Measuring Efflux and Permeability in Mycobacteria

Mycobacteria are intrinsically resistant to most antimicrobials, which is generally attributed to the impermeability of their cell wall that considerably limits drug uptake. Moreover, like in other pathogenic bacteria, active efflux systems have been widely characterized from diverse mycobacterial species in laboratory conditions, showing that they can promote resistance by extruding noxious compounds prior to their reaching their intended targets. Therefore, the intracellular concentration of a given compound is determined by the balance between permeability, influx, and efflux. Given the urgent need to discover and develop novel antimycobacterial compounds in order to design effective therapeutic strategies, the contributions to drug resistance made by the controlled permeability of the cell wall and the increased activity of efflux pumps must be determined. In this chapter, we will describe a method that allows (1) the measuring of permeability and the quantification of general efflux activity of mycobacteria, by the study of the transport (influx and efflux) of fluorescent compounds, such as ethidium bromide; and (2) the screening of compounds in search of agents that increase the permeability of the cell wall and efflux inhibitors that could restore the effectiveness of antimicrobials that are subject to efflux

Low-fat fresh sausage from rabbit meat: An alternative to traditional rabbit consumption

The study aimed at the development of fresh sausages using rabbit exclusively as raw material. The idea was to offer an innovative product to increase rabbit consumption. Also, to meet currently consumers' requirements, a low-fat version was made. Two final formulations, a control sausage and a low-fat version using konjac gum, were developed through an iterative process and stored in a MAP under refrigeration. Sensory, microbiological and physicochemical analyses were carried out on days 1, 6, 8 and 13 after packaging. The shelf-life of the sausages was determined according to a multivariate criterion. Results showed a significant reduction in fat content and energy value. Sensory analysis showed a decrease in characteristic aroma and flavour and an increase in rancid odour, while hardness and fragility decreased in the low-fat treatment. The shelf-life was 7 days for all treatments, concluding that the multivariate method was a powerful technique as physicochemical, microbiological and sensory criteria were considered

Co-delivery of free vancomycin and transcription factor decoy-nanostructured lipid carriers can enhance inhibition of methicillin resistant Staphylococcus aureus (MRSA)

Bacterial resistance to antibiotics is widely regarded as a major public health concern with last resort MRSA treatments like vancomycin now encountering resistant strains. TFDs (Transcription Factor Decoys) are oligonucleotide copies of the DNA-binding sites for transcription factors. They bind to and sequester the targeted transcription factor, thus inhibiting transcription of many genes. By developing TFDs with sequences aimed at inhibiting transcription factors controlling the expression of highly conserved bacterial cell wall proteins, TFDs present as a potential method for inhibiting microbial growth without encountering typical resistance mechanisms. However, the efficient protection and delivery of the TFDs inside the bacterial cells is a critical step for the success of this technology. Therefore, in our study, specific TFDs against S. aureus were complexed with two different types of nanocarriers: cationic nanostructured lipid carriers (cNLCs) and chitosan-based nanoparticles (CS-NCs). These TFD-carrier nanocomplexes were characterized for size, zeta potential and TFD complexation or loading efficiency in a variety of buffers. In vitro activity of the nanocomplexes was examined alone and in combination with vancomycin, first in methicillin susceptible strains of S. aureus with the lead candidate advancing to tests against MRSA cultures. Results found that both cNLCs and chitosan-based carriers were adept at complexing and protecting TFDs in a range of physiological and microbiological buffers up to 72 hours. From initial testing, chitosan-TFD particles demonstrated no visible improvements in effect when co-administered with vancomycin. However, co-delivery of cNLC-TFD with vancomycin reduced the MIC of vancomycin by over 50% in MSSA and resulted in significant decreases in viability compared with vancomycin alone in MRSA cultures. Furthermore, these TFD-loaded particles demonstrated very low levels of cytotoxicity and haemolysis in vitro. To our knowledge, this is the first attempt at a combined antibiotic/oligonucleotide-TFD approach to combatting MRSA and, as such, highlights a new avenue of MRSA treatment combining traditional small molecules drugs and bacterial gene inhibition

Mycobacterial Aminoglycoside Acetyltransferases: A Little of Drug Resistance, and a Lot of Other Roles

Aminoglycoside acetyltransferases are important determinants of resistance to aminoglycoside antibiotics in most bacterial genera. In mycobacteria, however, aminoglycoside acetyltransferases contribute only partially to aminoglycoside susceptibility since they are related with low level resistance to these antibiotics (while high level aminoglycoside resistance is due to mutations in the ribosome). Instead, aminoglycoside acetyltransferases contribute to other bacterial functions, and this can explain its widespread presence along species of genus Mycobacterium. This review is focused on two mycobacterial aminoglycoside acetyltransferase enzymes. First, the aminoglycoside 2′-N-acetyltransferase [AAC(2′)], which was identified as a determinant of weak aminoglycoside resistance in M. fortuitum, and later found to be widespread in most mycobacterial species; AAC(2′) enzymes have been associated with resistance to cell wall degradative enzymes, and bactericidal mode of action of aminoglycosides. Second, the Eis aminoglycoside acetyltransferase, which was identified originally as a virulence determinant in M. tuberculosis (enhanced intracellular survival); Eis protein in fact controls production of pro-inflammatory cytokines and other pathways. The relation of Eis with aminoglycoside susceptibility was found after the years, and reaches clinical significance only in M. tuberculosis isolates resistant to the second-line drug kanamycin. Given the role of AAC(2′) and Eis proteins in mycobacterial biology, inhibitory molecules have been identified, more abundantly in case of Eis. In conclusion, AAC(2′) and Eis have evolved from a marginal role as potential drug resistance mechanisms into a promising future as drug targets

Repurposing β-Lactams for the Treatment of <i>Mycobacterium kansasii</i> Infections: An In Vitro Study

Mycobacterium kansasii (Mkn) causes tuberculosis-like lung infection in both immunocompetent and immunocompromised patients. Current standard therapy against Mkn infection is lengthy and difficult to adhere to. Although β-lactams are the most important class of antibiotics, representing 65% of the global antibiotic market, they have been traditionally dismissed for the treatment of mycobacterial infections, as they were considered inactive against mycobacteria. A renewed interest in β-lactams as antimycobacterial agents has shown their activity against several mycobacterial species, including M. tuberculosis, M. ulcerans or M. abscessus; however, information against Mkn is lacking. In this study, we determined the in vitro activity of several β-lactams against Mkn. A selection of 32 agents including all β-lactam chemical classes (penicillins, cephalosporins, carbapenems and monobactams) with three β-lactamase inhibitors (clavulanate, tazobactam and avibactam) were evaluated against 22 Mkn strains by MIC assays. Penicillins plus clavulanate and first- and third-generation cephalosporins were the most active β-lactams against Mkn. Combinatorial time-kill assays revealed favorable interactions of amoxicillin–clavulanate and cefadroxil with first-line Mkn treatment. Amoxicillin–clavulanate and cefadroxil are oral medications that are readily available, and well tolerated with an excellent safety and pharmacokinetic profile that could constitute a promising alternative option for Mkn therapy

Overcoming the Prokaryote/Eukaryote Barrier in Tuberculosis Treatment: A Prospect for the Repurposing and Use of Antiparasitic Drugs

Antimicrobial resistance, the so-called silent pandemic, is pushing industry and academia to find novel antimicrobial agents with new mechanisms of action in order to be active against susceptible and drug-resistant microorganisms. In the case of tuberculosis, the need of novel anti-tuberculosis drugs is specially challenging because of the intricate biology of its causative agent, Mycobacterium tuberculosis. The repurposing of medicines has arisen in recent years as a fast, low-cost, and efficient strategy to identify novel biomedical applications for already approved drugs. This review is focused on anti-parasitic drugs that have additionally demonstrated certain levels of anti-tuberculosis activity; along with this, natural products with a dual activity against parasites and against M. tuberculosis are discussed. A few clinical trials have tested antiparasitic drugs in tuberculosis patients, and have revealed effective dose and toxicity issues, which is consistent with the natural differences between tuberculosis and parasitic infections. However, through medicinal chemistry approaches, derivatives of drugs with anti-parasitic activity have become successful drugs for use in tuberculosis therapy. In summary, even when the repurposing of anti-parasitic drugs for tuberculosis treatment does not seem to be an easy job, it deserves attention as a potential contributor to fuel the anti-tuberculosis drug pipeline