Phenotypic Screening Research Articles

Identification of DDX5 as a Potential Therapeutic Target of Osteosarcoma Using Thiazolone Probes.

Osteosarcoma (OS) is a rare malignant tumor that has predominantly affected children and adolescents in the past 50 years. The genomes of OS tumors exhibit a high degree of complexity, which leads to the great challenge of target identification for anti-OS. To date, no efficient therapeutic target for the treatment of OS has been validated in clinical practice. In our previous drug hunting for the treatment of OS by phenotypic screening, we found that thiazolone derivate (R)-8i was an effective and selective inhibitor against OS in MNNG/HOS cells and in vivo. However, the mechanism of action and specific molecular targets of (R)-8i remain unclear. In this study, we design and synthesize the photo-cross-linking probes based on the lead compound (R)-8i and identify DDX5 as a potential target protein using an activity-based protein profiling strategy. Further experiments including Western blot, shRNA knockdown experiments, cell colony formation, wound healing assays, and cellular thermal shift assays support that (R)-8i binds to DDX5 and induces its degradation, which affect cell proliferation and migration through the PI3K-AKT-mTOR signaling pathway. The research shows that DDX5 is a potential therapeutic target for the treatment of OS.

Identification of novel small molecule-based strategies of COL7A1 upregulation and readthrough activity for the treatment of recessive dystrophic epidermolysis bullosa

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare genetic disease caused by loss of function mutations in the gene coding for collagen VII (C7) due to deficient or absent C7 expression. This disrupts structural and functional skin architecture, leading to blistering, chronic wounds, inflammation, important systemic symptoms affecting the mouth, gastrointestinal tract, cornea, and kidney function, and an increased skin cancer risk. RDEB patients have an extremely poor quality of life and often die at an early age. A frequent class of mutations in RDEB is premature termination codons (PTC), which appear in homozygosity or compound heterozygosity with other mutations. RDEB has no cure and current therapies are mostly palliative. Using patient-derived keratinocytes and a library of 8273 small molecules and 20,160 microbial extracts evaluated in a phenotypic screening interrogating C7 levels, we identified three active chemical series. Two of these series had PTC readthrough activity, and one upregulated C7 mRNA, showing synergistic activity when combined with the reference readthrough molecule gentamicin. These compounds represent novel potential small molecule-based systemic strategies that could complement topical-based treatments for RDEB.

Pseudomonas fragariae sp. nov., a novel bacterial species causing leaf spots on strawberry (Fragaria×ananassa).

In Florida, angular leaf spot, caused by Xanthomonas fragariae, was the only known bacterial disease in strawberry, which is sporadic and affects the foliage and calyx. However, from the 2019-2020 to 2023-2024 Florida strawberry seasons, unusual bacterial-like symptoms were observed in commercial farms, with reports of up to 30 % disease incidence. Typical lesions were water-soaked and angular in early stages that later became necrotic with a circular-ellipsoidal purple halo, and consistently yielded colonies resembling Pseudomonas on culture media. Strains were pathogenic on strawberry, fluorescent, oxidase- and arginine-dihydrolase-negative, elicited a hypersensitive reaction on tobacco, and lacked pectolytic activity. Although phenotypic assays, such as fatty acid methyl profiles and Biolog protocols, placed the strains into the Pseudomonas group, there was a low similarity at the species level. Further analysis using 16S rRNA genes, housekeeping genes, and whole genome sequencing showed that the strains cluster into the Pseudomonas group but do not share more than 95 % average nucleotide identity compared to representative members. Therefore, the genomic and phenotypic analysis confirm that the strains causing bacterial spot in strawberry represent a new plant pathogenic bacterial species for which we propose the name Pseudomonas fragariae sp. nov. with 20-417T (17T=LMG 32456T=DSM 113340 T) as the type strain, in relation to Fragaria×ananassa, the plant species from which the pathogen was first isolated. Future work is needed to assess the epidemiology, cultivar susceptibility, chemical sensitivity, and disease management of this possible new emerging strawberry pathogen.

The phosphatase inhibitor BVT-948 can be used to efficiently screen functional sexual development proteins in the malaria parasite Plasmodium berghei

BackgroundStudying and discovering the molecular mechanism of Plasmodium sexual development is crucial for the development of transmission blocking drugs and malaria eradication. The aim of this study was to investigate the feasibility of using phosphatase inhibitors as a tool for screening proteins essential for Plasmodium sexual development and to discover proteins affecting the sexual development of malaria parasites. MethodsDifferences in protein phosphorylation among Plasmodium gametocytes incubated with BVT-948 under in vitro ookinete culture conditions were evaluated using phosphoproteomic methods. Gene Ontology (GO) analysis was performed to predict the mechanism by which BVT-948 affected gametocyte–ookinete conversion. The functions of 8 putative proteins involved in Plasmodium berghei sexual development were evaluated. Bioinformatic analysis was used to evaluate the possible mechanism of PBANKA_0100800 in gametogenesis and subsequent sexual development. ResultsThe phosphorylation levels of 265 proteins decreased while those of 67 increased after treatment with BVT-948. Seven of the 8 genes selected for phenotype screening play roles in P. berghei sexual development, and 4 of these were associated with gametocytogenesis. PBANKA_0100800 plays essential roles in gametocyte–ookinete conversion and transmission to mosquitoes. ConclusionsSeven proteins identified by screening affect P. berghei sexual development, suggesting that phosphatase inhibitors can be used for functional protein screening.

POU5F1B is responsible for the acquired resistance to dabrafenib in papillary thyroid cancer cells with the BRAF V600E mutation.

Dabrafenib, an inhibitor of the B-Raf proto-oncogene (BRAF) V600E mutant, has become the major drug for targeted therapy of papillary thyroid cancer (PTC) with the BRAF V600E mutant; however, acquired resistance is inevitable. To identify key transcription factors (TFs) involved in dabrafenib resistance and identify targets to reverse dabrafenib resistance. Dabrafenib-resistant PTC cell lines BCPAP/DabR and K1/DabR were established, and phenotypic assays were performed to validate the malignant phenotype. RNA sequencing and bioinformatics analyses were used to identify differentially expressed genes (DEGs) and screen TFs involved in resistant phenotype-related pathways. The role of the key TF POU5F1B in dabrafenib resistance was further validated using gene gain-and-loss assays. BCPAP/DabR and K1/DabR were resistant to dabrafenib, with a resistance index of 5-8. Resistant cells exhibited slower proliferation, strong migration, and spheroid-forming abilities. RNA sequencing screened 6233 DEGs in the resistant group, including 2687 protein-coding RNA (mRNA). Venn analysis indicated that three genes, E2F2, WNT4, and POU5F1B, were involved in resistant phenotype-related pathways and were included in the TF regulatory network. Four TFs of the three genes, POU5F1B, TBX4, FOXO4, and FOXP3, were validated, and POU5F1B showed the highest validated fold-change. Overexpression of POU5F1B in sensitive cells resulted in resistance to dabrafenib and induced a malignant phenotype, whereas silencing it sensitized the resistant cells and reversed the resistant phenotype. This study successfully established two dabrafenib-resistant PTC cell lines, and POU5F1B could be a potential target for reversing dabrafenib resistance.

Nontarget Analysis of Legacy and Emerging PFAS in a Lithium-Ion Power Battery Recycling Park and Their Possible Toxicity Measured Using High-Throughput Phenotype Screening.

Driven by the global popularity of electric vehicles and the shortage of critical raw materials for batteries, the spent lithium-ion power battery (LIPB) recycling industry has exhibited explosive growth in both quantity and scale. However, relatively little information is known about the environmental risks posed by LIPB recycling, in particular with regards to perfluoroalkyl and polyfluoroalkyl substances (PFAS). In this work, suspect screening and nontarget analysis were carried out to characterize PFAS in soil, dust, water and sediment from a LIPB recycling area. Twenty-five PFAS from nine classes were identified at confidence level 3 or above, including 13 legacy and 12 emerging PFAS, as well as two ultrashort-chain PFAS. Based on the target analysis of 16 PFAS, at least nine were detected in each environmental sample, indicating their widespread presence in a LIPB recycling area. Perfluorodecanoic acid, perfluorooctanesulfonic acid and trifluoromethanesulfonamide showed significant differences in the four phenotypic parameters (growth, movement, survival and fecundity) of Caenorhabditis elegans and were the most toxic substances in all target PFAS at an exposure concentration of 200 μM. Our project provides first-hand information on the existence and environmental risk of PFAS, facilitating the formulation of regulations and green development of the LIPB recycling industry.

Cuproptosis related ceRNA axis AC008083.2/miR-142-3p promotes the malignant progression of nasopharyngeal carcinoma through STRN3

Background CeRNA axis is an important way to regulate the occurrence and development of Nasopharyngeal carcinoma (NPC). Although the research on inducing cuproptosis of tumor cells is in the early stage of clinical practice, its mechanism of action is still of great significance for tumor treatment, including NPC. However, the regulation mechanism of cuproptosis in NPC by ceRNA network remains unclear. Methods The ceRNA network related to the survival of nasopharyngeal carcinoma related genes was constructed by bioinformatics. Dual-luciferase reporter assay and other experiments were used to prove the conclusion. Results Our findings indicate that the AC008083.2/miR-142-3p axis drives STRN3 to promote the malignant progression of NPC. By performing enrichment analysis and phenotypic assays, we demonstrated that the changes in the expressions of AC008083.2/miR-142-3p/NPC can affect the proliferation of NPC. Mechanistically, luciferase reporter gene assays suggested that AC008083.2 acts as a ceRNA of miR-142-3p to regulate the content of STRN3. Furthermore, the regulations of STRN3 and the malignant progression of NPC by AC008083.2 depends on miR-142-3p to some extent. Conclusions Our study reveals an innovative ceRNA regulatory network in NPC, which can be considered a new potential target for diagnosing and treating NPC.

Discovery of a potent melatonin-based inhibitor of quinone reductase-2 with neuroprotective and neurogenic properties

5-Methoxy-3-(5-methoxyindolin-2-yl)-1H-indole (3), whose structure was unambiguously elucidated by X-ray analysis, was identified as a multi-target compound with potential application in neurodegenerative diseases. It is a low nanomolar inhibitor of QR2 (IC50 = 7.7 nM), with greater potency than melatonin and comparable efficacy to the most potent QR2 inhibitors described to date. Molecular docking studies revealed the potential binding mode of 3 to QR2, which explains its superior potency compared to melatonin. Furthermore, compound 3 inhibits hMAO-A, hMAO-B and hLOX-5 in the low micromolar range and is an excellent ROS scavenger. In phenotypic assays, compound 3 showed neuroprotective activity in a cellular model of oxidative stress damage, it was non-toxic, and was able to activate neurogenesis from neural stem-cell niches of adult mice. These excellent biological properties, together with its both good in silico and in vitro drug-like profile, highlight compound 3 as a promising drug candidate for neurodegenerative diseases.

Phenotypic and Molecular Characterization of Escherichia coli Isolated from Food Animals for Antimicrobial Resistance

Background: The use of antimicrobials in food animals significantly contributes to the development of antimicrobial resistance in nature. This study aimed to determine the prevalence of ESBL-producing E. coli isolated from cattle, buffalo, sheep and goat fecal samples. Methods: In this study out of 2025 healthy animals from 10 different farms, fecal samples were randomly collected by rectal swabs from 200 animals (50 each from cattle, buffalo, sheep and goat). E. coli was isolated and further studied for AMR and ESBL production by phenotypic assays and PCR targeting ESBL genes. Result: The overall prevalence of E. coli was 81.50% with the highest isolation rate from cattle and goat samples. Out of 163 isolates, 45.39% were ESBL-producers. Genes encoding beta-lactam resistance viz. blaSHV, blaTEM, blaCTX-M and blaOXA were detected in 44.59% ESBL-positive isolates. The blaSHV was present in only cattle isolates, while blaCTX-M was present in E. coli isolated from all animals. The prevalence of beta-lactam genes was found as blaSHV (2.70%), blaTEM (12.16%), blaCTX-M (22.97%) and blaOXA (6.75%). A high degree of resistance to multiple antimicrobials was observed. Maximum susceptibility of E. coli was observed for trimethoprim, imipenem and chloramphenicol. This study demonstrated that food animals could be the source of ESBL-producing multidrug-resistant E. coli. The present findings are significant in the context of AMR monitoring in India.

Prevalence of red blood cell alloantibodies in pregnant women with sickle cell disease in Bamako.

Pregnancy in women with sickle cell disease (SCD) is associated with severe complications. Red blood cell (RBC) alloimmunisation is a worrying situation in pregnant women with SCD. This could increase the difficulty in finding a pheno-compatible red blood product. Our study aimed to determine the prevalence of RBC alloantibodies in pregnant women with SCD and to determine the risk factors for alloantibodies formation. We conducted a prospective study at the "Centre National de Transfusion Sanguine de Bamako" from August 2022 to January 2023. For each participant, we collected important information, including obstetrical and transfusion histories. We performed ABO group, Rh and Kell phenotyping, and antibody screening in all study participants. We performed statistical analysis. We recruited 95 pregnant women with SCD. In our study, 62% of our participant had a history of blood transfusion. Only 23% of our pregnant women with SCD had a history of miscarriage. The prevalence of RBC alloantibodies was 14%. The main antibodies detected were anti-E (38%) and pan-agglutinins (23%). Miscarriage history, blood transfusion history, and pregnancy number were the main risk factors for RBC alloimmunisation. The care of pregnant women with SCD is complex and requires collaboration between haematologists, clinicians and gynaecologists. National guidelines should be implemented to make ABO and D typing, Rh and Kell phenotyping and antibody screening routine for all pregnant women. This would facilitate early detection of high-risk situations. Particular attention should be paid to SCD pregnant women with miscarriage and blood transfusion histories.

Barcoded overexpression screens in gut Bacteroidales identify genes with roles in carbon utilization and stress resistance

A mechanistic understanding of host-microbe interactions in the gut microbiome is hindered by poorly annotated bacterial genomes. While functional genomics can generate large gene-to-phenotype datasets to accelerate functional discovery, their applications to study gut anaerobes have been limited. For instance, most gain-of-function screens of gut-derived genes have been performed in Escherichia coli and assayed in a small number of conditions. To address these challenges, we develop Barcoded Overexpression BActerial shotgun library sequencing (Boba-seq). We demonstrate the power of this approach by assaying genes from diverse gut Bacteroidales overexpressed in Bacteroides thetaiotaomicron. From hundreds of experiments, we identify new functions and phenotypes for 29 genes important for carbohydrate metabolism or tolerance to antibiotics or bile salts. Highlights include the discovery of a d-glucosamine kinase, a raffinose transporter, and several routes that increase tolerance to ceftriaxone and bile salts through lipid biosynthesis. This approach can be readily applied to develop screens in other strains and additional phenotypic assays.

Salmonella Prevalence and antibiotic resistance profile in raw poultry meat sold in North Lebanon: Insights from the COVID-19 pandemic and economic crisis

Salmonella-related foodborne illness is a significant public health concern, with the primary source of human infection being animal-based food products, particularly chicken meat. Lebanon is currently experiencing a dual crisis: the COVID-19 pandemic and an unprecedented economic crisis, which has resulted in substantial challenges to the public health system and food safety. This study aims to assess the prevalence and antibiotic resistance profile of Salmonella in raw poultry meat sold in North Lebanon during this dual crisis. A cross-sectional study was carried out between May 2021 and April 2022 across six different districts in North Lebanon. A total of 288 whole, unprocessed chickens were examined. The isolation and identification of Salmonella isolates were done based on cultural and biochemical properties. All isolates were subjected to antimicrobial susceptibility testing and phenotypic assays for Extended-Spectrum Beta-lactamase (ESBL) detection. The prevalence of Salmonella in raw poultry meat purchased in North Lebanon reached 18.05 % (52/288). The dry season and chilled chicken were significantly associated with an increased risk of Salmonella contamination (P < 0.05). Additionally, 34.61 % of the isolates were potential ESBL producers, and 57.69 % exhibited multidrug resistance (MDR). This study highlights the existence of MDR in chicken meat in North Lebanon, posing a potential health risk if undercooked chicken meat is consumed. This emphasizes the importance of the implementation of preventive strategies and hygienic procedures throughout the food chain to reduce the risk of Salmonella spp. contamination in chicken meats and its potential transmission to humans.

Modernizing the NCI60 Cell Line Screen for Phenotypic Drug Discovery in the 21st Century.

Over the past three decades, high-throughput phenotypic cancer cell line screens have revealed unanticipated small-molecule activities and illuminated connections between tumor genotypes and anticancer efficacy. Founded in 1984, the National Cancer Institute's "NCI60" screen laid the conceptual groundwork for the contemporary landscape of phenotypic drug discovery. NCI60 first operated as a primary bioactivity screen, but molecular characterization of the NCI60 cell line panel and development of a small-molecule sensitivity pattern recognition algorithm (called "COMPARE") have enabled subsequent studies into drug mechanisms of action and biomarker identification. In this issue of Cancer Research, Kunkel and colleagues report an updated version of the NCI60 screen, dubbed "HTS384" NCI60, that better aligns with current cell proliferation assay standards and has higher throughput. Changes include the use of a 384-well plate format, automated laboratory equipment, 3 days of compound exposure, and a CellTiter-Glo luminescent endpoint. To confirm that data from the HTS384 and classic NCI60 screen are comparable, the authors tested a library of 1,003 anticancer agents using both protocols and applied COMPARE to analyze patterns of cell line sensitivities. More than three dozen groups of targeted therapies showed high comparability between screens. Modernization of NCI60, and closer integration with other large-scale pharmacogenomic screens and molecular feature sets, will help this public screening service remain pertinent for cancer drug discovery efforts for years to come. See related article by Kunkel et al., p. 2403.

Identification of potent and reversible piperidine carboxamides that are species-selective orally active proteasome inhibitors to treat malaria

Malaria remains a global health concern as drug resistance threatens treatment programs. We identified a piperidine carboxamide (SW042) with anti-malarial activity by phenotypic screening. Selection of SW042-resistant Plasmodium falciparum (Pf) parasites revealed point mutations in the Pf_proteasome β5 active-site (Pfβ5). A potent analog (SW584) showed efficacy in a mouse model of human malaria after oral dosing. SW584 had a low propensity to generate resistance (minimum inoculum for resistance [MIR] >109) and was synergistic with dihydroartemisinin. Pf_proteasome purification was facilitated by His8-tag introduction onto β7. Inhibition of Pfβ5 correlated with parasite killing, without inhibiting human proteasome isoforms or showing cytotoxicity. The Pf_proteasome_SW584 cryoelectron microscopy (cryo-EM) structure showed that SW584 bound non-covalently distal from the catalytic threonine, in an unexplored pocket at the β5/β6/β3 subunit interface that has species differences between Pf and human proteasomes. Identification of a reversible, species selective, orally active series with low resistance propensity provides a path for drugging this essential target.

Molecular evaluation and phenotypic screening of brown and orange rust in Saccharum germplasm.

Brazil is the largest global producer of sugarcane and plays a significant role-supplier of sugar and bioethanol. However, diseases such as brown and orange rust cause substantial yield reductions and economic losses, due decrease photosynthesis and biomass in susceptible cultivars. Molecular markers associated with resistance genes, such as Bru1 (brown rust) and G1 (orange rust), could aid in predicting resistant genotypes. In this study, we sought to associate the phenotypic response of 300 sugarcane accessions with the genotypic response of Bru1 and G1 markers. The field trials were conducted in a randomized block design, and five six-month-old plants per plot were evaluated under natural disease conditions. Genotypic information about the presence or absence of Bru1 (haplotype 1) and G1 gene was obtained after extraction of genomic DNA and conventional PCR. Of the total accessions evaluated, 60.3% (181) showed resistance to brown rust in the field, and of these, 70.7% (128) had the Bru1 gene present. Considering the field-resistant accessions obtained from Brazilian breeding programs (116), the Bru1 was present in 77,6% of these accessions. While alternative resistance sources may exist, Bru1 likely confers enduring genetic resistance in current Brazilian cultivars. Regarding the phenotypic reaction to orange rust, the majority of accessions, 96.3% (288), were field resistant, and of these, 52.7% (152) carried the G1 marker. Although less efficient for predicting resistance when compared to Bru1, the G1 marker could be part of a quantitative approach when new orange rust resistance genes are described. Therefore, these findings showed the importance of Bru1 molecular markers for the early selection of resistant genotypes to brown rust by genetic breeding programs.

Phenotypic Screening of Advanced Breeding Lines for Blast Resistance in Rice (Oryza sativa L.)

Phenotypic Screening of Advanced Breeding Lines for Blast Resistance in Rice (Oryza sativa L.)

Combined Use of Phenotypic Screening and of a Novel Commercial Assay (REALQUALITY Carba-Screen) for the Rapid Molecular Detection of Carbapenemases: A Single-Center Experience.

Carbapenem resistance is a serious public health threat, causing numerous deaths annually primarily due to healthcare-associated infections. To face this menace, surveillance programs in high-risk patients are becoming a widespread practice. Here we report the performance of the combined use of a recently approved commercial multiplex real-time PCR assay (REALQUALITY Carba-Screen kit) with conventional phenotypic screening. In this three-month study, 479 rectal swabs from 309 patients across high-risk units were evaluated by combining the two approaches. Although the molecular assay showed a higher positivity rate than phenotypic screening (7.1% vs. 5%), it should be noted that the molecular method alone would have missed eight carbapenem-resistant isolates, while using only phenotypic screening would not have detected sixteen isolates. This demonstrates the complementary strengths of each method. Our study confirms the need for a combined approach to maximize the possible clinical impact of this kind of screening, ensuring a more comprehensive detection of resistant strains.

Von Willebrand disease.

von Willebrand disease (VWD) is the most common inherited bleeding disorder. The disorder is characterized by excessive mucocutaneous bleeding. The most common bleeding manifestations of this condition include nosebleeds, bruising, bleeding from minor wounds, menorrhagia or postpartum bleeding in women as well as bleeding after surgery. Other less frequent symptoms include gastrointestinal bleeding, haematomas or haemarthroses. VWD pathophysiology is complex and results from defects in von Willebrand factor (VWF) glycoprotein. Quantitative deficiencies are responsible for type 1 VWD with a partial decrease of VWF and type 3 with the complete absence of VWF. Qualitative abnormalities cause type 2 VWD, being further divided into types 2A, 2B, 2M and 2N. Although common, VWD is at risk of misdiagnosis, overdiagnosis and underdiagnosis owing to several factors, including complex diagnosis, variability of bleeding symptoms, presence of external variables (blood groups and other physiological modifiers such as exercise, thyroid hormones, oestrogens, and ageing), and lack of disease awareness among non-specialist health-care providers. Establishing the correct VWD diagnosis requires an array of specialized phenotypic assays and/or molecular genetic testing of the VWF gene. The management of bleeding includes increasing endogenous VWF levels with desmopressin or infusion of exogenous VWF concentrates (plasma-derived or recombinant). Fibrinolytic inhibitors, topical haemostatic agents and hormonal therapies are used as effective adjunctive measures.

Atypical phosphatase DUSP11 inhibition promotes nc886 expression and potentiates gemcitabine-mediated cell death through NF-kB modulation.

Pancreatic ductal adenocarcinoma (PDAC) represents one of the deadliest cancers among all solid tumors. First-line treatment relies on gemcitabine (Gem) and despite treatment improvements, refractoriness remains a universal challenge. Attempts to decipher how feedback-loops control signaling pathways towards drug resistance have gained attention in recent years, particularly focused on the role of phosphatases. In this study, a CRISPR/Cas9-based phenotypic screen was performed to identify members from the dual-specificity phosphatases (DUSP) family potentially acting on Gem response in PDAC cells. The approach revealed the atypical RNA phosphatase DUSP11 as a potential target, whose inhibition creates vulnerability of PDAC cells to Gem. DUSP11 genetic inhibition impaired cell survival and promoted apoptosis, synergistically enhancing Gem cytotoxicity. In silico transcriptome analysis of RNA-seq data from PDAC human samples identified NF-ĸB signaling pathway highly correlated with DUSP11 upregulation. Consistently, Gem-induced NF-ĸB phosphorylation was blocked upon DUSP11 inhibition in vitro. Mechanistically, we found that DUSP11 directly impacts nc886 expression and modulates PKR-NF-ĸB signaling cascade after Gem exposure in PDAC cells resulting in resistance to Gem-induced cell death. In conclusion, this study provides new insights on DUSP11 role in RNA biology and Gem response in PDAC cells.

Inside-out, antimicrobial resistance mediated by efflux pumps in clinical strains of Acinetobacter baumannii isolated from burn wound infections.

Acinetobacter baumannii belongs to the ESKAPE group. It is classified as a critical priority group by the World Health Organization and a global concern on account of its capacity to acquire and develop resistance mechanisms to multiple antibiotics. Data from the United States indicates 500 deaths annually. Resistance mechanisms of this bacterium include enzymatic pathways such as ß-lactamases, carbapenemases, and aminoglycoside-modifying enzymes, decreased permeability, and overexpression of efflux pumps. A. baumannii has been demonstrated to possess efflux pumps, which are classified as members of the MATE family, RND and MFS superfamilies, and SMR transporters. The aim of our work was to assess the distribution of efflux pumps and their regulatory gene expression in clinical strains of A. baumannii isolated from burned patients. METHODS: From the Clinical Microbiology Laboratory at the Instituto Nacional de Rehabilitación Luis Guillermo Ibarra Ibarra collection in Mexico, 199 strains were selected. Antibiotics susceptibilities were performed by broth microdilutions to determine minimal inhibitory concentrations. Phenotypic assays with efflux pump inhibitors were conducted using carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and phenylalanine-arginine ß-naphthylamide (PAßN) in conjunction with amikacin, ceftazidime, imipenem, meropenem and levofloxacin. A search was conducted for structural genes that are linked to efflux pumps, and the relative expression of the adeR, adeS, and adeL genes was analyzed. RESULTS: Among a total of 199 strains, 186 exhibited multidrug resistance (MDR). Fluoroquinolones demonstrated the highest resistance rates, while minocycline and amikacin displayed comparatively reduced resistance rates (1.5 and 28.1, respectively). The efflux activity of fluorquinolones exhibited the highest phenotypic detection (from 85 to 100%), while IMP demonstrated the lowest activity of 27% with PAßN and 43.3% with CCCP. Overexpression was observed in adeS and adeL, with adeR exhibiting overexpression. Concluding that clinical strains of A. baumannii fromour institution exhibited efflux pumps as one of the resistance mechanisms.