Analysis of antibiotics in fish samples

The use of antibiotics in food-producing animals has generated considerable interest because the widespread administration of these drugs may lead to the development of resistant human pathogens. A large increase in the demand for seafood products has occurred in the last century. This has led to a concomitant increase in high-intensity aquaculture methods, characterized by high stock density and volume, and the heavy use of formulated feeds containing antibiotics, among other substances. Therefore, accurate and sensitive determination of antibiotic residues is now a necessity. In order to protect human health, the European Union and other regulatory authorities worldwide have established maximum residue limits (MRL) for antibiotic residues in animal products entering the human food chain. This paper reviews the most recent methods for analysis of antibiotic residues in fish.     

Amycolamicin: A Novel Broad‐Spectrum Antibiotic Inhibiting Bacterial Topoisomerase

The abuse of antibacterial drugs imposes a selection pressure on bacteria that has driven the evolution of multidrug resistance in many pathogens. Our efforts to discover novel classes of antibiotics to combat these pathogens resulted in the discovery of amycolamicin (AMM). The absolute structure of AMM was determined by NMR spectroscopy, X‐ray analysis, chemical degradation, and modification of its functional groups. AMM consists of trans‐decalin, tetramic acid, two unusual sugars (amycolose and amykitanose), and dichloropyrrole carboxylic acid. The pyranose ring named as amykitanose undergoes anomerization in methanol. AMM is a potent and broad‐spectrum antibiotic against Gram‐positive pathogenic bacteria by inhibiting DNA gyrase and bacterial topoisomerase IV. The target of AMM has been proved to be the DNA gyrase B subunit and its binding mode to DNA gyrase is different from those of novobiocin and coumermycin, the known DNA gyrase inhibitors.     

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.     

Monitoring bacterial resistance to chloramphenicol and other antibiotics by liquid chromatography electrospray ionization tandem mass spectrometry using selected reaction monitoring

Antibiotic resistance is a growing problem worldwide. For this reason, clinical laboratories often determine the susceptibility of the bacterial isolate to a number of different antibiotics in order to establish the most effective antibiotic for treatment. Unfortunately, current susceptibility assays are time consuming. Antibiotic resistance often involves the chemical modification of an antibiotic to an inactive form by an enzyme expressed by the bacterium. Selected reaction monitoring (SRM) has the ability to quickly monitor and identify these chemical changes in an unprecedented time scale. In this work, we used SRM as a technique to determine the susceptibility of several different antibiotics to the chemically modifying enzymes β‐lactamase and chloramphenicol acetyltransferase, enzymes used by bacteria to confer resistance to major classes of commonly used antibiotics. We also used this technique to directly monitor the effects of resistant bacteria grown in a broth containing a specific antibiotic. Because SRM is highly selective and can also identify chemical changes in a multitude of antibiotics in a single assay, SRM has the ability to detect organisms that are resistant to multiple antibiotics in a single assay. For these reasons, the use of SRM greatly reduces the time it takes to determine the susceptibility or resistance of an organism to a multitude of antibiotics by eliminating the time‐consuming process found in other currently used methods. Copyright © 2013 John Wiley & Sons, Ltd.     

Targeting Antibiotic Resistance

Finding strategies against the development of antibiotic resistance is a major global challenge for the life sciences community and for public health. The past decades have seen a dramatic worldwide increase in human‐pathogenic bacteria that are resistant to one or multiple antibiotics. More and more infections caused by resistant microorganisms fail to respond to conventional treatment, and in some cases, even last‐resort antibiotics have lost their power. In addition, industry pipelines for the development of novel antibiotics have run dry over the past decades. A recent world health day by the World Health Organization titled “Combat drug resistance: no action today means no cure tomorrow” triggered an increase in research activity, and several promising strategies have been developed to restore treatment options against infections by resistant bacterial pathogens.     

In-vitro antibacterial properties of crude aqueous and n-hexane extracts of the husk of Cocos nucifera

The increasing numbers of cases of antibiotic resistance among pathogenic bacteria such as Vibrio species poses a major problem to the food and aquaculture industries, as most antibiotics are no longer effective in controlling pathogenic bacteria affecting these industries. Therefore, this study was carried out to assess the antibacterial potentials of crude aqueous and n-hexane extracts of the husk of Cocos nucifera against some selected Vibrio species and other bacterial pathogens including those normally implicated in food and wound infections. The crude extracts were screened against forty-five strains of Vibrio pathogens and twenty-five other bacteria isolates made up of ten Gram positive and fifteen Gram negative bacteria. The aqueous extract was active against 17 of the tested bacterial and 37 of the Vibrio isolates; while the n-hexane extract showed antimicrobial activity against 21 of the test bacteria and 38 of the test Vibrio species. The minimum inhibitory concentrations (MICs) of the aqueous and n-hexane extracts against the susceptible bacteria ranged between 0.6-5.0 mg/mL and 0.3-5.0 mg/mL respectively, while the time kill study result for the aqueous extract ranged between 0.12 Log?? and 4.2 Log?? cfu/mL after 8 hours interaction in 1 x MIC and 2 x MIC. For the n-hexane extract, the log reduction ranged between 0.56 Log?? and 6.4 Log?? cfu/mL after 8 hours interaction in 1 x MIC and 2 x MIC. This study revealed the huge potential of C. nucifera extracts as alternative therapies against microbial infections.     

New Antibiotics for Multidrug-Resistant Bacterial Strains: Latest Research Developments and Future Perspectives

The present work aims to examine the worrying problem of antibiotic resistance and the emergence of multidrug-resistant bacterial strains, which have now become really common in hospitals and risk hindering the global control of infectious diseases. After a careful examination of these phenomena and multiple mechanisms that make certain bacteria resistant to specific antibiotics that were originally effective in the treatment of infections caused by the same pathogens, possible strategies to stem antibiotic resistance are analyzed. This paper, therefore, focuses on the most promising new chemical compounds in the current pipeline active against multidrug-resistant organisms that are innovative compared to traditional antibiotics: Firstly, the main antibacterial agents in clinical development (Phase III) from 2017 to 2020 are listed (with special attention on the treatment of infections caused by the pathogens Neisseria gonorrhoeae, including multidrug-resistant isolates, and Clostridium difficile), and then the paper moves on to the new agents of pharmacological interest that have been approved during the same period. They include tetracycline derivatives (eravacycline), fourth generation fluoroquinolones (delafloxacin), new combinations between one β-lactam and one β-lactamase inhibitor (meropenem and vaborbactam), siderophore cephalosporins (cefiderocol), new aminoglycosides (plazomicin), and agents in development for treating drug-resistant TB (pretomanid). It concludes with the advantages that can result from the use of these compounds, also mentioning other approaches, still poorly developed, for combating antibiotic resistance: Nanoparticles delivery systems for antibiotics.     

A β‐Lactamase‐Imprinted Responsive Hydrogel for the Treatment of Antibiotic‐Resistant Bacteria

Antibiotics play important roles in infection treatment and prevention. However, the effectiveness of antibiotics is now threatened by the prevalence of drug‐resistant bacteria. Furthermore, antibiotic abuse and residues in the environment cause serious health issues. In this study, a stimuli‐responsive imprinted hydrogel was fabricated by using β‐lactamase produced by bacteria for deactivating antibiotics as the template molecule. The imprinted hydrogel could initially trap β‐lactamase excreted by drug‐resistant bacteria, thus making bacteria sensitive to antibiotics. After the bactericidal treatment, the “imprinted sites” on the hydrogel could be reversibly abolished with a temperature stimulus, which resulted in the reactivation of β‐lactamase to degrade antibiotic residues. We also present an example of the use of this antibacterial design to treat wound infection.     

Fluorogenic Probes/Inhibitors of β‐Lactamase and their Applications in Drug‐Resistant Bacteria

β‐Lactam antibiotics are generally perceived as one of the greatest inventions of the 20^th century, and these small molecular compounds have saved millions of lives. However, upon clinical application of antibiotics, the β‐lactamase secreted by pathogenic bacteria can lead to the gradual development of drug resistance. β‐Lactamase is a hydrolase that can efficiently hydrolyze and destroy β‐lactam antibiotics. It develops and spreads rapidly in pathogens, and the drug‐resistant bacteria pose a severe threat to human health and development. As a result, detecting and inhibiting the activities of β‐lactamase are of great value for the rational use of antibiotics and the treatment of infectious diseases. At present, many specific detection methods and inhibitors of β‐lactamase have been developed and applied in clinical practice. In this Minireview, we describe the resistance mechanism of bacteria producing β‐lactamase and further summarize the fluorogenic probes, inhibitors of β‐lactamase, and their applications in the treatment of infectious diseases. It may be valuable to design fluorogenic probes with improved selectivity, sensitivity, and effectiveness to further identify the inhibitors for β‐lactamases and eventually overcome bacterial resistance.     

Adjuvants Based on Hybrid Antibiotics Overcome Resistance in Pseudomonas aeruginosa and Enhance Fluoroquinolone Efficacy

The use of adjuvants that rescue antibiotics against multidrug‐resistant (MDR) pathogens is a promising combination strategy for overcoming bacterial resistance. While the combination of β‐lactam antibiotics and β‐lactamase inhibitors has been successful in restoring antibacterial efficacy in MDR bacteria, the use of adjuvants to restore fluoroquinolone efficacy in MDR Gram‐negative pathogens has been challenging. We describe tobramycin–ciprofloxacin hybrid adjuvants that rescue the activity of fluoroquinolone antibiotics against MDR and extremely drug‐resistant Pseudomonas aeruginosa isolates in vitro and enhance fluoroquinolone efficacy in vivo. Structure–activity studies reveal that the presence of both tobramycin and ciprofloxacin, which are separated by a C12 tether, is critical for the function of the adjuvant. Mechanistic studies indicate that the antibacterial modes of ciprofloxacin are retained while the role of tobramycin is limited to destabilization of the outer membrane in the hybrid.     

Plants as sources of new antimicrobials and resistance-modifying agents

Covering: up to November 2011Infections caused by multidrug-resistant bacteria are an increasing problem due to the emergence and propagation of microbial drug resistance and the lack of development of new antimicrobials. Traditional methods of antibiotic discovery have failed to keep pace with the evolution of resistance. Therefore, new strategies to control bacterial infections are highly desirable. Plant secondary metabolites (phytochemicals) have already demonstrated their potential as antibacterials when used alone and as synergists or potentiators of other antibacterial agents. The use of phytochemical products and plant extracts as resistance-modifying agents (RMAs) represents an increasingly active research topic. Phytochemicals frequently act through different mechanisms than conventional antibiotics and could, therefore be of use in the treatment of resistant bacteria. The therapeutic utility of these products, however, remains to be clinically proven. The aim of this article is to review the advances in in vitro and in vivo studies on the potential chemotherapeutic value of phytochemical products and plant extracts as RMAs to restore the efficacy of antibiotics against resistant pathogenic bacteria. The mode of action of RMAs on the potentiation of antibiotics is also described.     

Chimeric streptogramin-tyrocidine antibiotics that overcome streptogramin resistance

Streptogramin antibiotics are comprised of two distinct chemical components: the type A polyketides and the type B cyclic depsipeptides. Clinical resistance to the type B streptogramins can occur via enzymatic degradation catalyzed by the lyase Vgb or by target modification through the action of Erm ribosomal RNA methyltransferases. We have prepared through chemical and chemo-enzymatic approaches a series of chimeric antibiotics composed of elements of type B streptogramins and the membrane-active antibiotic tyrocidine that evade these resistance mechanisms. These new compounds show broad antibiotic activity against gram-positive bacteria including a number of important pathogens, and chimeras appear to function by a mechanism that is distinct from their parent antibiotics. These results allow for the development of a brand new class of antibiotics with the ability to evade type B streptogramin-resistance mechanisms.     

Viability study of clinical bacterial strains by capillary electrophoresis and flow cytometry approaches

One of the challenges medicine faces is the constantly growing resistance of pathogens to various classes of antibiotics. In this study, we investigated the use of capillary electrophoresis (CE) to characterize and assess the physiological states of three clinical bacterial strains—methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive S. aureus (MSSA), and Escherichia coli extended-spectrum β-lactamases (ESβL)—exposed to different antibiotics. All chosen bacteria are the leading causes of healthcare-associated and hospital-acquired invasive infections in adults. In the first part of the research, it was determined the optimal incubation time of the tested strains with antibiotics, represented as an optimal time of 24 h. In the second part, we have compared two approaches: flow cytometry (FC) as a standard method and CE as a proposed alternative approach. The viability of clinical strains treated with different class antibiotics calculated in CE measurements was strongly correlated (>0.83 for MSSA, >0.92 for ESβL and MRSA) with the viability obtained on the basis of FC measurements. As a result, CE has a chance to become a modern diagnostic method used in clinical practice. The CE cutoff was found to be 50%; above this value, the strain shows resistance to the action of the antibiotic.     

A Glycosylated Cationic Block Poly(β-peptide) Reverses Intrinsic Antibiotic Resistance in All ESKAPE Gram-Negative Bacteria

Carbapenem-resistant Gram-negative bacteria (GNB) are heading the list of pathogens for which antibiotics are the most critically needed. Many antibiotics are either unable to penetrate the outer-membrane or are excluded by efflux mechanisms. Here, we report a cationic block β-peptide (PAS8-b-PDM12) that reverses intrinsic antibiotic resistance in GNB by two distinct mechanisms of action. PAS8-b-PDM12 does not only compromise the integrity of the bacterial outer-membrane, it also deactivates efflux pump systems by dissipating the transmembrane electrochemical potential. As a result, PAS8-b-PDM12 sensitizes carbapenem- and colistin-resistant GNB to multiple antibiotics in vitro and in vivo. The β-peptide allows the perfect alternation of cationic versus hydrophobic side chains, representing a significant improvement over previous antimicrobial α-peptides sensitizing agents. Together, our results indicate that it is technically possible for a single adjuvant to reverse innate antibiotic resistance in all pathogenic GNB of the ESKAPE group, including those resistant to last resort antibiotics.     

Late-Stage Modification of Aminoglycoside Antibiotics Overcomes Bacterial Resistance Mediated by APH(3’) Kinases

The continuous emergence of antimicrobial resistance is causing a threat to patients infected by multidrug-resistant pathogens. In particular, the clinical use of aminoglycoside antibiotics, broad-spectrum antibacterials of last resort, is limited due to rising bacterial resistance. One of the major resistance mechanisms in Gram-positive and Gram-negative bacteria is phosphorylation of these amino sugars at the 3’-position by O-phosphotransferases [APH(3’)s]. Structural alteration of these antibiotics at the 3’-position would be an obvious strategy to tackle this resistance mechanism. However, the access to such derivatives requires cumbersome multi-step synthesis, which is not appealing for pharma industry in this low-return-on-investment market. To overcome this obstacle and combat bacterial resistance mediated by APH(3’)s, we introduce a novel regioselective modification of aminoglycosides in the 3’-position via palladium-catalyzed oxidation. To underline the effectiveness of our method for structural modification of aminoglycosides, we have developed two novel antibiotic candidates overcoming APH(3’)s-mediated resistance employing only four synthetic steps.     

Functional analysis of the lipoglycodepsipeptide antibiotic ramoplanin

The peptide antibiotic ramoplanin is highly effective against several drug-resistant gram-positive bacteria, including vancomycin-resistant Enterococcus faecium (VRE) and methicillin-resistant Staphylococcus aureus (MRSA), two important opportunistic human pathogens. Ramoplanin inhibits bacterial peptidoglycan (PG) biosynthesis by binding to Lipid intermediates I and II at a location different than the N-acyl-D-Ala-D-Ala dipeptide site targeted by vancomycin. Lipid I/II capture physically occludes these substrates from proper utilization by the late-stage PG biosynthesis enzymes MurG and the transglycosylases. Key structural features of ramoplanin responsible for antibiotic activity and PG molecular recognition have been discovered by antibiotic semisynthetic modification in conjunction with NMR analyses. These results help define a minimalist ramoplanin pharmacophore and introduce the possibility of generating ramoplanin-derived peptide or peptidomimetic antibiotics for use against VRE, MRSA, and related pathogens.     

Targeting virulence: salmochelin modification tunes the antibacterial activity spectrum of β-lactams for pathogen-selective killing of Escherichia coli

New antibiotics are required to treat bacterial infections and counteract the emergence of antibiotic resistance. Pathogen-specific antibiotics have several advantages over broad-spectrum drugs, which include minimal perturbation to the commensal microbiota. We present a strategy for targeting antibiotics to bacterial pathogens that utilises the salmochelin-mediated iron uptake machinery of Gram-negative Escherichia coli. Salmochelins are C-glucosylated derivatives of the siderophore enterobactin. The biosynthesis and utilisation of salmochelins are important for virulence because these siderophores allow pathogens to acquire iron and evade the enterobactin-scavenging host-defense protein lipocalin-2. Inspired by the salmochelins, we report the design and chemoenzymatic preparation of glucosylated enterobactin–β-lactam conjugates that harbour the antibiotics ampicillin (Amp) and amoxicillin (Amx), hereafter GlcEnt–Amp/Amx. The GlcEnt scaffolds are based on mono- and diglucosylated Ent where one catechol moiety is functionalized at the C5 position for antibiotic attachment. We demonstrate that GlcEnt–Amp/Amx provide up to 1000-fold enhanced antimicrobial activity against uropathogenic E. coli relative to the parent β-lactams. Moreover, GlcEnt–Amp/Amx based on a diglucosylated Ent (DGE) platform selectively kill uropathogenic E. coli that express the salmochelin receptor IroN in the presence of non-pathogenic E. coli and other bacterial strains that include the commensal microbe Lactobacillus rhamnosus GG. Moreover, GlcEnt–Amp/Amx evade the host-defense protein lipocalin-2, and exhibit low toxicity to mammalian cells. Our work establishes that siderophore–antibiotic conjugates provide a strategy for targeting virulence, narrowing the activity spectrum of antibiotics in clinical use, and achieving selective delivery of antibacterial cargos to pathogenic bacteria on the basis of siderophore receptor expression.     

In situ monitoring of antibiotic susceptibility of bacterial biofilms in a microfluidic device

Antibiotic resistance of biofilms is a growing public health concern due to overuse and improper use of antibiotics. Thus, determining an effective minimal concentration of antibiotics to eradicate bacterial biofilms is crucial. Here we present a simple, novel one-pot assay for the analysis of antibiotic susceptibility of bacterial biofilms using a microfluidics system where continuous concentration gradients of antibiotics are generated. The results of minimal biofilm eradication concentration (MBEC) clearly confirm that the concentration required to eradicate biofilm-grown Pseudomonas aeruginosa is higher than the minimal inhibitory concentration (MIC) that has been widely used to determine the lowest concentration of antibiotics against planktonically grown bacteria.     

A Glycosylated Cationic Block Poly(β‐peptide) Reverses Intrinsic Antibiotic Resistance in All ESKAPE Gram‐Negative Bacteria

Carbapenem‐resistant Gram‐negative bacteria (GNB) are heading the list of pathogens for which antibiotics are the most critically needed. Many antibiotics are either unable to penetrate the outer‐membrane or are excluded by efflux mechanisms. Here, we report a cationic block β‐peptide (PAS8‐b‐PDM12) that reverses intrinsic antibiotic resistance in GNB by two distinct mechanisms of action. PAS8‐b‐PDM12 does not only compromise the integrity of the bacterial outer‐membrane, it also deactivates efflux pump systems by dissipating the transmembrane electrochemical potential. As a result, PAS8‐b‐PDM12 sensitizes carbapenem‐ and colistin‐resistant GNB to multiple antibiotics in vitro and in vivo. The β‐peptide allows the perfect alternation of cationic versus hydrophobic side chains, representing a significant improvement over previous antimicrobial α‐peptides sensitizing agents. Together, our results indicate that it is technically possible for a single adjuvant to reverse innate antibiotic resistance in all pathogenic GNB of the ESKAPE group, including those resistant to last resort antibiotics.