopy. The bacterial capture efficiency of these functionalized surfaces was studied for two common bacteria, Escherichia coli and Salmonella typhimurium. Binding of the phages to a solid surface affected their biofunctionality as expressed by the capture efficiency and rate of host membrane disruption. Moreover, the size and shape of the bacteriophage and positioning of its specific binding proteins significantly affected its bacterial capture capability in the immobilized state. Symmetric bacteriophages were found to be a better choice for antibacterial surfaces compared to more asymmetric tailed bacteriophages. Immobilized phages were found to disrupt the membranes of attached bacteria and are thus proposed as a candidate for antimicrobial surfaces.     

Disarming the invader

Type III secretion systems are used by many gram-negative bacterial pathogens of animals and plants to deliver essential virulence factors into targeted host cells. The identification of chemical compounds that block the function of these systems is the first step toward developing chemical attenuation as an effective method for the treatment of infectious disease.     

Rapid detection of Staphylococcus aureus by a combination of monoclonal antibody-coated latex and capillary electrophoresis

The rapid detection of pathogenic bacteria is extremely important in biotechnology and clinical diagnosis. CE has been utilized in the field of bacterial analysis for many years, but to some extent, simultaneous separation and identification of certain microbes from complex samples by CE coupled with UV detector is still a challenge. In this paper, we propose a new strategy for rapid separation and identification of Staphylococcus aureus (S. aureus) in bacterial mixtures by means of specific mAb-coated latex coupled with CZE. An appropriate set of conditions that selectively isolated S. aureus from the microorganisms Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae were established. S. aureus could be differentiated from the others by unique peaks in the electropherograms. The validity was also confirmed by LIF with antibodies specific to both the latex and the microbial cells. The LOD is as low as 9.0 x 10(5) colony forming unit/mL. We have also utilized this technology to identify S. aureus in a stool sample coming from a healthy volunteer spiked successfully with S. aureus. This CZE-UV technique can be applied to rapid diagnosis of enteritis caused by S. aureus or other bacterial control-related fields needing rapid identification of target pathogens from microbial mixtures. In theory, this method is suitable for the detection of any bacterium as long as corresponding bacterium-specific antibody-coated latex is available.     

Microbore liquid chromatography with dual electrochemical detection for the determination of serotonin and 5-hydroxyindoleacetic acid in rat brain dialysates.

A microbore liquid chromatographic assay with dual electrochemical detection is described for the determination of serotonin and its metabolite 5-hydroxyindoleacetic acid in rat brain dialysates. The concentration of serotonin in these samples is usually in the low nanomolar range (fmol per 20 microliters range). To optimize separation and detection, several adaptations were made to the system with respect to the injection valve, flow-rate of the pump, connections between injector, column and detector, and cell volume of the detector. These aspects are discussed, as well as the procedure developed for optimal peak identification of serotonin and correct estimation of 5-hydroxyindoleacetic acid. The assay allows the measurement of basal serotonin release without the use of a re-uptake inhibitor added to the perfusion fluid.     

New approaches to the microscopic imaging of Trypanosoma brucei.

Protozoan parasites are fearsome pathogens responsible for a substantial proportion of human mortality, morbidity, and economic hardship. The principal disease agents are members of the orders Apicomplexa (Plasmodium, Toxoplasma, Eimeria) and Kinetoplastida (Trypanosomes, Leishmania). The majority of humans are at risk from infection from one or more of these organisms, with profound effects on the economy, social structure and quality of life in endemic areas; Plasmodium itself accounts for over one million deaths per annum, and an estimated 4 x 10(7) disability-adjusted life years (DALYs), whereas the Kinetoplastida are responsible for over 100,000 deaths per annum and 4 x 10(6) DALYs. Current control strategies are failing due to drug resistance and inadequate implementation of existing public health strategies. Trypanosoma brucei, the African Trypanosome, has emerged as a favored model system for the study of basic cell biology in Kinetoplastida, because of several recent technical advances (transfection, inducible expression systems, and RNA interference), and these advantages, together with genome sequencing efforts are widely anticipated to provide new strategies of therapeutic intervention. Here we describe a suite of methods that have been developed for the microscopic analysis of T. brucei at the light and ultrastructural levels, an essential component of analysis of gene function and hence identification of therapeutic targets.     

Innate immune sensing of coronavirus and viral evasion strategies

The innate immune system is the first line of the host defense program against pathogens and harmful substances. Antiviral innate immune responses can be triggered by multiple cellular receptors sensing viral components. The activated innate immune system produces interferons (IFNs) and cytokines that perform antiviral functions to eliminate invading viruses. Coronaviruses are single-stranded, positive-sense RNA viruses that have a broad range of animal hosts. Coronaviruses have evolved multiple means to evade host antiviral immune responses. Successful immune evasion by coronaviruses may enable the viruses to adapt to multiple species of host organisms. Coronavirus transmission from zoonotic hosts to humans has caused serious illnesses, such as severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and coronavirus disease-2019 (COVID-19), resulting in global health and economic crises. In this review, we summarize the current knowledge of the mechanisms underlying host sensing of and innate immune responses against coronavirus invasion, as well as host immune evasion strategies of coronaviruses.Subject terms: Infection, Pattern recognition receptors, Immune evasion     

Mass spectrometric identification of toxins of biological origin

The chemical/biological (CB) threat spectrum encompasses a wide range of potential agents including chemical warfare agents, biological warfare agents and toxins of biological origin that fall between these two main agent categories. These proteinaceous and non-proteinaceous toxins, commonly referred to as mid-spectrum agents, range in molecular mass from a few hundred to more than a hundred thousand daltons. The large number of potential candidates as well as the structural diversity of possible mid-spectrum agents makes identification of these compounds a challenge. The NATO defense community has recognized these challenges and has a working group that is developing identification protocols and evaluating methods through a series of international analytical exercises. Identification strategies rely heavily on recent advances that have been made in both mass spectrometry (MS) and liquid chromatography (LC), with LC-MS typically being employed as the primary method for separation/identification. While this paper focuses on the application of these and related instrumental analytical techniques for the identification of mid-spectrum agents, the approach described could be applied in the fields of toxicology, forensic science and environmental analysis. Areas for future research have been identified and application of developed mid-spectrum identification methods to the ongoing biological and toxin weapons convention (BTWC) are anticipated.     

A simple multisensor detector based on tin dioxide in capillary gas chromatography

The use of an array of metal oxide film sensors as a detector for volatile organic compounds in capillary gas chromatography is described. The results of determination of the sensitivity, response time, and linearity range of the analytical signals from sensors are presented; these parameters are compared with similar characteristics of commercial gas chromatographic detectors. An approach is proposed to the identification of organic compounds by the shape and relative size of responses of individual sensors. It is demonstrated that the multisensor detector is applicable to solving typical analytical problems of the quantitative analysis of multicomponent mixtures.     

Pulsed discharge emission detector: an element-selective detector for gas chromatography

An element-specific pulsed discharge emission detector (PDED) has been coupled directly with a vacuum UV monochromator so that vacuum UV atomic emissions from Cl, Br, I and S can be observed. The observed sensitivities for the elements are in the range of mid to high pg/s, but can be lowered by direct absorption of the radiation using a vacuum UV radiation photomultiplier tube. A helium pulsed discharge photoionization detector (He-PDPID) was run simultaneously in parallel with the PDED. The chromatograms recorded with the two detectors had similar peak shapes, suggesting that there is no peak tailing in the PDED. The ratio of the detector responses PDED/He-PDPID can be used for qualitative identification of the Cl-, Br-, I- or S-containing compounds.     

Identification of pesticide residues in real matrices by combining retention indices and specific multidetection responses

A previous paper reported the possibility of using the ratio between the responses of two different specific detectors to a single compound (detector response ratio, DRR) as an identification criterion for that compound, in combination with its retention index. This article gives some applications of DRR in the identification of some pesticides in four different fruits (apples, apricots, strawberries, and peaches) in a range varying from 0.02 to 1.3 ppm, by means of their ECD/FPD DRRs. After clean-up GC analyses were performed on a single column with dual parallel detection; a personal computer system was used for data analysis.     

Determination of free intracellular amino acids in single mouse peritoneal macrophages after naphthalene-2,3-dicarboxaldehyde derivatization by capillary zone electrophoresis with electrochemical detection.

A method is described for the direct identification and quantification of amino acids in individual mouse peritoneal macrophages by capillary zone electrophoresis with electrochemical detection after on-column derivatization with naphthalene-2,3-dicarboxaldehyde (NDA) and CN-. In this method, individual macrophages and then the lysing/ derivatizing buffer are injected into the front end of the separation capillary by electromigration with the aid of an inverted microscope. The front end of the separation capillary is used as a chamber to lyse the macrophage and derivatize its contents, which minimizes dilution of amino acids of a single macrophage during derivatization. Six amino acids (serine, alanine, taurine, glycine, glutamic acid, and aspartic acid) in single mouse peritoneal macrophages have been identified. Quantitation has been accomplished through the use of calibration curves, where the concentration ratios of these standard amino acids are similar to the concentration ratios of amino acids in macrophages. Cellular levels of the amino acids in these cells range from 0.27 +/- 0.20 fmol/ cell for alanine to 6.4 +/- 4.6 fmol/cell for taurine.