Farnesol abrogates biofilm- and efflux pump- associated genes in drug resistant Candida auris strains

Background: Candida auris, a decade old Candida species, has been identified globally as a significant nosocomial multidrug resistant (MDR) pathogen responsible for causing invasive outbreaks. Biofilms and over expression of efflux pumps such as Major Facilitator Superfamily and ATP Binding Cassette are known to cause multidrug resistance in Candida species, including C. auris. Therefore, targeting these factors may prove an effective approach to combat MDR in C. auris. Methods: In this study, 25 clinical isolates of C. auris from different hospitals of South Africa were used. Antifungal susceptibility profile of all the isolates against commonly used drugs was determined following CLSI recommended guidelines. Rhodamine-6-G extracellular efflux and intracellular accumulation assays were used to study active drug efflux mechanism. We further studied the role of farnesol in modulating development of biofilms and drug efflux in C. auris. Down-regulation of biofilm- and efflux pump- associated genes by farnesol was also investigated. CLSM analysis for examining C. auris biofilm architecture among treated and untreated isolates. Results: Most of the isolates (twenty-two) were found resistant to FLZ whereas five were resistant to AmB. All the isolates were found capable of biofilm formation and ornamented with active drug efflux mechanism. The MIC for planktonic cells ranged from 62.5-125 mM and for sessile cells was 125 mM (0 h and 4 h biofilm) and 500 mM (12 h and 24 h biofilm), CLSM studies also confirmed these findings. Farnesol also blocked efflux pumps and down-regulated biofilm- and efflux pump- associated genes. Conclusion: Modulation of biofilm- and efflux pump- associated genes by farnesol represent a promising approach in combating C. auris infection.

Silencing of an efflux pump coding gene decreases the efflux rate of pyrazinoic acid in Mycobacterium smegmatis

Background: Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (MTB). The recommended treatment for TB is based on the use of first-line drugs, including pyrazinamide (PZA). PZA is also a drug used in the treatment of multidrug-resistant TB (MDR-TB) because of its main effect against the latent stage. The main cause of resistance to PZA is mutations in the pncA gene, which compromise the activity of the encoded enzyme pyrazinamidase (PZAse), which hydrolyzes PZA into POA, the active antituberculosis molecule. The mechanism of action of PZA requires that POA is expelled from the bacterium by an efflux mechanism. After that, if the extracellular medium is sufficiently acidic, POA is protonated and returns to the cytosol, releasing the proton and repeating the cycle, resulting lethal to the bacteria. The efflux pump responsible for extruding the POA to the extracellular environment is not yet known. Mycobacterium smegmatis is naturally resistant to PZA and has a 900-fold faster POA efflux rate than MTB, and has the advantage to be a faster growing mycobacterium. Methods: In the present study we have silenced the transcription of several genes encoding efflux pumps in M. smegmatis by CRISPRi (CRISPR interference). These genes (MSMEG_0250, MSMEG_3815, MSMEG_0241, MSMEG_5046 and MSMEG_0410) were homologous to efflux pump genes in MTB. POA efflux rate was measured, and a quantitative Wayne's test was performed after silencing each gene. Results: Silencing of MSMEG_0250, resulted in an approximately 5-fold decrease in the POA efflux rate in M. smegmatis (P<0.0001). None of the other silenced genes showed a notable decrease in the POA efflux rate.

PHENOTYPIC DETECTION OF EFFLUX MECHANISM IN AMIKACIN-RESISTANT ACINETOBACTER ISOLATES FROM TWO DIFFERENT STATES IN SOUTH INDIA

Acinetobacter has already gained resistance to the majority of antibiotics available. Aminoglycosides are commonly used to treat invasive infections. Aminoglycoside resistance is associated with decreased drug absorption, aminoglycoside modification, and aminoglycoside efflux. The aim of this study was to detect the presence of an efflux mechanism in amikacin-resistant Acinetobacter isolated from hospital wards using Carbonyl Cyanide 3- Chlorophenylhydrazone (CCCP). One hundred isolates of Acinetobacter were isolated from tertiary care hospitals in two distinct South Indian states. Antibacterial susceptibility patterns were discovered between 2017 and 2019. Amikacin minimum inhibitory concentration (MIC) for resistant Acinetobacter isolates was determined using Clinical and Laboratory Standards Institute (CLSI) standards. The efflux system activity was determined using CCCP. Among 100 Acinetobacter baumannii isolates, 49 isolates with amikacin resistance were found. The MIC’s of Acinetobacter ranged between 2 – 1024 μg/mL for the amikacin studied. After treatment with the efflux pump inhibitor, 38.77% of isolates became less resistant to amikacin, as determined by phenotypic detection of efflux pumps, showing a decrease in antibiotic MICs of at least four fold. The data demonstrated the importance of efflux pump activity conferring amikacin resistance on Acinetobacter clinical isolates.

Detection of Inducible Resistance to Clindamycin among Methicillin Resistant and Sensitive strains of Staphylococcus aureus from India

The resistance to MLSB antibiotics, i.e. Macrolide-Lincosamide-Streptogramin B (MLSB), is an increasing problem among Methicillin-resistant Staphylococci. The resistance to macrolides can be by efflux mechanism or via inducible or constitutive resistance. Unfortunately, routine clindamycin susceptibility testing fails to detect the inducible resistance, which commonly results in treatment failure and necessitates incorporating a simple D-test to detect such resistance. A retrospective observational study was performed on S. aureus isolates from patients. The strains were subjected to antibiotic susceptibility testing followed by detection of mecA gene by a polymerase chain reaction and, the ‘D-test’ was performed to know the inducible resistance to clindamycin. A total of 235 isolates were identified as S. aureus. Antibiotic susceptibility test indicated 190 MRSA and 45 are sensitive to MLSB (MS). Inducible clindamycin resistance was found among 48 (20.4%) isolates and constitutive resistance in 104 (44.2%). MRSA strains had higher inducible and constitutive resistance than MSSA strains (22.1%, 51.6% and 13.3%, 13.3%, respectively). Clindamycin is a commonly used antibiotic in patients with MRSA infections to spare higher-end anti-MRSA antibiotics like linezolid and vancomycin. To detect inducible clindamycin to avoid treatment failures; the study showed the importance of incorporating the D-test in routine testing.

Structure and efflux mechanism of the yeast pleiotropic drug resistance transporter Pdr5

Pdr5, a member of the extensive ABC transporter superfamily, is representative of a clinically relevant subgroup involved in pleiotropic drug resistance. Pdr5 and its homologues drive drug efflux through uncoupled hydrolysis of nucleotides, enabling organisms such as baker’s yeast and pathogenic fungi to survive in the presence of chemically diverse antifungal agents. Here, we present the molecular structure of Pdr5 solved with single particle cryo-EM, revealing details of an ATP-driven conformational cycle, which mechanically drives drug translocation through an amphipathic channel, and a clamping switch within a conserved linker loop that acts as a nucleotide sensor. One half of the transporter remains nearly invariant throughout the cycle, while its partner undergoes changes that are transmitted across inter-domain interfaces to support a peristaltic motion of the pumped molecule. The efflux model proposed here rationalises the pleiotropic impact of Pdr5 and opens new avenues for the development of effective antifungal compounds.

Lipid-based Nanocarriers loaded with Taxanes for the Management of Breast Cancer: Promises and Challenges

: Breast cancer is the leading cause of deaths worldwide among women. Taxanes (most propitious class of diterpenes) have shown dynamic potentials in the treatment of early and metastatic breast cancer. However, challenges like poor bioavailability, low tissue-permeability, compromised aqueous solubility, and dose-dependent side-effects limit the clinical applications of these drugs. Henceforth, to overcome these challenges, various nanotechnology-based drug delivery systems are being explored for the delivery of taxanes in the management of breast cancer. One such promising nanocarrier category is lipid-based nanocarriers, which employ the meritorious features of a variety of lipids, both of natural and synthetic origin. It is also known that lipid uptake plays a significant role in breast cancer cells proliferation and tumor genesis. However, lipid-based nanocarriers could be a great choice to nanoencapsulate the poorly soluble and permeable taxanes for breast cancer management. These systems have an immense promise of bioavailability enhancement, spatial and temporal taxane delivery, improved efficacy, reduced dosing frequency, and even mild inhibition of the P-gp efflux mechanism. Apart from these promises, these carriers are not yet available for the benefit of the end-user. The present review will not only discuss the merits, progress, and promises of these systems but also ponder upon the various challenges faced by these carriers to reach the clinics for the benefit of the patients afflicted with breast cancer.

Structure and efflux mechanism of the yeast pleiotropic drug resistance transporter Pdr5

Pdr5, a member of the extensive ABC transporter superfamily, is representative of a clinically relevant subgroup involved in pleiotropic drug resistance. Through the coupling of nucleotide hydrolysis with drug efflux, Pdr5 homologues enable pathogenic species to survive in the presence of chemically diverse antifungal agents. Our structural and functional results reveal details of an ATP-driven conformational cycle, which mechanically drives drug translocation through an amphipathic channel, and a clamping switch within a conserved linker loop that acts as a nucleotide sensor. One half of the transporter remains nearly invariant throughout the cycle, while its partner undergoes changes that are transmitted across interdomain interfaces to support a peristaltic motion of the pumped molecule. The efflux model proposed here rationalises the pleiotropic impact of Pdr5 and opens avenues for the development of effective antifungal compounds.

Demonstrating the involvement of an active efflux mechanism in the intestinal absorption of chlorogenic acid and quinic acid using a Caco-2 bidirectional permeability assay

The intestinal absorption of chlorogenic acid and quinic acid was investigated using an in vitro bidirectional Caco-2 permeability assay and LC-MS/MS.