Antibacterial potential and mechanism of action of botanicals and phytochemicals from Stachytarpheta cayennensis (Verbenaceae) against Gram-negative multidrug-resistant phenotypes expressing efflux pumps

AbstractNew antimicrobial agents are urgently needed, especially to eliminate multidrug resistant Gram-negative bacteria that stand for most antibiotic-resistant threats. In the following study, we present superior properties of an engineered antimicrobial peptide, OMN6, a 40-amino acid cyclic peptide based on Cecropin A, that presents high efficacy against Gram-negative bacteria with a bactericidal mechanism of action. The target of OMN6 is assumed to be the bacterial membrane in contrast to small molecule-based agents which bind to a specific enzyme or bacterial site. Moreover, OMN6 mechanism of action is effective on Acinetobacter baumannii laboratory strains and clinical isolates, regardless of the bacteria genotype or resistance-phenotype, thus, is by orders-of-magnitude, less likely for mutation-driven development of resistance, recrudescence, or tolerance. OMN6 displays an increase in stability and a significant decrease in proteolytic degradation with full safety margin on erythrocytes and HEK293T cells. Taken together, these results strongly suggest that OMN6 is an efficient, stable, and non-toxic novel antimicrobial agent with the potential to become a therapy for humans.

Download Full-text

An alkylaminoquinazoline restores antibiotic activity in Gram-negative resistant isolates

Microbiology ◽

10.1099/mic.0.045716-0 ◽

2011 ◽

Vol 157 (2) ◽

pp. 566-571 ◽

Cited By ~ 17

Author(s):

Abdallah Mahamoud ◽

Jacqueline Chevalier ◽

Milad Baitiche ◽

Elissavet Adam ◽

Jean-Marie Pagès

Keyword(s):

Efflux Pump ◽

Bacterial Species ◽

Efflux Pumps ◽

Multidrug Resistant ◽

Side Chain ◽

Structural Class ◽

Gram Negative ◽

Activity Spectrum ◽

Efflux Pump Inhibitors ◽

Drug Efflux Pump

To date, various bacterial drug efflux pump inhibitors (EPIs) have been described. They exhibit variability in their activity spectrum with respect to antibiotic structural class and bacterial species. Among the various 4-alkylaminoquinazoline derivatives synthesized and studied in this work, one molecule, 1167, increased the susceptibility of important human-pathogenic, resistant, Gram-negative bacteria towards different antibiotic classes. This 4-(3-morpholinopropylamino)-quinazoline induced an increase in the activity of chloramphenicol, nalidixic acid, norfloxacin and sparfloxacin, which are substrates of the AcrAB-TolC and MexAB-OprM efflux pumps that act in these multidrug-resistant isolates. In addition, 1167 increased the intracellular concentration of chloramphenicol in efflux pump-overproducing strains. The rate of restoration depended on the structure of the antibiotic, suggesting that different sites in the efflux pumps may be involved. A molecule exhibiting a morpholine functional group and a propyl extension of the side chain was more active.

Download Full-text

Clinical Status of Efflux Resistance Mechanisms in Gram-Negative Bacteria

Antibiotics ◽

10.3390/antibiotics10091117 ◽

2021 ◽

Vol 10 (9) ◽

pp. 1117

Author(s):

Anne Davin-Regli ◽

Jean-Marie Pages ◽

Aurélie Ferrand

Keyword(s):

Clinical Status ◽

Efflux Pumps ◽

Multidrug Resistant ◽

Resistance Mechanisms ◽

Gram Negative Bacteria ◽

Easy Method ◽

Gram Negative ◽

Healthcare Associated ◽

Rational Use Of Antibiotics ◽

Antibiotic Efflux

Antibiotic efflux is a mechanism that is well-documented in the phenotype of multidrug resistance in bacteria. Efflux is considered as an early facilitating mechanism in the bacterial adaptation face to the concentration of antibiotics at the infectious site, which is involved in the acquirement of complementary efficient mechanisms, such as enzymatic resistance or target mutation. Various efflux pumps have been described in the Gram-negative bacteria most often encountered in infectious diseases and, in healthcare-associated infections. Some are more often involved than others and expel virtually all families of antibiotics and antibacterials. Numerous studies report the contribution of these pumps in resistant strains previously identified from their phenotypes. The authors characterize the pumps involved, the facilitating antibiotics and those mainly concerned by the efflux. However, today no study describes a process for the real-time quantification of efflux in resistant clinical strains. It is currently necessary to have at hospital level a reliable and easy method to quantify the efflux in routine and contribute to a rational choice of antibiotics. This review provides a recent overview of the prevalence of the main efflux pumps observed in clinical practice and provides an idea of the prevalence of this mechanism in the multidrug resistant Gram-negative bacteria. The development of a routine diagnostic tool is now an emergency need for the proper application of current recommendations regarding a rational use of antibiotics.

Download Full-text

Alkylaminoquinolines inhibit the bacterial antibiotic efflux pump in multidrug-resistant clinical isolates

Biochemical Journal ◽

10.1042/bj20030963 ◽

2003 ◽

Vol 376 (3) ◽

pp. 801-805 ◽

Cited By ~ 75

Author(s):

Monique MALLÉA ◽

Abdallah MAHAMOUD ◽

Jacqueline CHEVALIER ◽

Sandrine ALIBERT-FRANCO ◽

Pierre BROUANT ◽

...

Keyword(s):

Antibiotic Resistance ◽

Efflux Pump ◽

Efflux Pumps ◽

Multidrug Resistant ◽

Clinical Isolates ◽

Microbial Pathogens ◽

Gram Negative Bacteria ◽

Gram Negative ◽

High Level ◽

Antibiotic Efflux

Over the last decade, MDR (multidrug resistance) has increased worldwide in microbial pathogens by efflux mechanisms, leading to treatment failures in human infections. Several Gram-negative bacteria efflux pumps have been described. These proteinaceous channels are capable of expelling structurally different drugs across the envelope and conferring antibiotic resistance in various bacterial pathogens. Combating antibiotic resistance is an urgency and the blocking of efflux pumps is an attractive response to the emergence of MDR phenotypes in infectious bacteria. In the present study, various alkylaminoquinolines were tested as potential inhibitors of drug transporters. We showed that alkylaminoquinolines are capable of restoring susceptibilities to structurally unrelated antibiotics in clinical isolates of MDR Gram-negative bacteria. Antibiotic efflux studies indicated that 7-nitro-8-methyl-4-[2´-(piperidino)ethyl]aminoquinoline acts as an inhibitor of the AcrAB–TolC efflux pump and restores a high level of intracellular drug concentration. Inhibitory activity of this alkylaminoquinoline is observed on clinical isolates showing different resistance phenotypes.

Download Full-text

The Action of Efflux Pump Genes in Conferring Drug Resistance to Klebsiella Species and Their Inhibition

Journal of Health and Allied Sciences NU ◽

10.1055/s-0041-1731914 ◽

2021 ◽

Author(s):

Priyanka Ashwath ◽

Akhila Dharnappa Sannejal

Keyword(s):

Drug Resistance ◽

Antimicrobial Agents ◽

Efflux Pump ◽

Acquired Resistance ◽

Efflux Pumps ◽

Multidrug Resistant ◽

Resistance Mechanisms ◽

Klebsiella Species ◽

Gram Negative ◽

Multidrug Efflux Pumps

AbstractNosocomial infections caused by Klebsiella species are characterized by high rates of morbidity and mortality. The emergence of the multidrug-resistant (MDR) and extensive drug-resistant (XDR) Gram-negative bacteria reduces the antibiotic efficacy in the treatment of infections caused by the microorganisms. Management of these infections is often difficult, due to the high frequency of strains resistant to multiple antimicrobial agents. Multidrug efflux pumps play a major role as a mechanism of antimicrobial resistance in Gram-negative pathogens. Efflux systems are significant in conferring intrinsic and acquired resistance to the bacteria. The emergence of increasing drug resistance among Klebsiella pneumoniae nosocomial isolates has limited the therapeutic options for treatment of these infections and hence there is a constant quest for an alternative. In this review, we discuss various resistance mechanisms, focusing on efflux pumps and related genes in conferring resistance to Klebsiella. The role of various efflux pump inhibitors (EPIs) in restoring the antibacterial activity has also been discussed. In specific, antisense oligonucleotides as alternative therapeutics in combatting efflux-mediated resistance in Klebsiella species have focused upon.

Download Full-text

Phytochemical analysis and antibiotic-modulating activity of Cocos nucifera, Glycine max and Musa sapientum methanol extracts against multidrug resistant Gram-negative bacteria

Investigational Medicinal Chemistry and Pharmacology ◽

10.31183/imcp.2020.00053 ◽

2021 ◽

Vol 4 (2) ◽

pp. 1-12

Author(s):

Carine M.N. Ngaffo ◽

Simplice B. Tankeo ◽

Michel-Gael F. Guefack ◽

Paul Nayim ◽

Brice E.N. Wamba ◽

...

Keyword(s):

Glycine Max ◽

Bioactive Compounds ◽

Cocos Nucifera ◽

Efflux Pumps ◽

Multidrug Resistant ◽

Significant Activity ◽

Gram Negative ◽

E Coli ◽

Bactericidal Effects ◽

Different Parts

Background: The rapid emergence of multidrug resistant (MDR) bacteria is occurring worldwide, endangering the efficacy of antibiotics, which have transformed medicine and saved millions of lives. Antibiotic-resistant infections are already widespread in the Sub-Saharan Africa and across the globe. To extend the search for new and more efficient antimicrobial drugs from natural sources, this work has been carried out to study the phytochemical composition and the antibacterial activities of some Cameroonian dietary plants (Cocos nucifera, Glycine max and Musa sapientum) against several MDR Gram-negative strains including Escherichia coli, Enterobacter aerogenes, Providencia stuartii, Klebsiella pneumoniae, Pseudomonas aeruginosa species expressing efflux pumps. Methods: Phytochemical screening of plant extracts was performed using qualitative standard methods and the antimicrobial assays of these extracts alone and in combination with antibiotics were done using serial 96-wells microplate dilution essays. Results: Each plant extract contained at least three mean classes of secondary metabolites. Glycine max, epicarps, leaves and bark of C. nucifera as well as mesocarps of M. sapientum contained each alkaloids, polyphenols, flavonoids, and triterpenes. Moreover, steroids were also found in G. max, steroids and saponins in epicarps and saponins in bark of C. nucifera. Meanwhile epicarps from M. sapientum contained only polyphenols, flavonoids and saponins. Antibacterial assays showed that different parts of C. nucifera were more active than other extracts. Their minimal inhibitory concentrations (MICs) varied from 128 to 2048 µg/mL. The bark part presented the highest antibacterial potential inhibiting the growth of 90% of strains with significant activity (100≤MIC≤512 µg/mL) against 50% of them (three E. coli, four E. aerogenes and three K. pneumoniae). It showed bactericidal effects (MBC/MIC≤4) on 45% of the same bacterial species. It was followed by epicarps and leaves parts which exhibited an inhibitory power against 75% and 60% of bacteria with significant activity on 40% and 20% of them respectively. They also showed bactericidal effects on E. coli ATCC8739 for epicarps extract and E. coli ATCC8739 and P. stuartii NEA16 for leaves extract. Extracts from G. max were less active and those from mesocarps and epicarps of M. sapientum did not showed any activity on all studied bacteria. Bark and epicarps extracts of C. nucifera potentiated the activities of all used antibiotics against at least 70% of bacteria while leaves extract exhibited this effect improving the activities of 67% of antibiotics with improvement activity factors (IAF) ranging from 2 to 256 suggesting that they contain bioactive compounds which could be considered as efflux pumps inhibitors. Extracts from G. max, epicarps and mesocarps of M. sapientum enhanced the inhibitory potential of 56%, 34% and 23% of antibiotics respectively against at least 70% of studied bacteria. These increases of activities also characterize synergistic effects between antibiotics and bioactive compounds of plants. Conclusion: The findings of this work suggest that infections by resistant bacteria can be treated using different parts of C. nucifera as an alternative to commonly used antibiotics.

Download Full-text

Antibiotic-potentiation activities of four Cameroonian dietary plants against multidrug-resistant Gram-negative bacteria expressing efflux pumps

BMC Complementary and Alternative Medicine ◽

10.1186/1472-6882-14-258 ◽

2014 ◽

Vol 14 (1) ◽

Cited By ~ 33

Author(s):

Francesco K Touani ◽

Armel J Seukep ◽

Doriane E Djeussi ◽

Aimé G Fankam ◽

Jaurès A K Noumedem ◽

...

Keyword(s):

Efflux Pumps ◽

Multidrug Resistant ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Dietary Plants

Download Full-text

Synergy between Active Efflux and Outer Membrane Diffusion Defines Rules of Antibiotic Permeation into Gram-Negative Bacteria

mBio ◽

10.1128/mbio.01172-17 ◽

2017 ◽

Vol 8 (5) ◽

Cited By ~ 64

Author(s):

Ganesh Krishnamoorthy ◽

Inga V. Leus ◽

Jon W. Weeks ◽

David Wolloscheck ◽

Valentin V. Rybenkov ◽

...

Keyword(s):

Outer Membrane ◽

Efflux Pumps ◽

Antibacterial Activities ◽

Multidrug Resistant ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Active Efflux ◽

Membrane Barrier ◽

Biological Determinants ◽

Resistant Strains

ABSTRACT Gram-negative bacteria are notoriously resistant to antibiotics, but the extent of the resistance varies broadly between species. We report that in significant human pathogens Acinetobacter baumannii, Pseudomonas aeruginosa, and Burkholderia spp., the differences in antibiotic resistance are largely defined by their penetration into the cell. For all tested antibiotics, the intracellular penetration was determined by a synergistic relationship between active efflux and the permeability barrier. We found that the outer membrane (OM) and efflux pumps select compounds on the basis of distinct properties and together universally protect bacteria from structurally diverse antibiotics. On the basis of their interactions with the permeability barriers, antibiotics can be divided into four clusters that occupy defined physicochemical spaces. Our results suggest that rules of intracellular penetration are intrinsic to these clusters. The identified specificities in the permeability barriers should help in the designing of successful therapeutic strategies against antibiotic-resistant pathogens. IMPORTANCE Multidrug-resistant strains of Gram-negative pathogens rapidly spread in clinics. Significant efforts worldwide are currently directed to finding the rules of permeation of antibiotics across two membrane envelopes of these bacteria. This study created the tools for analysis of and identified the major differences in antibacterial activities that distinguish the permeability barriers of P. aeruginosa, A. baumannii, Burkholderia thailandensis, and B. cepacia. We conclude that synergy between active efflux and the outer membrane barrier universally protects Gram-negative bacteria from antibiotics. We also found that the diversity of antibiotics affected by active efflux and outer membrane barriers is broader than previously thought and that antibiotics cluster according to specific biological determinants such as the requirement of specific porins in the OM, targeting of the OM, or specific recognition by efflux pumps. No universal rules of antibiotic permeation into Gram-negative bacteria apparently exist. Our results suggest that antibiotic clusters are defined by specific rules of permeation and that further studies could lead to their discovery. IMPORTANCE Multidrug-resistant strains of Gram-negative pathogens rapidly spread in clinics. Significant efforts worldwide are currently directed to finding the rules of permeation of antibiotics across two membrane envelopes of these bacteria. This study created the tools for analysis of and identified the major differences in antibacterial activities that distinguish the permeability barriers of P. aeruginosa, A. baumannii, Burkholderia thailandensis, and B. cepacia. We conclude that synergy between active efflux and the outer membrane barrier universally protects Gram-negative bacteria from antibiotics. We also found that the diversity of antibiotics affected by active efflux and outer membrane barriers is broader than previously thought and that antibiotics cluster according to specific biological determinants such as the requirement of specific porins in the OM, targeting of the OM, or specific recognition by efflux pumps. No universal rules of antibiotic permeation into Gram-negative bacteria apparently exist. Our results suggest that antibiotic clusters are defined by specific rules of permeation and that further studies could lead to their discovery.

Download Full-text

Ever-Adapting RND Efflux Pumps in Gram-Negative Multidrug-Resistant Pathogens: A Race against Time

Antibiotics ◽

10.3390/antibiotics10070774 ◽

2021 ◽

Vol 10 (7) ◽

pp. 774

Author(s):

Martijn Zwama ◽

Kunihiko Nishino

Keyword(s):

Legionella Pneumophila ◽

Bacterial Infections ◽

Efflux Pump ◽

Efflux Pumps ◽

Multidrug Resistant ◽

Bacterial Strains ◽

Gram Negative ◽

Over Expression ◽

Wide Range ◽

Resistance Nodulation Division

The rise in multidrug resistance (MDR) is one of the greatest threats to human health worldwide. MDR in bacterial pathogens is a major challenge in healthcare, as bacterial infections are becoming untreatable by commercially available antibiotics. One of the main causes of MDR is the over-expression of intrinsic and acquired multidrug efflux pumps, belonging to the resistance-nodulation-division (RND) superfamily, which can efflux a wide range of structurally different antibiotics. Besides over-expression, however, recent amino acid substitutions within the pumps themselves—causing an increased drug efflux efficiency—are causing additional worry. In this review, we take a closer look at clinically, environmentally and laboratory-evolved Gram-negative bacterial strains and their decreased drug sensitivity as a result of mutations directly in the RND-type pumps themselves (from Escherichia coli, Salmonella enterica, Neisseria gonorrhoeae, Pseudomonas aeruginosa, Acinetobacter baumannii and Legionella pneumophila). We also focus on the evolution of the efflux pumps by comparing hundreds of efflux pumps to determine where conservation is concentrated and where differences in amino acids can shed light on the broad and even broadening drug recognition. Knowledge of conservation, as well as of novel gain-of-function efflux pump mutations, is essential for the development of novel antibiotics and efflux pump inhibitors.

Download Full-text

Antibiotic-potentiation activities of three animal species extracts, Bitis arietans, Helix aspersa, and Aristaeomorpha foliacea and mode of action against MDR Gram-negative bacteria phenotypes

Investigational Medicinal Chemistry and Pharmacology ◽

10.31183/imcp.2020.00048 ◽

2020 ◽

Vol 4 (1) ◽

pp. 1-15

Author(s):

Michel-Gael F. Guefack ◽

Simplice B. Tankeo ◽

Carine M.N. Ngaffo ◽

Paul Nayim ◽

Brice E.N. Wamba ◽

...

Keyword(s):

Animal Species ◽

Efflux Pumps ◽

Multidrug Resistant ◽

Proton Pumps ◽

Gram Negative Bacteria ◽

Significant Activity ◽

Bacterial Strains ◽

Gram Negative ◽

E Coli ◽

Aristaeomorpha Foliacea

Abstract Background: In recent years, drug resistance to human pathogenic bacteria has been commonly reported from all over the world. As antimicrobial activities of most medicinal plants and antibiotics have been already explored, it is more important to make investigations on animal species mainly invertebrates which could constitute an efficient source of antimicrobial molecules. This work was aimed at contributing to the fight against microbial resistance through the study of antibacterial potential of three animal species (Helix aspersa, Bitis arietans, Aristaeomorpha foliacea) on several multidrug-resistant (MDR) Gram-negative strains overexpressing efflux pumps including Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae, Pseudomonas aeruginosa. Methods: The microdilution technique was used to evaluate the antibacterial activities of the tested samples by determining their minimal inhibitory concentrations (MICs), as well as the effect of their combination with antibiotics. Studies on the mechanisms of action of the most active sample, dried Bitis arietans extract, was carried out using standard methods for evaluating the effects of this extract on bacterial H+-ATPases-mediated proton pumps and on bacterial growth kinetics. In this latter case, the optical density was read spectrophotometrically. Results: Zoochemical screening indicated the presence of protein constituents and alkaloids and the absence of other metabolites in all tested extracts. Dried B. arietans showed the best antibacterial activity by inhibiting the growth of 90% of studied bacterial strains with MICs ranging from 128 to 2048 μg/ml. Moreover, this extract presented a significant activity (100≤MIC≤512 µg/ml) against 35% of bacteria that are E. coli (ATCC8739, AG100ATet, MC4100), E. aerogenes EA27, K. pneumoniae ATCC11296, P. aeruginosa (PA01, PA124) and a moderate activity (512<MIC≤2048 µg/ml) against 55% of studied bacteria. It was followed by fresh B. arietans which inhibited the growth of 65% of bacteria with significant activity on three bacteria (E. coli ATCC8739, E. aerogenes ATCC13048 and K. pneumoniae ATCC11296. These two extracts showed bactericidal effects on many strains. The other extracts samples selectively exhibited an antibacterial activity against less than 40% of strains. All samples potentiated the activity of at least 56% of used antibiotics against at least 70% of studied bacterial strains. B. arietans extracts at MIC/2 and MIC/4 mostly improved the activities of more than 78% of antibiotics on at least 70% of bacteria with improvement activity factors (IAF) ranging from 2 – 128 suggesting that this animal contains bioactive compounds which could act as efflux pumps inhibitors. Bacterial growth kinetic study showed that when treated with dried B. arietans extract (the most active sample) at different concentrations MIC/2, MIC and 2xMIC, the growth of tested bacteria (E. coli ATCC8739) decreased respectively when the concentrations increased. Furthermore, this extract inhibited the H+-ATPase-mediated proton pumps of this bacterium increasing the pH values. Conclusion: Results obtained in the present work provide interesting data for the use of dried B. arietans extract and invertebrates in general in the traditional therapy for the treatment of bacterial infections involving multidrug-resistant phenotypes. Keywords: Gram-negative bacteria; multidrug resistance; efflux pumps; infectious diseases; animal species; secondary metabolites.

Download Full-text