Overexpression Of Efflux Pumps Research Articles

Exploring Antibiotic Resistance Gene Expression in Acinetobacter baumannii Using Microarray Technology

Background: Antibiotic resistance is a significant challenge in healthcare, particularly in nosocomial infections caused by Acinetobacter baumannii. Efflux pumps play a crucial role in mediating antibiotic resistance in A. baumannii, yet comprehensive evaluation of these pumps and acquired resistance determinants is lacking. Here, we present the development and validation of an oligonucleotide-based DNA microarray for assessing gene expression of efflux pumps and detecting acquired antibiotic resistance determinants in A. baumannii. Objective: The primary objective of this study was to develop a robust microarray platform capable of simultaneously assessing the expression of efflux pump genes and detecting acquired resistance determinants in A. baumannii. Additionally, we aimed to validate the microarray's performance using mutants overexpressing or deficient in efflux pumps and single-step mutants obtained on various antibiotics. Methods: The DNA microarray consisted of probes targeting 78 genes, including 17 efflux systems, 15 resistance determinants, and 19 housekeeping genes. Comparative analysis of mutants, along with quantitative reverse transcriptase PCR validation, was conducted to confirm the microarray's accuracy in detecting efflux pump overexpression. Results: Validation experiments revealed overexpression of RND efflux pumps AdeABC and AdeIJK in mutants obtained on gentamicin, cefotaxime, or tetracycline, as well as identification of a novel efflux pump, AdeFGH, overexpressed in a mutant exposed to chloramphenicol. Clinical isolates showed overexpression of AdeABC and chromosomally encoded cephalosporinase, along with several acquired resistance genes, accounting for the multidrug-resistant phenotype. Conclusion: The developed microarray demonstrates high sensitivity and specificity in detecting efflux pump expression and acquired resistance determinants in A. baumannii. Its potential utility in identifying antibiotic resistance and novel efflux systems highlights its importance in clinical settings.

Extending the Potency and Lifespan of Antibiotics: Inhibitors of Gram-Negative Bacterial Efflux Pumps.

Efflux is a natural process found in all prokaryotic and eukaryotic cells that removes a diverse range of substrates from inside to outside. Many antibiotics are substrates of bacterial efflux pumps, and modifications to the structure or overexpression of efflux pumps are an important resistance mechanism utilized by many multidrug-resistant bacteria. Therefore, chemical inhibition of bacterial efflux to revitalize existing antibiotics has been considered a promising approach for antimicrobial chemotherapy over two decades, and various strategies have been employed. In this review, we provide an overview of bacterial multidrug resistance (MDR) efflux pumps, of which the resistance nodulation division (RND) efflux pumps are considered the most clinically relevant in Gram-negative bacteria, and describe over 50 efflux inhibitors that target such systems. Although numerous efflux inhibitors have been identified to date, none have progressed into clinical use because of formulation, toxicity, and pharmacokinetic issues or a narrow spectrum of inhibition. For these reasons, the development of efflux inhibitors has been considered a difficult and complex area of research, and few active preclinical studies on efflux inhibitors are in progress. However, recently developed tools, including but not limited to computational tools including molecular docking models, offer hope that further research on efflux inhibitors can be a platform for research and development of new bacterial efflux inhibitors.

A laboratory-based predictive pathway for the development of Neisseria gonorrhoeae high-level resistance to corallopyronin A, an inhibitor of bacterial RNA polymerase.

The continued emergence of multi-antibiotic-resistant strains of Neisseria gonorrhoeae necessitates the development of new antibiotics that are effective against this human pathogen. We previously described that the RNA polymerase-targeting antibiotic corallopyronin A (CorA) has potent activity against a large collection of clinical strains that express different antibiotic resistance phenotypes including when such gonococci are in a biofilm state. Herein, we tested whether a CorA-sensitive gonococcal strain could develop spontaneous resistance. Our finding that CorA resistance could only be achieved by a multi-step process involving over-expression of the MtrCDE efflux pump and single amino acid changes in RpoB and RpoC suggests that such resistance may be difficult for gonococci to evolve if this antibiotic is used in the future to treat gonorrheal infections that are refractory to cure by other antibiotics.

Genetic determinants of antimicrobial resistance in polymyxin B resistant Pseudomonas aeruginosa isolated from airways of patients with cystic fibrosis.

Pseudomonas aeruginosa is the main pathogen associated with pulmonary exacerbation in patients with cystic fibrosis (CF). CF is a multisystemic genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator gene, which mainly affects pulmonary function. P. aeruginosa isolated from individuals with CF in Brazil is not commonly associated with multidrug resistance (MDR), especially when compared to global occurrence, where the presence of epidemic clones, capable of expressing resistance to several drugs, is often reported. Due to the recent observations of MDR isolates of P. aeruginosa in our centers, combined with these characteristics, whole-genome sequencing was employed for analyses related to antimicrobial resistance, plasmid identification, search for phages, and characterization of CF clones. All isolates in this study were polymyxin B resistant, exhibiting diverse mutations and reduced susceptibility to carbapenems. Alterations in mexZ can result in the overexpression of the MexXY efflux pump. Mutations in oprD, pmrB, parS, gyrA and parC may confer reduced susceptibility to antimicrobials by affecting permeability, as observed in phenotypic tests. The phage findings led to the assumption of horizontal genetic transfer, implicating dissemination between P. aeruginosa isolates. New sequence types were described, and none of the isolates showed an association with epidemic CF clones. Analysis of the genetic context of P. aeruginosa resistance to polymyxin B allowed us to understand the different mechanisms of resistance to antimicrobials, in addition to subsidizing the understanding of possible relationships with epidemic strains that circulate among individuals with CF observed in other countries.

Reversing Anticancer Drug Resistance by Synergistic Combination of Chemotherapeutics and Membranolytic Antitumor β-Peptide Polymer.

Multidrug resistance is the main obstacle to cancer chemotherapy. Overexpression of drug efflux pumps causes excessive drug efflux from cancer cells, ultimately leading to drug resistance. Hereby, we raise an effective strategy to overcome multidrug resistance using a synergistic combination of membranolytic antitumor β-peptide polymer and chemotherapy drugs. This membrane-active β-peptide polymer promotes the transmembrane transport of chemotherapeutic drugs by increasing membrane permeability and enhances the activity of chemotherapy drugs against multidrug-resistant cancer cells. As a proof-of-concept demonstration, the synergistic combination of β-peptide polymer and doxorubicin (DOX) is substantially more effective than DOX alone against drug-resistant cancer both in vitro and in vivo. Notably, the synergistic combination maintains a potent anticancer activity after continuous use. Collectively, this combination therapy using membrane lytic β-peptide polymer appears to be an effective strategy to reverse anticancer drug resistance.

Synergistic Antibacterial Activity of Curcuma domestica Val. Extract with Tetracycline Against Multidrug-resistant Acinetobacter baumannii

Infections caused by multidrug-resistant (MDR) bacteria are on the rise globally. MDR is facilitated by overexpression of efflux pump and permeability changes of membrane. Acinetobacter baumannii is a pathogenic germ that causes a major problem in infection, also, there has been an increase in the incidence of resistance to various antibiotics. The present study highlights the synergistic of ethanolic extract of Curcuma domestica (EECD) rhizome with tetracycline against multidrug-resistant A. baumannii (MDR-Ab). Assessment of Minimum Inhibitory Concentration (MIC) was determined by microdilution using 96-well plates. The synergistic effect of EECD and tetracycline was determined by checkerboard method. The effect of EECD and tetracycline combination was investigated by bacteriolytic activity and inhibition of efflux pump by Ethidium Bromide (EtBr) accumulation assay. EECD presented the MIC value 250 µg/mL against MDR-Ab. Fractional Inhibitory Concentration Index (FICI) value of EECD and tetracycline combination was 0.4, which showed their synergistic effect. Additionally, the combination of EECD and tetracycline could inhibit the efflux pump in MDR-Ab. This combination can also compromise cell integrity by altering membrane permeability thus lysing the bacteria cells. According to these results, EECD and tetracycline combination has synergistic effects at some sites of action, and thus could be used as a breakthrough to overcome infection problems due to MDR-Ab.
 
 Keywords: Acinetobacter baumannii, antibacterial, Curcuma domestica, multidrug-resistant, tetracycline

Characterization of antibiotic and disinfectant susceptibility in biofilm-forming Acinetobacter baumannii: A focus on environmental isolates.

The clinical role of Acinetobacter baumannii has been highlighted in numerous infectious syndromes with a high mortality rate, due to the high prevalence of multidrug-resistant (MDR) isolates. The treatment and eradication of this pathogen is hindered by biofilm-formation, providing protection from noxious environmental factors and antimicrobials. The aim of this study was to assess the antibiotic susceptibility, antiseptic susceptibility and biofilm-forming capacity using phenotypic methods in environmental A.baumannii isolates. One hundred and fourteen (n = 114) isolates were collected, originating from various environmental sources and geographical regions. Antimicrobial susceptibility testing was carried out using the disk diffusion method, while antiseptic susceptibility was performed using the agar dilution method. Determination of biofilm-forming capacity was carried out using a microtiter-plate based method. Resistance in environmental A. baumannii isolates were highest for ciprofloxacin (64.03%, n = 73), levofloxacin (62.18%, n = 71) and trimethoprim-sulfamethoxazole (61.40%, n = 70), while lowest for colistin (1.75%, n= 2). Efflux pump overexpression was seen in 48.25% of isolates (n = 55), 49.12% (n = 56) were classified as MDR. 6.14% (n = 7), 9.65% (n = 11), 24.65% (n = 28) and 59.65% (n = 68) of isolates were non-biofilm producers, weak, medium, and strong biofilm producers, respectively. No significant differences were observed between non-MDR vs. MDR isolates regarding their distribution of biofilm-producers (P = 0.655). The MIC ranges for the tested antiseptics were as follows: benzalkonium chloride 16-128 μg mL-1, chlorhexidine digluconate 4-128 μg mL-1, formaldehyde 64-256 μg mL-1 and triclosan 2-16 μg mL-1, respectively. The conscientious use of antiseptics, together with periodic surveillance, is essential to curb the spread of these bacteria, and to maintain current infection prevention capabilities.

Pan-azole-resistant Meyerozyma guilliermondii clonal isolates harbouring a double F126L and L505F mutation in Erg11.

Meyerozyma guilliermondii is a yeast species responsible for invasive fungal infections. It has high minimum inhibitory concentrations (MICs) to echinocandins, the first-line treatment of candidemia. In this context, azole antifungal agents are frequently used. However, in recent years, a number of azole-resistant strains have been described. Their mechanisms of resistance are currently poorly studied. The aim of this study was consequently to understand the mechanisms of azole resistance in several clinical isolates of M. guilliermondii. Ten isolates of M. guilliermondii and the ATCC 6260 reference strain were studied. MICs of azoles were determined first. Whole genome sequencing of the isolates was then carried out and the mutations identified in ERG11 were expressed in a CTG clade yeast model (C. lusitaniae). RNA expression of ERG11, MDR1 and CDR1 was evaluated by quantitative PCR. A phylogenic analysis was developed and performed on M. guilliermondii isolates. Lastly, invitro experiments on fitness cost and virulence were carried out. Of the ten isolates tested, three showed pan-azole resistance. A combination of F126L and L505F mutations in Erg11 was highlighted in these three isolates. Interestingly, a combination of these two mutations was necessary to confer azole resistance. An overexpression of the Cdr1 efflux pump was also evidenced in one strain. Moreover, the three pan-azole-resistant isolates were shown to be genetically related and not associated with a fitness cost or a lower virulence, suggesting a possible clonal transmission. In conclusion, this study identified an original combination of ERG11 mutations responsible for pan-azole-resistance in M. guilliermondii. Moreover, we proposed a new MLST analysis for M. guilliermondii that identified possible clonal transmission of pan-azole-resistant strains. Future studies are needed to investigate the distribution of this clone in hospital environment and should lead to the reconsideration of the treatment for this species.

Genomic epidemiology and ceftazidime-avibactam high-level resistance mechanisms of Pseudomonas aeruginosa in China from 2010 to 2022

ABSTRACT Ceftazidime-avibactam (CZA) resistance is a huge threat in the clinic; however, the underlying mechanism responsible for high-level CZA resistance in Pseudomonas aeruginosa (PA) isolates remains unknown. In this study, a total of 5,763 P. aeruginosa isolates were collected from 2010 to 2022 to investigate the ceftazidime-avibactam (CZA) high-level resistance mechanisms of Pseudomonas aeruginosa (PA) isolates in China. Fifty-six PER-producing isolates were identified, including 50 isolates carrying bla PER-1 in PA, and 6 isolates carrying bla PER-4. Of these, 82.1% (46/56) were classified as DTR-PA isolates, and 76.79% (43/56) were resistant to CZA. Importantly, bla PER-1 and bla PER-4 overexpression led to 16-fold and >1024-fold increases in the MICs of CZA, respectively. WGS revealed that the bla PER-1 gene was located in two different transferable IncP-2-type plasmids and chromosomes, whereas bla PER-4 was found only on chromosomes and was carried by a class 1 integron embedded in a Tn6485-like transposon. Overexpression of efflux pumps may be associated with high-level CZA resistance in bla PER-1-positive strains. Kinetic parameter analysis revealed that PER-4 exhibited a similar kcat/Km with ceftazidime and a high (∼3359-fold) IC50 value with avibactam compared to PER-1. Our study found that overexpression of PER-1 combined with enhanced efflux pump expression and the low affinity of PER-4 for avibactam contributes to high-level resistance to CZA. Additionally, the Tn6485-like transposon plays a significant role in disseminating bla PER. Urgent active surveillance is required to prevent the further spread of high-level CZA resistance in DTR-PA isolates.

Candida glabrata hospital isolate from Lebanon reveals micafungin resistance associated with increased chitin and resistance to a cell-surface-disrupting agent

This study aimed to identify the resistance mechanisms to micafungin and fluconazole in a clinical isolate of Candida glabrata. The isolate was whole-genome sequenced to identify amino acid changes in key proteins involved in antifungal resistance, and the isolate was further characterised by pathogenicity-related phenotypic assays that supported the sequencing results. Amino acid substitutions were detected in 8 of 17 protein candidates. Many of these substitutions were novel, including in CHS3, CHS3B, and KRE5, which are involved in the development of micafungin resistance. Regarding fluconazole resistance, overexpression of efflux pumps was observed. Our isolate did not exhibit an increased virulence potential compared with the control strain; however, a significant increase in chitin content and potential to resist the cell surface disruptant sodium dodecyl sulphate was observed. This clinical Candida glabrata isolate experienced a change in cell wall architecture, which correlates with the development of micafungin resistance.

A chemical screen identifies structurally diverse metal chelators with activity against the fungal pathogen Candida albicans.

Candida albicans, one of the most prevalent human fungal pathogens, causes diverse diseases extending from superficial infections to deadly systemic mycoses. Currently, only three major classes of antifungal drugs are available to treat systemic infections: azoles, polyenes, and echinocandins. Alarmingly, the efficacy of these antifungals against C. albicans is hindered both by basal tolerance toward the drugs and the development of resistance mechanisms such as alterations of the drug's target, modulation of stress responses, and overexpression of efflux pumps. Thus, the need to identify novel antifungal strategies is dire. To address this challenge, we screened 3,049 structurally-diverse compounds from the Boston University Center for Molecular Discovery (BU-CMD) chemical library against a C. albicans clinical isolate and identified 17 molecules that inhibited C. albicans growth by >80% relative to controls. Among the most potent compounds were CMLD013360, CMLD012661, and CMLD012693, molecules representing two distinct chemical scaffolds, including 3-hydroxyquinolinones and a xanthone natural product. Based on structural insights, CMLD013360, CMLD012661, and CMLD012693 were hypothesized to exert antifungal activity through metal chelation. Follow-up investigations revealed all three compounds exerted antifungal activity against non-albicans Candida, including Candida auris and Candida glabrata, with the xanthone natural product CMLD013360 also displaying activity against the pathogenic mould Aspergillus fumigatus. Media supplementation with metallonutrients, namely ferric or ferrous iron, rescued C. albicans growth, confirming these compounds act as metal chelators. Thus, this work identifies and characterizes two chemical scaffolds that chelate iron to inhibit the growth of the clinically relevant fungal pathogen C. albicansIMPORTANCEThe worldwide incidence of invasive fungal infections is increasing at an alarming rate. Systemic candidiasis caused by the opportunistic pathogen Candida albicans is the most common cause of life-threatening fungal infection. However, due to the limited number of antifungal drug classes available and the rise of antifungal resistance, an urgent need exists for the identification of novel treatments. By screening a compound collection from the Boston University Center for Molecular Discovery (BU-CMD), we identified three compounds representing two distinct chemical scaffolds that displayed activity against C. albicans. Follow-up analyses confirmed these molecules were also active against other pathogenic fungal species including Candida auris and Aspergillus fumigatus. Finally, we determined that these compounds inhibit the growth of C. albicans in culture through iron chelation. Overall, this observation describes two novel chemical scaffolds with antifungal activity against diverse fungal pathogens.

Regulation of overexpressed efflux pump encoding genes by cinnamon oil and trimethoprim to abolish carbapenem-resistant Acinetobacter baumannii clinical strains

Resistance mechanisms are a shelter for Acinetobacter baumannii to adapt to our environment which causes difficulty for the infections to be treated and WHO declares this organism on the top of pathogens priority for new drug development. The most common mechanism that develops drug resistance is the overexpression of the efflux pump, especially Resistance-nodulation-cell division (RND) family, to almost most antibiotics. The study is designed to detect RND efflux pump genes in A. baumannii, and its correlation to multidrug resistance, in particular, the carbapenems resistance Acinetobacterbaumannii (CRAB), and using different inhibitors that restore the antibiotic susceptibility of imipenem. Clinical A. baumannii isolates were recovered from different Egyptian hospitals in Intensive care unit (ICU). The expression of genes in two strains was analyzed using RT-PCR before and after inhibitor treatment. About 100 clinical A. baumannii isolates were recovered and identified and recorded as MDR strains with 75% strains resistant to imipenem. adeB, adeC, adeK, and adeJ were detected in thirty- seven the carbapenems resistance Acinetobacterbaumannii (CRAB) strains. Cinnamomum verum oil, Trimethoprim, and Omeprazole was promising inhibitor against 90% of the carbapenems resistance Acinetobacterbaumannii (CRAB) strains with a 2-6-fold decrease in imipenem MIC. Downregulation of four genes was associated with the addition of those inhibitors to imipenem for two the carbapenems resistance Acinetobacterbaumannii (CRAB) (ACN15 and ACN99) strains, and the effect was confirmed in 24 h killing kinetics. Our investigation points to the carbapenems resistance Acinetobacterbaumannii (CRAB) strain’s prevalence in Egyptian hospitals with the idea to revive the imipenem activity using natural and chemical drugs as inhibitors that possessed high synergistic activity.

Structure-Based In Silico Approaches Reveal IRESSA as a Multitargeted Breast Cancer Regulatory, Signalling, and Receptor Protein Inhibitor.

Breast cancer begins in the breast cells, mainly impacting women. It starts in the cells that line the milk ducts or lobules responsible for producing milk and can spread to nearby tissues and other body parts. In 2020, around 2.3 million women across the globe received a diagnosis, with an estimated 685,000 deaths. Additionally, 7.8 million women were living with breast cancer, making it the fifth leading cause of cancer-related deaths among women. The mutational changes, overexpression of drug efflux pumps, activation of alternative signalling pathways, tumour microenvironment, and cancer stem cells are causing higher levels of drug resistance, and one of the major solutions is to identify multitargeted drugs. In our research, we conducted a comprehensive screening using HTVS, SP, and XP, followed by an MM/GBSA computation of human-approved drugs targeting HER2/neu, BRCA1, PIK3CA, and ESR1. Our analysis pinpointed IRESSA (Gefitinib-DB00317) as a multitargeted inhibitor for these proteins, revealing docking scores ranging from -4.527 to -8.809 Kcal/mol and MM/GBSA scores between -49.09 and -61.74 Kcal/mol. We selected interacting residues as fingerprints, pinpointing 8LEU, 6VAL, 6LYS, 6ASN, 5ILE, and 5GLU as the most prevalent in interactions. Subsequently, we analysed the ADMET properties and compared them with the standard values of QikProp. We extended our study for DFT computations with Jaguar and plotted the electrostatic potential, HOMO and LUMO regions, and electron density, followed by a molecular dynamics simulation for 100 ns in water, showing an utterly stable performance, making it a suitable drug candidate. IRESSA is FDA-approved for lung cancer, which shares some pathways with breast cancers, clearing the hurdles of multitargeted drugs against breast and lung cancer. This has the potential to be groundbreaking; however, more studies are needed to concreate IRESSA's role.

The Role of Efflux Pumps transporter in Multi-drug Resistant Tuberculosis: Mycobacterial memberane protein(MmpL5).

The overexpression of efflux pumps (Eps) was reported to contribute to multidrug resistant tuberculosis (MDR-TB). Increases in Eps that expel structurally unrelated drugs contribute to reduced susceptibility by decreasing the intracellular concentration of antibiotics. In the present study, an association of mycobacterial membrane protein (MmpS5-MmpL5) Ep and its gene regulator (Rv0678) was investigated in MDR-tuberculosis isolates. MTB strains were isolated from patients at two different intervals, i.e., once when they had persistent symptoms despite 3-15 ≥ months of treatment and once when they had started new combination therapy ≥2-3 months. Sputum specimens were subjected to Xpert MTB/rifampicin test and then further susceptibility testing using proportional method and multiplex polymerase chain reaction (PCR) were performed on them. The isolates were characterized using both 16S-23S RNA and hsp65 genes spacer (PCR-restriction fragment length polymorphism). Whole-genome sequencing (WGS) was investigated on two isolates from culture-positive specimen per patient. The protein structure was simulated using the SWISS-MODEL. The input format used for this web server was FASTA (amino acid sequence). Protein structure was also analysis using Ramachandran plot. WGS documented deletion, insertion, and substitution in transmembrane transport protein MmpL5 (Rv0676) of Eps. Majority of the studied isolates (n = 12; 92.3%) showed a unique deletion mutation at three positions: (a) from amino acid number 771 (isoleucine) to 776 (valine), (b) from amino acid number 785 (valine) to 793 (histidine), and (c) from amino acid number 798 (leucine) to 806 (glycine)." One isolate (7.6%) had no deletion mutation. In all isolates (n = 13; 100%), a large insertion mutation consisting of 94 amino acid was observed "from amino acid number 846 (isoleucine) to amino acid number 939 (leucine)". Thirty-eight substitutions in Rv0676 were detected, of which 92.3% were identical in the studied isolates. WGS of mycobacterial membrane proteins (MmpS5; Rv0677) and its gene regulator (Rv0678) documented no deletion, insertion, and substitution. No differences were observed between MmpS5-MmpL5 and its gene regulator in isolates that were collected at different intervals. Significant genetic mutation like insertion, deletion, and substitution within transmembrane transport protein MmpL5 (Rv0676) can change the functional balance of Eps and cause a reduction in drug susceptibility. This is the first report documenting a unique amino acid mutation (insertion and deletion ≥4-94) in Rv0676 among drug-resistant MTB. We suggest the changes in Mmpl5 (Rv0676) might occurred due to in-vivo sub-therapeutic drug stress within the host cell. Changes in MmpL5 are stable and detected through subsequent culture-positive specimens.

Small molecular adjuvants repurpose antibiotics towards Gram-negative bacterial infections and multispecies bacterial biofilms.

Gram-negative bacterial infections pose a significant challenge due to two major resistance elements, including the impermeability of the outer membrane and the overexpression of efflux pumps, which contribute to antibiotic resistance. Additionally, the coexistence of multispecies superbugs in mixed species biofilms further complicates treatment, as these infections are refractory to most antibiotics. To address this issue, combining obsolete antibiotics with non-antibiotic adjuvants that target bacterial membranes has shown promise in combating antibacterial resistance. However, the clinical translation of this cocktail therapy has been hindered by the toxicity associated with these membrane active adjuvants, mainly due to a limited understanding of their structure and mechanism of action. Towards this goal, herein, we have designed a small molecular adjuvant by tuning different structural parameters, such as the balance between hydrophilic and hydrophobic groups, spatial positioning of hydrophobicity and hydrogen bonding interactions, causing moderate membrane perturbation in bacterial cells without any toxicity to mammalian cells. Moderate membrane perturbation not only enhances the internalization of antibiotics, but also increases the intracellular concentration of drugs by hampering the efflux machinery. This revitalises the efficacy of various classes of antibiotics by 32-512 fold, without inducing toxicity. The leading combination not only exhibits potent bactericidal activity against A. baumannii biofilms but also effectively disrupts mature multispecies biofilms composed of A. baumannii and methicillin-resistant Staphylococcus aureus (MRSA), which is typically resistant to most antibiotics. Importantly, the combination therapy demonstrates good biocompatibility and excellent in vivo antibacterial efficacy (>99% reduction) in a skin infection model of A. baumannii. Interestingly, A. baumannii shows reduced susceptibility to develop resistance against the leading combination, underscoring its potential for treating multi-drug resistant infections.

Chemotherapy Resistance in Cancer: Mechanism and Roadmap to Evade Exploring Apoptosis

Chemotherapy resistance indicates the non-responsiveness of cancer cells to the cytotoxic and inhibitory effects of chemo drugs attributed to either intrinsic or extrinsic resistance mechanisms in cancer cells. Studies so far indicate that drug resistance can be triggered by a multitude of factors such as the over-expression of drug efflux pumps, DNA repair mechanisms, modifications in the drug target such as point mutations and gene amplification, over-expression of anti-apoptotic proteins and down-regulation of pro-apoptotic signals, presence of cancer stem cells and immune-suppressive cells, excessive cytokine production, tumor heterogeneity, epigenetic changes, activation of alternate pro-survival signaling pathways, etc. Both host and tumor-related factors can contribute to therapy resistance. Currently, chemo resistance poses the foremost setback in the successful treatment of cancer, and it exerts significant stress on the available medical resources. Besides the costs associated with the treatment, patients go through severe emotional and physical trauma. Chemotherapy resistance is also a major contributor to accelerated metastasis and invasion. Dose-escalation is not always practical since the associated side effects may increase apart from increasing the treatment costs. Several studies are ongoing to address this issue productively, such as therapeutic molecules designed to restore the apoptotic machinery. Site-specific delivery of pro-apoptotic agents such as small molecules, antibodies, peptides, etc. targeting the apoptosis pathway is also thoroughly studied. Moreover, the efficacy of combination strategies is also a topic of research.

7-Year (2015-21) longitudinal surveillance of lefamulin in vitro activity against bacterial pathogens collected worldwide from patients with respiratory tract infections including pneumonia and characterization of resistance mechanisms.

Lefamulin (Xenleta™), a pleuromutilin antibiotic, was approved for the oral and IV treatment of community-acquired bacterial pneumonia (CABP) in adults in 2019/2020. This study evaluated the in vitro activity of lefamulin and comparators against 19 584 unique bacterial isolates collected from patients with community-acquired respiratory tract infections and hospitalized patients with pneumonia within the global SENTRY Antimicrobial Surveillance Program during 2015-21. Isolates were susceptibility tested by the CLSI broth microdilution method, and resistance mechanisms were investigated in isolates with elevated lefamulin MICs. Lefamulin exhibited potent antibacterial activity against the most common and typical CABP pathogens tested, including Streptococcus pneumoniae [MIC50/90, 0.06/0.25 mg/L; 99.9% susceptible (S)], Staphylococcus aureus (MIC50/90, 0.06/0.12 mg/L; 99.6% S), Haemophilus influenzae (MIC50/90, 0.5/2 mg/L; 99.1% S) and Moraxella catarrhalis (MIC50/90, 0.06/0.12 mg/L; 100.0% S). Potent activity was also observed against the less common pneumonia pathogens: β-haemolytic (MIC50/90 of 0.03/0.06 mg/L) and viridans group Streptococcus spp. (MIC50/90 of 0.06/0.25 mg/L) and Haemophilus parainfluenzae (MIC50/90 of 1/4 mg/L). Lefamulin's activity was not adversely affected by resistance to macrolides, penicillin, tetracyclines, fluoroquinolones and other resistance phenotypes. Non-susceptibility/resistance to lefamulin was rare and primarily determined by ribosomal protection through vga(A) variants in S. aureus, overexpression of AcrAB-TolC efflux pump in H. influenzae or modifications in L3, L4 and 23SrRNA in Streptococcus spp. Based on the coverage of the most important CABP pathogens and lacking cross-resistance, lefamulin may represent a valuable empirical treatment option for ambulatory and hospitalized patients with CABP, particularly in settings with high prevalence of resistance.

Prenylated isoflavonoids from Fabaceae against the NorA efflux pump in Staphylococcus aureus

Overexpression of NorA efflux pumps plays a pivotal role in the multidrug-resistance mechanism in S. aureus. Here, we investigated the activities of prenylated isoflavonoids, present in the legume plant family (Fabaceae), as natural efflux pump inhibitors (EPIs) in fluoroquinolone-resistant S. aureus. We found that four prenylated isoflavonoids, namely neobavaisoflavone, glabrene, glyceollin I, and glyceollin III, showed efflux pump inhibition in the norA overexpressing S. aureus. At sub-inhibitory concentrations, neobavaisoflavone (6.25 µg/mL, 19 µM) and glabrene (12.5 µg/mL, 39 µM), showed up to 6 times more Eth accumulation in norA overexpressing S. aureus than in the control. In addition, these two compounds boosted the MIC of fluoroquinolones up to eightfold. No fluoroquinolone potentiation was observed with these isoflavonoids in the norA knockout strain, indicating NorA as the main target of these potential EPIs. In comparison to the reported NorA EPI reserpine, neobavaisoflavone showed similar potentiation of fluoroquinolone activity at 10 µM, higher Eth accumulation, and less cytotoxicity. Neobavaisoflavone and glabrene did not exhibit membrane permeabilization effects or cytotoxicity on Caco-2 cells. In conclusion, our findings suggest that the prenylated isoflavonoids neobavaisoflavone and glabrene are promising phytochemicals that could be developed as antimicrobials and resistance-modifying agents to treat fluoroquinolone-resistant S. aureus strains.

Phenotypic characteristics of environmental Pseudomonas aeruginosa: an in vitro study on epidemiological aspects

Pseudomonas aeruginosa is one of the most commonly isolated species among non-fermenting Gram-negative bacteria, both from clinical samples and from environmental sources. The survival of the species in harsh conditions is ensured by the production of a protective biofilm; assessment of biofilm-forming capacity aids future pathogen eradication strategies. The aim of our present study was to assess the relationship between antibiotic resistance, biofilm-forming capacity and other phenotypic virulence factors in environmental P. aeruginosa isolates. One hundred and fourteen (n = 114) isolates were included in the study, which were obtained from various geographical regions and environmental origins. Antimicrobial susceptibility testing was carried out using standard protocols. Biofilm-forming capacity and pyocyanin pigment production were tested using microtiter plate-based methods. Swarming, swimming and twitching motility, and siderophore-production were assessed using agar-plate based methodologies. Resistance in environmental isolates were highest for levofloxacin/ciprofloxacin 49.12% (n = 56), ceftazidime 42.98% (n = 49) and cefepime 35.96% (n = 41), while lowest for colistin 0% (n = 0); overexpression of RND-type efflux pumps was seen in 33.33% (n = 33) of isolates. 21.93% (n = 25) met the criteria to be classified as multidrug resistant (MDR). 17.54% (n = 20) of isolates were weak/non-biofilm producers, while (25.45%, n = 29) and (57.01%, n = 65) were moderate and strong biofilm producers, respectively. No significant differences were noted in biofilm-formation (OD570 values non-MDR [mean ± SD]: 0.396 ± 0.138 vs. MDR: 0.348 ± 0.181; p > 0.05) or pyocyanin pigment production (OD686 values non-MDR: 0.403 ± 0.169 vs. MDR: 0.484 ± 0.125; p > 0.05) between MDR and non-MDR environmental P. aeruginosa. Highest motility values were observed for swarming motility, followed by swimming and twitching motility; no relevant differences (p > 0.05) in motility were noted in the context of MDR status or biofilm-formation in the tested isolates. P. aeruginosa is an opportunistic pathogen with high medical importance, being a causative agent of recalcitrant infections, which are becoming difficult to treat with the onset of MDR. Further studies are warranted to assess biofilm-forming capacity, and to provide insights into the mechanisms underlying biofilm-formation both in isolates of clinical and environmental origins.

Co-targeting HSP90 alpha and CDK7 overcomes resistance against HSP90 inhibitors in BCR-ABL1+ leukemia cells

HSP90 has emerged as an appealing anti-cancer target. However, HSP90 inhibitors (HSP90i) are characterized by limited clinical utility, primarily due to the resistance acquisition via heat shock response (HSR) induction. Understanding the roles of abundantly expressed cytosolic HSP90 isoforms (α and β) in sustaining malignant cells’ growth and the mechanisms of resistance to HSP90i is crucial for exploiting their clinical potential. Utilizing multi-omics approaches, we identified that ablation of the HSP90β isoform induces the overexpression of HSP90α and extracellular-secreted HSP90α (eHSP90α). Notably, we found that the absence of HSP90α causes downregulation of PTPRC (or CD45) expression and restricts in vivo growth of BCR-ABL1+ leukemia cells. Subsequently, chronic long-term exposure to the clinically advanced HSP90i PU-H71 (Zelavespib) led to copy number gain and mutation (p.S164F) of the HSP90AA1 gene, and HSP90α overexpression. In contrast, acquired resistance toward other tested HSP90i (Tanespimycin and Coumermycin A1) was attained by MDR1 efflux pump overexpression. Remarkably, combined CDK7 and HSP90 inhibition display synergistic activity against therapy-resistant BCR-ABL1+ patient leukemia cells via blocking pro-survival HSR and HSP90α overexpression, providing a novel strategy to avoid the emergence of resistance against treatment with HSP90i alone.