Lacticin 3147--biosynthesis, molecular analysis, immunity, bioengineering and applications

The continuing problem of the emergence of multidrug resistance in pathogens has resulted in renewed efforts to identify novel antimicrobials that could be used in clinical settings. Lantibiotics are bacterially produced gene encoded antimicrobial peptides which have been the focus of extensive investigation in recent years because of their broad spectrum of activity. Lantibiotics (lanthionine-containing antibiotics), which have traditionally been regarded as antimicrobials for use in food or veterinary medicine, may provide at least part of the solution to these problems. Lacticin 3147 is a two peptide lantibiotic (consisting of the peptides Ltnα and Ltnβ) which is active at low concentrations against many pathogens. It has been the subject of extensive research, which has generated significant insights into the mechanisms of lacticin 3147 biosynthesis, immunity, structure function relationships and the consequences of molecular bioengineering. The merits of employing lacticin 3147 to control spoilage microbes as well as its potential in the elimination of food, human and veterinary pathogens have also been highlighted. Here we review the knowledge which has been gained with respect to lacticin 3147 since its discovery in 1995.     

The Nargenicin Family of Oxa-Bridged Macrolide Antibiotics

The nargenicin family of antibiotic macrolides comprise a group of bacterial natural products with a rare ether bridged cis-decalin moiety and a narrow spectrum of activity. Most family members were identified almost four decades ago and were placed on the shelf due to the numbers of broad-spectrum compounds available at the time. However, in light of rising rates of antimicrobial resistance, there has been a renewed interest in the use of narrow-spectrum antimicrobials. Here, we review the history of this family of compounds, including synthetic approaches, and highlight the recently uncovered genetic basis for nargenicin production. Given the renewed pharmaceutical interest in these compounds, we also investigate structure–activity relationships among these molecules, with a view to the future development of members of this unusual antibiotic family.     

Antibiotic use in food animals: controlling the human health impact

Resistance to antimicrobial drugs has compromised control of many bacterial pathogens. For foodborne pathogens, the most likely source of resistance is use of antimicrobials in food-producing animals. To control the human health impact from use of antimicrobials in animals, the U.S. Food and Drug Administration (FDA) recently announced plans to assess the microbial safety of all antimicrobials intended for use in food-producing animals. This paper describes the history of antimicrobial use and regulation in animals, the public health concern, the current animal drug approval process in the United States, the international perspective, and FDA's proposed procedures to evaluate the human health impact of the antimicrobial effects associated with animal drugs intended for use in food-producing animals. The primary public health goal of the improved regulatory paradigm is to ensure that significant human antimicrobial therapies are not lost due to use of antimicrobials in food animals.     

Identification and design of antimicrobial peptides for therapeutic applications

Indiscriminate use of antibiotics has led to a rapid increase of antibiotic resistance among microbes which has increased the need to develop novel antimicrobial agents to fight various infectious diseases. Peptide antibiotics signify a novel class of therapeutic agents and have been isolated from a wide variety of multi-cellular organisms. Peptide antibiotics have shown broad-spectrum antimicrobial activity and they not only kill different bacteria, but also kill various fungi, parasites, protozoans and cancerous cells. Peptides bear several properties that make them particularly attractive such as their small size, rapid activity and a low chance for development of resistance. Because of these distinct properties, the focus for research on antimicrobial peptides has increased tremendously in the recent years. Despite their potential, only selected cationic antimicrobial peptides have been able to enter in clinical trials. Therefore, there is a pressing need to develop new approaches to identify novel antimicrobial peptide therapeutics replacing conventional antibiotics. Recent findings strongly suggest that one can design a new generation of antimicrobials peptides with a wide range of systemic and topical applications against bacterial infections. In this review, we focus on the identification and design of novel antimicrobial peptides for therapeutic applications based on different approaches and strategies. This review also highlights some recent advances in the study of the molecular basis of anti-microbial activity in these peptides, their current pharmacological and clinical development and future directions and applications.     

Induced and photoinduced DNA damage by quinolones: ciprofloxacin, ofloxacin and nalidixic acid determined by comet assay

Quinolones are degraded by light with loss of their antimicrobial activity, generating active species or radicals. Evidence exists that some fluoroquinolones (lomefloxacin, fleroxacin and enoxacin) induce damage to the cellular membrane and DNA cleavage by photosensitization. In this study, the genotoxic potential of the quinolones ofloxacin, nalidixic acid and ciprofloxacin (three antimicrobials frequently used in therapy) was evaluated upon irradiation with UV light by using the comet assay on cells of the Jurkat line. The results demonstrate that there are significant differences between the control groups (positive control with 50 microM H2O2, negative controls without drug and with and without irradiation) and the groups of irradiated quinolones (ofloxacin 2.76 x 10(-5) M, nalidixic acid 2.15 x 10(-4) M and ciprofloxacin 2.01 x 10(-5) M), indicating that, at the dose of irradiation employed (necessary to produce 50% photodegradation), the photodecomposition of the quinolones enhanced DNA damage. The unirradiated drugs also exhibited genotoxicity significantly different from that of the negative control.     

Antimicrobial activity and cytotoxic effects of Magnolia dealbata and its active compounds

Multi-drug resistance is of great concern for public health worldwide and necessitates the search for new antimicrobials from sources such as plants. Several Magnolia (Magnoliaceae) species have been reported to exert antimicrobial effects on sensitive and multidrug-resistant microorganisms. However, the antimicrobial properties of Magnolia dealbata have not been experimentally evaluated. The antimicrobial effects of an ethanol extract of Magnolia dealbata seeds (MDE) and its active compounds honokiol (HK) and magnolol (MG) were tested against the phytopathogen Clavibacter michiganensis subsp. michiganensis and several human multi-drug resistant pathogens using the disk-diffusion assay. The effects of MDE and its active compounds on the viability of human peripheral blood mononuclear cells (PBMC) were evaluated using MTT assay. MDE and its active compounds had antimicrobial activity (inhibition zone > 10 mm) against C. michiganensis, Pseudomonas aeruginosa, Acinetobacter baumannii, Acinetobacter lwoffii, Candida albicans, Candida tropicalis and Trichosporon belgeii. The results suggest that M. dealbata and its active compounds have selective antimicrobial effects against drug-resistant fungal and Gram (-) bacteria and exert minimal toxic effects on human PMBC.     

Electrophoretic behaviour of quinolones in capillary electrophoresis. Effect of pH and evaluation of ionization constants.

Quinolones are a family of antibacterial agents that are used extensively in both human and veterinary clinics. Their antibacterial activity is pH-dependent, and therefore an examination of protonation equilibria in quinolone solution is essential. In this work, dissociation constants of quinolones in water were obtained using capillary electrophoresis (CE). The method is based on measuring the electrophoretic mobility of the solute as a function of pH. Mobility and pH data are fitted using different models. These developed equations have two advantages. They permit the determination of pKa of analytes with the advantages of CE and also permit the prediction of the effect of pH on the electrophoretic behaviour of substances and then the prediction of the pH optimum for the separation methods, using the minimum of experimental measurements.     

An overview of sample preparation procedures for LC-MS multiclass antibiotic determination in environmental and food samples

Antibiotics are a class of pharmaceuticals that are of great interest due to the large volumes of these substances that are consumed in both human and veterinary medicine, and due to their status as the agents responsible for bacterial resistance. They can be present in foodstuffs and in environmental samples as multicomponent chemical mixtures that exhibit a wide range of mechanisms of action. Moreover, they can be transformed into different metabolites by the action of microorganisms, as well as by other physical or chemical means, resulting in mixtures with higher ecotoxicities and risks to human health than those of the individual compounds. Therefore, there is growing interest in the availability of multiclass methods for the analysis of antimicrobial mixtures in environmental and food samples at very low concentrations. Liquid chromatography (LC) has become the technique of choice for multiclass analysis, especially when coupled to mass spectrometry (LC-MS) and tandem MS (LC-MS²). However, due to the complexity of the matrix, in most cases an extraction step for sample clean-up and preconcentration is required before analysis in order to achieve the required sensitivities. This paper reviews the most recent developments and applications of multiclass antimicrobial determination in environmental and food matrices, emphasizing the practical aspects of sample preparation for the simultaneous extraction of antimicrobials from the selected samples. Future trends in the application of LC-MS-based techniques to multiclass antibiotic analysis are also presented.     

Determination of antimicrobial residues and metabolites in the aquatic environment by liquid chromatography tandem mass spectrometry

Antimicrobials are used in large quantities in human and veterinary medicine. Their environmental occurrence is of particular concern due to the potential spread and maintenance of bacterial resistance. After intake by the organisms, the unchanged drug and its metabolized forms are excreted and enter wastewater treatment plants where they are mostly incompletely eliminated, and are therefore eventually released into the aquatic environment. The reliable detection of several antimicrobials in different environmental aqueous compartments is the result of great improvements achieved in analytical chemistry. This article provides an overview of the more outstanding analytical methods based on liquid chromatography tandem mass spectrometry, developed and applied to determine antimicrobial residues and metabolites present in surface, waste, and ground waters.      

A review of analytical methods for the determination of aminoglycoside and macrolide residues in food matrices

The development of antibiotic resistance in bacteria has been attributed to the overuse of antimicrobials in human medicine. Another route by which humans are exposed to antibiotics is through the animal foods we eat. In modern agricultural practice, veterinary drugs are being used on a large scale, administered for treating infection or prophylactically to prevent infection. Hence, there is pressure on analytical scientists to detect and confirm the presence of antimicrobials in foods of animal origin.The aminoglycosides and macrolides are two families of antibiotics, each with important applications in veterinary medicine. These antibiotics are widely used in the treatment of bacterial disease, e.g., aminoglycosides for mastitis and macrolides for enteric infections. They have also been used as feed additives for growth promotion. As a result, legislation has been laid down by the European commission in which member states must meet strict criteria for monitoring residues (including antimicrobials). Testing for low levels of aminoglycosides and macrolides in foods is a priority and hence the development of fast, reliable, sensitive methods for their extraction and subsequent analysis is of great interest.This paper reviews analytical methods for both extracting and determining these classes of antibiotics in various food matrices focusing in particular on the last 10 years. Extraction and clean-up methods such as deproteinisation, and solid-phase extraction are described. Various screening methods are also covered including thin layer chromatography (TLC), enzyme immunoassay, capillary electrophoresis (CE) and microbiological assays. Finally, liquid chromatography (LC) methods are discussed which are combined with mass spectrometry (MS) when sensitivity requirements are stringent.     

Chromatographic analysis of penicillins in pharmaceutical formulations and biological fluids

Natural penicillin (benzylpenicillin) is the oldest antibiotic observed by Alexander Fleming in 1928. To broaden its spectrum of activity, natural penicillin was modified, giving rise to a group of antibiotics under the name 'penicillins'. Although an increasing number of bacteria appear to be resistant to them, penicillins are used to treat a variety of bacterial infections including Gram-positive, Gram-negative aerobic and anaerobic bacteria. Consequently, they are widely used in human and veterinary medicine to prevent and treat diseases. This review covers the analytical methodologies, mainly chromatographic, employed to the penicillins determination in pharmaceutical formulations, biological fluids and in production-scale fermentations reported in the literature. Results of published assays are comparatively presented focusing on sample preparation regarding isolation and purification, chromatographic conditions and method validation. Information on chemical structure, spectrum of activity and action mechanism of common penicillins has also been given.     

More QACs,more questions: Recent advances in structure activity relationships and hurdles in understanding resistance mechanisms

Quaternary ammonium compounds (QACs) are a class of antimicrobials that have been around for over a century; nevertheless, they have found continued renewal in the structures to which they can be appended. Ranging from antimicrobial polymers to adding novel modes of action to existing antibiotics, QACs have found ongoing use due to their potent properties. However, resistance against QACs has begun to emerge, and the mechanism of resistance is still only partially understood. In this review, we aim to summarize the current state of the field and what is known about the mechanisms of resistance so that the QACs of the future can be designed to be evermore efficacious and utilized to unearth the remaining mysteries that surround bacteria’s resistance to them.     

Structure and Functional Characterisation of a Distinctive β-Lactamase from an Environmental Strain EMB20 of <Emphasis Type="Italic">Bacillus cereus</Emphasis>

The rampant use and misuse of antibiotics in human medicine, agriculture and veterinary have become the key contributors to global antimicrobial resistance. One of the significant resistance mechanisms that inactivates antibiotics and impedes treatment of bacterial infections is the expression of β-lactamases. Rising evidence of newer variants of β-lactamases in the environment is therefore a serious threat to the presently available antibiotic armoury. The present work describes the purification of a variant β-lactamase isolated from a soil strain EMB20 of Bacillus cereus. The lactamase was purified using three-phase partitioning and gel filtration chromatography to a 30-fold purification and 15% recovery yield. Contrary to the general trend, the lactamase was not a metalloenzyme, but its activity was enhanced in the presence of Mg²⁺ and Mn²⁺. The EMB20 lactamase exhibited improved stability against inhibitors and denaturing agents such as urea and GdmCl as compared to its commercial analogue. The improved stability of EMB20 lactamase was further validated by circular dichroism and fluorescence spectroscopy. This study reemphasizes the rising prevalence of environmental lactamase variants. Decoding the structure–function correlation of such lactamases in the presence of inhibitors will provide insights into the response of this enzyme towards inhibitors as well as its substrates.     

Multiclass method for the quantification of 92 veterinary antimicrobial drugs in livestock excreta,wastewater, and surface water by liquid chromatography with tandem mass spectrometry

A simple multiresidue method was developed for detecting and quantifying 92 veterinary antimicrobial drugs from eight classes (β‐lactams, quinolones, sulfonamides, tetracyclines, lincomycins, macrolides, chloramphenicols, and pleuromutilin) in livestock excreta and water by liquid chromatography with tandem mass spectrometry. The feces samples were extracted by ultrasound‐assisted extraction with a mixture of acetonitrile/water (80:20, v/v) and edetate disodium, followed by a cleanup using solid‐phase extraction with an amino cartridge. Water samples were purified with hydrophilic–lipophilic balance solid‐phase extraction column. Urine samples were extracted with acetonitrile and edetate disodium. Detection of veterinary antimicrobial drugs was achieved by liquid chromatography with tandem mass spectrometry using both positive and negative electrospray ionization mode. The recovery values of veterinary antimicrobial drugs in feces, urine, and water samples were 75–99, 85–110, and 85–101% and associated relative standard deviations were less than 15, 10, and 8%, respectively. The limits of quantification in feces, urine, and water samples were 0.5–1, 0.5–1, and 0.01–0.05 μg/L, respectively. This method was applied to determine real samples obtained from local farms and provides reliable quantification and identification results of 92 veterinary antimicrobial drugs in livestock excreta and water.     

Implementation of terbium-sensitized luminescence in sequential-injection analysis for automatic analysis of orbifloxacin

Orbifloxacin (ORBI) is a third-generation fluoroquinolone developed exclusively for use in veterinary medicine, mainly in companion animals. This antimicrobial agent has bactericidal activity against numerous gram-negative and gram-positive bacteria. A few chromatographic methods for its analysis have been described in the scientific literature. Here, coupling of sequential-injection analysis and solid-phase spectroscopy is described in order to develop, for the first time, a terbium-sensitized luminescent optosensor for analysis of ORBI. The cationic resin Sephadex-CM C-25 was used as solid support and measurements were made at 275/545 nm. The system had a linear dynamic range of 10-150 ng mL(-1), with a detection limit of 3.3 ng mL(-1) and an R.S.D. below 3% (n = 10). The analyte was satisfactorily determined in veterinary drugs and dog and horse urine.     

Prediction of electrophoretic behaviour of a series of quinolones in aqueous methanol.

Quinolones are a family of antibacterial agents used in human and veterinary clinics. The examination of protonation equilibria is essential because their antibacterial activity is pH-dependent. In this work, dissociation constants of quinolones in MeOH-water mixtures were obtained using capillary electrophoresis. The method is based on a model that relates electrophoretic mobility of the solute with pH. The effect of pH, pKa and activity coefficient on electrophoretic behaviour was considered. Standard pH values for buffer solutions were previously determined in MeOH-water mixtures, and the pH can thus be measured in these media as in water. This model is also used to obtain the optimum conditions for the separation of a series of substances because it allows one to predict the resolution between adjacent peaks from a few experimental data.     

New Derivatives of 5-Substituted Uracils: Potential Agents with a Wide Spectrum of Biological Activity

Pyrimidine nucleoside analogues are widely used to treat infections caused by the human immunodeficiency virus (HIV) and DNA viruses from the herpes family. It has been shown that 5-substituted uracil derivatives can inhibit HIV-1, herpes family viruses, mycobacteria and other pathogens through various mechanisms. Among the 5-substituted pyrimidine nucleosides, there are not only the classical nucleoside inhibitors of the herpes family viruses, 2′-deoxy-5-iodocytidine and 5-bromovinyl-2′-deoxyuridine, but also derivatives of 1-(benzyl)-5-(phenylamino)uracil, which proved to be non-nucleoside inhibitors of HIV-1 and EBV. It made this modification of nucleoside analogues very promising in connection with the emergence of new viruses and the crisis of drug resistance when the task of creating effective antiviral agents of new types that act on other targets or exhibit activity by other mechanisms is very urgent. In this paper, we present the design, synthesis and primary screening of the biological activity of new nucleoside analogues, namely, 5′-norcarbocyclic derivatives of substituted 5-arylamino- and 5-aryloxyuracils, against RNA viruses.     

Pharmacophore generation of 2-substituted benzothiazoles as AdeABC efflux pump inhibitors in A. baumannii

RND family efflux pumps are important for multidrug resistance in Gram-negative bacteria. To date no efflux pump inhibitors for clinical use have been found, so developing the specific inhibitors of this pump system will be beneficial for the treatment of infections caused by these multidrug-resistant pathogens. A set of BSN-coded 2-substituted benzothiazoles were tested alone and in combination with ciprofloxacin (CIP) against the RND family efflux pump AdeABC overexpressor Acinetobacter baumannii SbMox-2 strain. The results indicated that the BSN compounds did not have antimicrobial activity when tested alone. However, if they were applied in combination with CIP, it was observed that the antibiotic had antimicrobial activity against the tested pathogen, possessing a minimum inhibitory concentration value that could be utilized in clinical treatment. A 3D-common features pharmacophore model was applied by using the HipHop method and the generated pharmacophore hypothesis revealed that the hydrogen bond acceptor property of nitrogen in the thiazole ring and the oxygen of the amide substituted at the second position of the benzothiazole ring system were significant for binding to the target protein. Moreover, three hydrophobic aromatic features were found to be essential for inhibitory activity.     

Quinolone Antibiotic Photodynamic Production of 8-Oxo-7,8-dihydro-2'-deoxyguanosine in Cultured Liver Epithelial Cells

To study the basis for the phototoxicity of quinolones, a class of synthetic antibacterials, the photodynamic ability to mediate 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dG) formation in cultured cells was measured for lome-floxacin (LMX), which is strongly associated with clinical phototoxicity in humans, and ciprofloxacin (CFX), which has few reports of phototoxicity. Adult rat liver (ARL-18) cells were exposed to the quinolones in the presence of UVA and DNA was extracted and analyzed by HPLC with electrochemical detection. Low levels of 8-oxo-dG were found in the DNA of nonirradiated ARL-18 cells and this was increased up to 6-fold in the presence of either LMX (50–400 uAf) or up to 3.6-fold in the presence of CFX (50–400 µM) and UVA (20 J/cm²) when compared to the UVA control. Comparing separate experiments with LMX and CFX, LMX produced greater levels of 8-oxo-dG either after dark exposure or after UVA exposure at 20 J/cm². Also, LMX and CFX were both shown to photodegrade in the presence of UVA, and it was determined that UVA photoinstability alone does not reflect phototoxic potential. These data suggest that the photodynamic potential of LMX and CFX to produce 8-oxo-dG may relate to their human clinical phototoxicity profile. We suggest that the observed clinical phototoxicity is mediated through a UVA photodynamic effect on the quinolone to form reactive oxygen species in the presence of molecular oxygen. The findings indicate that 8-oxo-dG formation can serve as a marker for the potential phototoxicity of new quinolones.     

Preparation and antimicrobial properties of LL‐37 peptide immobilized lignin/caprolactone polymer film

The use of biopolymers has gained priority in tissue engineering and biotechnology, both as dressing material and for enhancing treatment efficiency. There is a demand for new biopolymers designed with protease inhibitors and antimicrobials. LL‐37 is an important antimicrobial peptide in human skin and exhibits a broad spectrum of antimicrobial activity against bacteria, fungi, and viral pathogens. Using lignin which is an abundant carbohydrate polymer in nature and a polyacrylic acid, we prepared a lignin/caprolactone biodegradable film by plastifying caprolactone and polyacyrlic acid. Lignin/caprolactone biodegradable film was activated with CDI and then immobilized LL‐37 peptide. The structure was elucidated in terms of its functional groups by attenuated total reflectance‐fourier transform infrared spectroscopy (ATR‐FTIR), and the morphology of the lignin/caprolactone biodegradable film was characterized by scanning electron microscopy (SEM) before and after the immobilization process. The amount of LL‐37 immobilized was determined by ELISA method. It was found that 97% of LL‐37 peptide was successfully immobilized onto the lignin/caprolactone biodegradable film. Antimicrobial activity was determined in the lignin/caprolactone biodegradable film samples by quantitative antimicrobial activity method. According to the results, LL‐37 immobilized lignin/caprolactone biodegradable film samples were effective on test organisms; Gram‐positive Staphylococcus aureus and Gram‐negative Escherichia coli. In bio‐compatibility assays, the ability to support tissue cell integration was detected by using 3 T3 mouse fibroblasts. Samples were examined under transverse microscope, non‐immobilized sample showed a huge cellular death, whereas LL‐37 immobilized lignin/caprolactone biodegradable film had identical cellular growth with the control group. This dual functional lignin/caprolactone biodegradable film with enhanced antibacterial properties and increased tissue cell compatibility may be used to design new materials for various types of biological applications.