Drug Susceptibility Assay Research Articles

Use of stable isotope combined with intact cell lipidomic by routine MALDI mass spectrometry analysis for rapid drug susceptibility assay in mycobacteria.

Rapid, accurate, and easy-to-perform diagnostic assays are required to address the current need for the diagnosis of resistant pathogens. That is particularly the case for mycobacteria, such as the human pathogen Mycobacterium tuberculosis, which requires up to 2weeks for the determination of the drug susceptibility profile using the conventional broth microdilution method. To address this challenge, we investigated the incorporation of deuterium, the stable isotope of hydrogen, into lipids as a read out of the drug susceptibility profile. Deuterium is incorporated into newly synthesized proteins or lipids in place of hydrogen as bacterial cells grow, increasing the mass of the macromolecules, which can then be observed via matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). As proof-of-concept, we used the non-pathogenic Mycobacterium smegmatis mc2155 strain, which is susceptible to the aminoglycoside antibiotic kanamycin, and M.smegmatis mc2155 containing the empty vector pVV16, which is kanamycin-resistant. Bacteria were incubated in a culture medium containing 50% of deuterium oxide (D2O) and either 1 or 2 times the minimal inhibitory concentration (MIC50) of kanamycin. Lipids were then analyzed using the MBT lipid Xtract matrix combined with routine MALDI mass spectrometry in the positive ion mode to evaluate the changes in the lipid profile. Using this approach, we were able to distinguish susceptible from resistant bacteria in less than 5h, a process that would take 72 h using the conventional broth microdilution method. We therefore propose a solution for the rapid determination of drug susceptibility profiles using a phenotypic assay combining D2O stable isotope labelling and lipid analysis by routine MALDI mass spectrometry.

Protein target similarity is positive predictor of in vitro antipathogenic activity: a drug repurposing strategy for Plasmodium falciparum

Drug discovery is an intricate and costly process. Repurposing existing drugs and active compounds offers a viable pathway to develop new therapies for various diseases. By leveraging publicly available biomedical information, it is possible to predict compounds’ activity and identify their potential targets across diverse organisms. In this study, we aimed to assess the antiplasmodial activity of compounds from the Repurposing, Focused Rescue, and Accelerated Medchem (ReFRAME) library using in vitro and bioinformatics approaches. We assessed the in vitro antiplasmodial activity of the compounds using blood-stage and liver-stage drug susceptibility assays. We used protein sequences of known targets of the ReFRAME compounds with high antiplasmodial activity (EC50 < 10 uM) to conduct a protein-pairwise search to identify similar Plasmodium falciparum 3D7 proteins (from PlasmoDB) using NCBI protein BLAST. We further assessed the association between the compounds' in vitro antiplasmodial activity and level of similarity between their known and predicted P. falciparum target proteins using simple linear regression analyses. BLAST analyses revealed 735 P. falciparum proteins that were similar to the 226 known protein targets associated with the ReFRAME compounds. Antiplasmodial activity of the compounds was positively associated with the degree of similarity between the compounds’ known targets and predicted P. falciparum protein targets (percentage identity, E value, and bit score), the number of the predicted P. falciparum targets, and their respective mutagenesis index and fitness scores (R2 between 0.066 and 0.92, P < 0.05). Compounds predicted to target essential P. falciparum proteins or those with a druggability index of 1 showed the highest antiplasmodial activity.

Impact of sphingolipid synthesis inhibition on the drug susceptibility patterns of Trichophyton species

The well known dermatophyte infections caused by Trichophyton species are an ambiguous problem to treat using the present arsenal of antifungals. This study expounds on the effect of inhibition of sphingolipid pathway on Trichophyton growth. Findings from the drug susceptibility assays suggest sphingolipid inhibition severely restricts the growth of T. interdigitale and T. tonsurans. The observed synergistic effects of combinations of sphingolipid inhibitor and conventional drugs provide a promising treatment strategy against Trichophyton infection.

MSMEG_0311 is a conserved essential polar protein involved in mycobacterium cell wall metabolism

Cell wall synthesis and cell division are two closely linked pathways in a bacterial cell which distinctly influence the growth and survival of a bacterium. This requires an appreciable coordination between the two processes, more so, in case of mycobacteria with an intricate multi-layered cell wall structure. In this study, we investigated a conserved gene cluster using CRISPR-Cas12 based gene silencing technology to show that knockdown of most of the genes in this cluster leads to growth defects. Investigating conserved genes is important as they likely perform vital cellular functions and the functional insights on such genes can be extended to other mycobacterial species. We characterised one of the genes in the locus, MSMEG_0311. The repression of this gene not only imparts severe growth defect but also changes colony morphology. We demonstrate that the protein preferentially localises to the polar region and investigate its influence on the polar growth of the bacillus. A combination of permeability and drug susceptibility assay strongly suggests a cell wall associated function of this gene which is also corroborated by transcriptomic analysis of the knockdown where a number of cell wall associated genes, particularly iniA and sigF regulon get altered. Considering the gene is highly conserved across mycobacterial species and appears to be essential for growth, it may serve as a potential drug target.

Rapid detection of drug-resistant Mycobacterium tuberculosis by Modified MODS assay suitable for resource-poor settings.

Cross contamination and biosafety are concerns with the microscopic observation drug susceptibility assay. To address these issues, we modified the MODS technique in the current study. Two hundred and seventy-five samples were processed on LJ media and drug susceptibility was performed by the Indirect agar proportion method. A modified MODS test was done in tissue culture bottles. GenoType MTBDRplus assay was performed to detect the resistance and mutational pattern associated with the resistances. Sensitivity, specificity, positive predictive value, and negative predictive value for the detection of tuberculosis by modified MODS were 97.44%, 80.00%, 97.44%, and 80.00% respectively. The perfect agreement was seen between modified MODS and the Indirect agar proportion method for drug susceptibility testing of isoniazid (kappa = 0.923) and rifampicin (kappa = 1). The contamination rate, cost and TAT for modified MODS were less as compared to the solid media. In the case of MDR-TB isolates S531L (66.66%) was the most prevalent mutation in the rpoB gene followed by S315T2 mutation (58.33%) and T8C (41.66%) in katG and inhA gene respectively. In hetero-resistant strains, C-15T mutation (37.50%) was the most common followed by A-16G (12.50%) in the inhA gene. In INH mono-resistant strains only two mutations were observed i.e., S-315T1(50%) and C-15T (50%) in the katG and inhA genes respectively. Modified MODS proved to be cost-effective and user-friendly, with minimal risk to the handler and no cross-contamination between samples were observed. Hence, it can be used in low-income countries for early detection of tuberculosis and its resistance.

In vitro drug susceptibility using a parasite-rescue and transformation assay of Leishmania (Mundinia) martiniquensis and Leishmania (Mundinia) orientalis amastigotes against antileishmanial drugs

Leishmaniasis is an emerging infectious disease in Thailand, with Leishmania martiniquensis and Leishmania orientalis identified as the primary causative agents among immunocompetent and immunocompromised individuals. Variations in drug susceptibility among different Leishmania species have been reported in different regions. Therefore, drug susceptibility assays are essential to assess the effectiveness of antileishmanial drugs used or potentially used in the affected areas. This study investigated the in vitro drug sensitivity of L. martiniquensis and L. orientalis, along with two reference species causing VL, namely L. donovani and L. infantum, against six antileishmanial drugs. Using a parasite-rescue and transformation assay, the results demonstrated that the IC50 values of amphotericin B (AmB), miltefosine (MIL), and sodium stibogluconate (Sb(III)) against all Leishmania species tested were within the sensitive range of each drug. On the contrary, the IC50 values of artemisinin (ART) and dihydroartemisinin (DHA), drugs primarily used for malaria treatment, were outside the sensitive range of the Leishmania species tested except L. infantum. This in vitro study highlights that AmB could effectively exhibit good sensitivity against the intracellular amastigotes of L. martiniquensis and L. orientalis. Also, MIL and Sb(III) could be considered alternative drugs for antileishmanial treatment in Thailand.

Generation of protoplasts provides a powerful experimental research tool for biological and pathogenicity studies of Pythium insidiosum.

Pythiosis is a high-mortality infectious condition in humans and animals. The etiologic agent is Pythium insidiosum. Patients present with an ocular, vascular, cutaneous/subcutaneous, or gastrointestinal infection. Antifungal medication often fails to fight against P. insidiosum. The effective treatment is limited to radical surgery, resulting in organ loss. Fatal outcomes are observed in advanced cases. Pythiosis needs to be studied to discover novel methods for disease control. Genome data of P. insidiosum is publicly available. However, information on P. insidiosum biology and pathogenicity is still limited due to the lack of a cost-effective animal model and molecular tools. We aimed to develop a high-efficiency protocol for generating P. insidiosum protoplast, and used it to set up an animal model, in vitro drug susceptibility assay, and DNA transformation for this pathogen. P. insidiosum protoplast was successfully generated to establish a feasible pythiosis model in embryonic chicken eggs and an efficient in vitro drug susceptibility assay. DNA transformation is a critical method for gene manipulation necessary for functional genetic studies in pathogens. Attempts to establish a DNA transformation method for P. insidiosum using protoplast were partly successful. Significant work needs to be done for genetically engineering a more robust selection marker to generate stable transformants at increased efficiency. This study is the first to report an efficient P. insidiosum protoplast production for clinical and research applications. Such advances are crucial to speeding up the pathogen's biology and pathogenicity exploration.

In vitro activities of contezolid (MRX-I) against drug-sensitive and drug-resistant Mycobacterium tuberculosis.

A novel oxazolidinone for the treatment of Mycobacterium tuberculosis has been developed, but the activity of contezolid (MRX-I) still needs to be clarified. In this study, we isolated Mycobacterium tuberculosis from 48 clinical patients with pulmonary tuberculosis. Roche drug susceptibility tests identified drug-sensitive and 39 drug-resistant M. tuberculosis isolates. Drug susceptibility assays indicated that MRX-I exhibited anti-Mycobacterium tuberculosis activity against both drug-sensitive and drug-resistant isolates, with an advantage against drug-resistant isolates. The results also showed that the anti-Mycobacterium tuberculosis activity was comparable to that of linezolid. IMPORTANCE Currently, Mycobacterium tuberculosis has exhibited increased drug resistance, leading to ineffective drug treatment in many patients with tuberculosis. Among the anti-Mycobacterium tuberculosis drugs, oxazolidinones have been gradually developed. Contezolid (MRX-I) has been newly developed in China with advantages versus the first oxazolidinone antibiotic approved by the Food and Drug Administration for clinical use, but the anti-M. tuberculosis activity needs to be further clarified. In this study, in vitro activities of MRX-I against M. tuberculosis were tested. The drug susceptibility assays indicated that MRX-I exhibited anti-M. tuberculosis activity comparable to that of linezolid, with an advantage against drug-resistant isolates.

To Test or Not? Xpert MTB/RIF as an Alternative to Smear Microscopy to Guide Line Probe Assay Testing for Drug-Resistant Tuberculosis.

Xpert MTB/RIF (Xpert) revolutionized tuberculosis (TB) diagnosis. Laboratory decision making on whether widely-used reflex drug susceptibility assays (MTBDRplus, first-line resistance; MTBDRsl, second-line) are conducted is based on smear status, with smear-negative specimens often excluded. We performed receiver operator characteristic (ROC) curve analyses using bacterial load information (smear microscopy grade, Xpert-generated semi-quantitation categories and minimum cycle threshold [CTmin] values) from Xpert rifampicin-resistant sputum for the prediction of downstream line probe assay results as "likely non-actionable" (no resistance or susceptible results generated). We evaluated actionable-to-non-actionable result ratios and pay-offs with missed resistance versus LPAs done universally. Smear-negatives were more likely than smear-positive specimens to generate a non-actionable MTBDRplus (23% [133/559] versus 4% [15/381]) or MTBDRsl (39% [220/559] versus 12% [47/381]) result. However, excluding smear-negatives would result in missed rapid diagnoses (e.g., only 49% [264/537] of LPA-diagnosable isoniazid resistance would be detected if smear-negatives were omitted). Testing smear-negatives with a semi-quantitation category ≥ "medium" had a high ratio of actionable-to-non-actionable results (12.8 or a 4-fold improvement versus testing all using MTBDRplus, 4.5 or 3-fold improvement for MTBDRsl), which would still capture 64% (168/264) and 77% (34/44) of LPA-detectable smear-negative resistance, respectively. Use of CTmins permitted optimization of this ratio with higher specificity for non-actionable results but decreased resistance detected. Xpert quantitative information permits identification of a smear-negative subset in whom the payoffs of the ratio of actionable-to-non-actionable LPA results with missed resistance may prove acceptable to laboratories, depending on context. Our findings permit the rational expansion of direct DST to certain smear-negative sputum specimens.

Doravirine responses to HIV-1 viruses bearing mutations to NRTIs and NNRTIs under in vitro selective drug pressure.

The NNRTI doravirine has been recently approved for the first-line treatment of HIV-infected patients, eliciting favourable responses against viruses bearing the K103N, Y181C and G190A mutations. This study used in vitro drug selections to elaborate the breadth of doravirine responses against viruses bearing NNRTI and NRTI resistance-associated mutations (RAMs). WT clinical isolates (n = 6) and viruses harbouring common NRTI and NNRTI RAMs (n = 6) were serially passaged in escalating concentrations of doravirine, doravirine/islatravir, doravirine/lamivudine and rilpivirine over 24 weeks. Genotypic analysis ascertained the appearance and accumulation of NNRTI RAMs. Phenotypic drug susceptibility assays assessed resistance conferred by acquired NNRTI RAMs. For WT viruses, doravirine pressure led to the appearance of V108I or V106A/I/M RAMs after 8 weeks, conferring low-level (∼2-fold) resistance. After 24 weeks, the accumulation of three to six secondary RAMs, including F227L, M230L, L234I and/or Y318, resulted in high-level (>100-fold) resistance to doravirine. Notably, viruses with these doravirine RAMs remained susceptible to rilpivirine and efavirenz. This contrasted with rilpivirine where acquisition of E138K, L100I and/or K101E resulted in >50-fold cross-resistance to all NNRTIs. Doravirine selection of viruses bearing common NRTI and NNRTI RAMs showed delayed acquisition of RAMs compared with WT virus. Pairing doravirine with islatravir or lamivudine attenuated the development of NNRTI RAMs. Doravirine showed favourable resistance profiles against viruses harbouring NRTI and NNRTI RAMs. The high barrier to resistance to doravirine coupled with the long intracellular half-life of islatravir may provide the opportunity for long-acting treatment options.

Evaluation of novel PCR-CTPP for simultaneous detection of Mycobacterium tuberculosis complex and identification of RpoB H526D point mutation

Background: Tuberculosis (TB) is a chronic and highly contagious disease caused of the acid-fast bacilli, Mycobacterium tuberculosis complex (MTC). Currently, it has been reported that rifampicin-mono resistant M. tuberculosis containing histidine to aspartate replacement at residue 526 (H526D) of the beta-subunit RNA polymerase enzyme (RpoB) increased the cell wall permeability and approximately eight times more susceptible to vancomycin. Objectives: To develop and evaluate the Polymerase chain reaction with confronting two-pair primers (PCR-CTPP) for simultaneous detection of MTC and identification of RpoB H526D mutation. Materials and methods: PCR-CTPP was implemented for TB diagnosis. Reaction and profile were optimized and applied for detection in a total of 308 clinical samples. Sensitivity and specificity of PCR-CTPP was calculated in comparison to the acid-fast bacilli (AFB) staining and standard culture method. In addition, microscopic observation drug susceptibility (MODS) assay was modified for vancomycin susceptibility determination in 15 clinical isolates. Results: Limit of detection of PCR-CTPP was approximately 2×103 bacilli and no cross-detection to other mycobacteria. PCR-CTPP was evaluated in 308 clinical samples. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of PCR-CTPP versus standard culture method were 90.95%, 79.31%, 91.78% and 77.53%, respectively. In addition, comparison of developed PCR-CTPP versus AFB staining were also represented. MODS was performed in fifteen samples, two multidrug-resistant (MDR) strains containing RpoB H526D were susceptible against vancomycin. Conclusion: The established PCR-CTPP is highly sensitive, specific for investigation of MTC and identification of RpoB H526D mutation. This method could be useful for TB diagnosis together with precision medicine application in vancomycin susceptibility determination in TB patients in the future.

Abstract 3895: New potential mechanism of mnx1-as1 in the regulation of the carboplatin chemoresistant phenotype in ovarian cancer cell lines

Abstract Introduction: Ovarian cancer (OC) is one of the most lethal diseases in the female population worldwide, being considered by 2020 the eighth most common type of cancer and the eighth cause of cancer death in that population. The most widely used chemotherapy treatment is based on the application of platinum based drugs, carboplatin (CBDCA) being one of the most common, in combination with taxanes and cisplatin. Unfortunately, a high percentage of the population relapses shortly after chemotherapy treatment, presenting a drug resistant phenotype. This phenotype reduces overall survival in patients with OC. In this context, various mechanisms have been investigated to understand and study these chemoresistant phenotypes. In the last decade, lncRNAs have been identified in multiple biological processes, including the development of drug-resistant phenotypes in malignant tumors, however, little is known about their role in carboplatin resistance in OC. This study seeks to characterize the effect of lncRNA expression on the chemoresistant phenotype and its potential molecular mechanism involved. Materials and Methods: Ovarian cancer cell lines corresponding to: A2780-R-CBDCA, A2780-P, UCI-101, OVCAR3 and SKOV3 were used. Drug susceptibility assays were performed using MTT. Relative expression was performed via RTqPCR. RNAseq was performed on a CBDCA resistance model in vitro using the Illumina HiSeq 4000 platform. Gene Ontology (GO) was analyzed using ClueGO. Gene set enrichment analysis (GSEA) was performed to define enriched biological pathways. Triplexator was used to predict the triplex forming oligonucleotides (TFO) and triplex target sites (TTS) that may be involved in the formation of RNA-DNA triplexes. Results: RNAseq analysis showed 156 differentially expressed protein coding genes (DEGs). The most enriched and deregulated signaling pathway was Wnt/β-catenin. In this context, several regulators of this pathway were found to be altered, including sFRP1, sFRP4, PCDHB6, CTNNA2, DACT1, PCDHB6, WNT3A, and CER1. In addition, 17 lncRNAs involved in CDBCA resistance were found to be deregulated. In this sense, MNX1-AS1 showed high levels of expression in CBDCA resistant cell lines. Interestingly, it has been shown that MNX1-AS1 has the ability to form a triplex with the PCDHB6 gene, which is a tumor suppressor gene and whose expression was found to be decreased in CBDCA resistant cell lines. Conclusions: Our results indicate that MNX1-AS1 expression could contribute to the modulation of platinum drug resistance through regulation of PCDHB6 expression via triplexes formation in OC. This finding could explain a new mechanism in CBDCA resistance in OC. Citation Format: Tamara Alejandra Viscarra Alvarez, Kurt Leopoldo Buchegger Mena, Daniela Inés León Garrido, Carmen Gloria Ili Gangas, Jorge Sapunar Zenteno, Marcela Berrios Flores, Ramón Silva Pezoa, Sindy Paola Cabarca Barreto, Priscilla Brebi Mieville. New potential mechanism of mnx1-as1 in the regulation of the carboplatin chemoresistant phenotype in ovarian cancer cell lines. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3895.

In Vitro Antimycobacterial Activity of Human Lactoferrin-Derived Peptide, D-hLF 1-11, against Susceptible and Drug-Resistant Mycobacterium tuberculosis and Its Synergistic Effect with Rifampicin.

Tuberculosis is a highly contagious disease caused by the Mycobacterium tuberculosis complex (MTBC). Although TB is treatable, multidrug-resistant, extensively drug-resistant, and totally drug-resistant forms of M. tuberculosis have become a new life-threatening concern. New anti-TB drugs that are capable of curing these drug-resistant strains are urgently needed. The purpose of this study is to determine the antimycobacterial activity of D-enantiomer human lactoferricin 1-11 (D-hLF 1-11) against mycobacteria in vitro using a 3-(4,5-dimethylthiazol-2-yl)-2,5-dephenyltetrazolium bromide colorimetric assay, resazurin microplate assay, and microscopic observation drug susceptibility assay. Three previously described antimicrobial peptides, protegrin-1, AK 15-6, and melittin, with potent anti-TB activity, were included in this study. The findings suggest that D-hLF 1-11 can inhibit the growth of M. tuberculosis with a minimum inhibitory concentration of 100-200 µg/mL in susceptible, isoniazid (INH)-monoresistant, rifampicin (RF)-monoresistant, and MDR strains. The peptide can also inhibit some nontuberculous mycobacteria and other MTBC in similar concentrations. The antibiofilm activity of D-hLF 1-11 against the biofilm-forming M. abscessus was determined by crystal violet staining, and no significant difference is observed between the treated and untreated biofilm control. The checkerboard assay was subsequently carried out with M. tuberculosis H37Rv and the results indicate that D-hLF 1-11 displays an additive effect when combined with INH and a synergistic effect when combined with RF, with fractional inhibitory concentration indices of 0.730 and 0.312, respectively. The red blood cell hemolytic assay was initially applied for the toxicity determination of D-hLF 1-11, and negligible hemolysis (&lt;1%) was observed, despite a concentration of up to 4 mg/mL being evaluated. Overall, D-hLF 1-11 has potential as a novel antimycobacterial agent for the future treatment of drug-sensitive and drug-resistant M. tuberculosis infections.

Genetic Diversity, Antibiotic Resistance, and Virulence Gene Features of Methicillin-Resistant Staphylococcus aureus Epidemics in Guiyang, Southwest China.

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most common pathogens of community- and hospital-acquired infections, and its prevalence is increasing globally. Guiyang is the capital city of Guizhou Province, Southwest China; as the transport and tourism centre of Southwest China, Guizhou Province is bordered by Yunnan, Sichuan, Chongqing, and Guangxi Provinces. Although MRSA prevalence is increasing, little is known about its aspects in the area. The purpose of this study was to analyse MRSA molecular characteristics, antimicrobial resistance, and virulence genes in Guiyang. In total, 209 MRSA isolates from four hospitals (2019-2020) were collected and analysed by antimicrobial susceptibility testing and molecular classification by the MLST, spa, and SCCmec typing methods. Isolate antibiotic resistance rates were detected by a drug susceptibility assays. PCR amplification was used to detect the virulence gene-carrying status. Twenty-four STs, including 4 new STs (ST7346, ST7347, ST7348, and ST7247) and 3 new allelic mutations, were identified based on MLST. The major prevalent ST type and clone complex were ST59 (49.8%) and CC59 (62.7%), respectively. Spa type t437 (42.1%) and SCCmec IV (55.5%) were identified by spa and SCCmec typing methods as the most important types. Drug sensitivity data showed that the multidrug resistance rate was 79.0%. There were significant differences in multidrug resistance rates and virulence gene-carrying rates for seb, hla, hlb, cna and bap between ST59 and non-ST59 types. ST59-SCCmecIV-t437 is a major epidemic clone in Guiyang that should be monitored by local medical and health institutions. The situation differs from other adjacent or middle provinces of China, which may be due to the special geographical location of the region and the trend in antibiotic use or lifestyle. This study provides empirical evidence for local medical and health departments to prevent and control the spread of MRSA.

Evaluation of three alternatives cost-effective culture media for Mycobacterium tuberculosis detection and drug susceptibility determination using the microscopic observation drug susceptibility (MODS) assay

Tuberculosis phenotypic detection assays are commonly used in low-resource countries. Therefore, reliable detection methods are crucial for early diagnosis and treatment. The microscopic observation drug susceptibility (MODS) assay is a culture-based test to detect Mycobacterium tuberculosis and characterize drug resistance in 7–10 days directly from sputum. The use of MODS is limited by the availability of supplies necessary for preparing the enriched culture. In this study, we evaluated three dry culture media that are easier to produce and cheaper than the standard one used in MODS [1]: an unsterilized powder-based mixed (Boldú et al., 2007) [2], a sterile-lyophilized medium, and (Sengstake et al., 2017) [3] an irradiated powder-based mixed. Mycobacterial growth and drug susceptibility were evaluated for rifampin, isoniazid, and pyrazinamide (PZA). The alternative cultures were evaluated using 282 sputum samples with positive acid-fast smears. No significant differences were observed in the positivity test rates. The positivity time showed high correlations (Rho) of 0.925, 0.889, and 0.866 between each of the three alternative media and the standard. Susceptibility testing for MDR and PZA showed an excellent concordance of 1 compared to the reference test. These results demonstrate that dry culture media are appropriate and advantageous for use in MODS in low-resource settings.

P472 The mechanism of action of antifungal activity of Zanthoxylum armatum fruit's oil against Candida cells does not involve ROS generation

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PMObjectiveTo explore the antifungal activity of Zanthoxylum armatum fruit's oil against different Candida species and its mechanism of action.MethodsThe Z. armatum fruit's oil activity was assessed against C. albicans, C. krusei, C. glabrata, C. parapsilosis, C. tropicalis, and C. guilliermondii through different drug susceptibility assays including, MIC, agar diffusion and spot assay. The mechanism of action was explored through sterol analysis, germ tube inhibition, epithelial cells adherence, and ROS generation.ResultsThe oil from the fruits of Z. armatum was subjected to GC-MS analysis, and linalool (72%) was found as the major component. The drug susceptibility measured through different methods, including minimum inhibitory concentration (MIC), where end-point was 3% v/v for different species tested, and the same pattern was observed in agar diffusion and spot assay. The antifungal activity was found to be fungicidal in nature and the major reason appeared to be the reduction in ergosterol levels inside cells. It resulted in lowered germ tube formation, an important indicator in virulence of C. albicans. The oil reduced adherence of Candida cells to buccal epithelium significantly, which is the first step in invasion, biofilm formation, and damage to oral epithelial cells. Interestingly, unlike most antifungals, where reactive oxygen species generation mediated killing is involved, was not found significant in the present study.ConclusionsThe Z. armatum fruits oil exerts its antifungal activity by inhibiting ergosterol formation and reduced germ tube formation.

Chemotherapy drug potency assessment method of ovarian cancer cells by digital holography microscopy

Drug potency assessment plays a crucial role in cancer chemotherapy. The selection of appropriate chemotherapy drugs can reduce the impact on the patient's physical condition and achieve a better therapeutic effect. Various methods have been used to achieve in vitro drug susceptibility assays, but there are few studies on calculating morphology and texture parameters quantitatively based on phase imaging for drug potency assessment. In this study, digital holography microscopy was used to get phase imaging of ovarian cancer cells after adding three different drugs, namely, Cisplatin, Adriamycin, and 5-fluorouracil. Based on the reconstructed phase imaging, four parameters of ovarian cancer cells changed with time, such as the average height, projected area, cluster shade, and entropy, were calculated. And the half-inhibitory concentration of cells under the effect of different drugs was calculated according to these four parameters. The half-inhibitory concentration, which can directly reflect the drug potency, is associated with the morphological and texture features extracted from phase images by numerical fitting. So, a new method for calculating the half-inhibitory concentration was proposed. The result shows that the morphological and texture feature parameters can be used to evaluate the sensitivity of ovarian cancer cells to different drugs by fitting the half-inhibitory concentration numerically. And the result provides a new idea for drug potency assessment methods before chemotherapy for ovarian cancer.

β-Nitrostyrene derivatives as broad range potential antifungal agents targeting fungal cell wall

The prevalence of multidrug resistance has been increasingly witnessed during the past few decades. Resistance of human pathogenic fungi against the currently available antifungal agents has increased the frequency of fungal infections and associated mortality rates. The discovery of novel lead antifungal agents is important to challenge multidrug resistance. The present study examined the antifungal potential of chemically synthesized β-Nitrostyrene derivatives. Among the eight β-Nitrostyrene derivatives used in this study, SS45, SS46 and SS47 showed strong antifungal potential. The results show that β-Nitrostyrene derivatives inhibited the growth of different species of human pathogenic Candida, particularly the highly prevalent C. albicans, C. glabrata and the emerging pathogenic C. auris species. Moreover, β-Nitrostyrene derivatives also show strong antifungal activities against drug-resistant clinical isolates and drug transporter overexpressing fungal species. The drug susceptibility assays revealed that β-Nitrostyrene derivatives are fungicidal and show the synergy of action when combined with antifungal drugs caspofungin and fluconazole. The transcriptomic study performed on C. albicans in the presence of β-Nitrostyrene derivatives revealed the differential expression of genes related to cell wall metabolism. Mechanistically, β-Nitrostyrene derivatives impact cell wall morphology, enhance ROS generation and modulate drug efflux. Collectively this study reveals that β-Nitrostyrene derivatives have strong antifungal potential with a particular mode of activity similar to known cell wall perturbing antifungal agents and thus can be exploited as promising potential antifungal agents for further studies.

Susceptibility of Drug Resistant Hepatitis B Virus Mutants to Besifovir.

Currently, interferon alpha and nucleos(t)ide analogues (NAs) are clinically available to treat hepatitis B virus (HBV) infection. Several NAs, including lamivudine (LMV), adefovir (ADV), entecavir (ETV) and tenofovir (TDF or TAF) have been approved and administered to chronic hepatitis B (CHB) patients. NAs inhibit HBV DNA synthesis by targeting the reverse transcriptase (RT) domain of HBV polymerase. Several mutations in the RT domain which lead to drug resistance against NAs have been reported, even for TDF and TAF which are highly potent with very low resistance rate. Besifovir (BFV) is a new antiviral dGMP analogue able to be used as a new NA drug for the control of CHB infection. Drug resistance to BFV is not well known due to its shorter duration of clinical use. Recently, we reported that rtL180M (M) and rtM204V (V) mutations, already resistant to LMV, are associated with BFV resistance. However, the susceptibility to BFV of previously known HBV mutants resistant to various drugs has not been studied. To investigate this, we performed in vitro drug susceptibility assays using natural and artificial mutants that are associated with resistance to LMV, ADV, ETV or TDF. As a result, LMV-resistant mutants were not susceptible to BFV and ETV-resistant clones showed partial resistance against BFV as well. However, ADV-resistant mutants were highly sensitive to BFV. In case of tenofovir-resistant mutations, the HBV mutants harboring primary mutations to tenofovir resistance were susceptible to BFV. Therefore, our study revealed that BSV may serve as an alternative drug for patients with ADV-, ETV-, TDF- or TAF-resistance.

Abstract 2630: Heterogeneity inducible tumor stem cells relate distant metastasis and the treatment resistance

Abstract Regarding metastatic potential of cancer cells and the refractory status, regulatory and epigenetic factors underlie the cancer treatment resistance by inducing cellular heterogeneity. Circulating tumor cells (CTCs) and their stemness are critical for metastases formation associated with poor survival rates. In colorectal cancer (CRC), several stem-cell markers, such as CD44v9 and LGR5, the positive cells cause intertumoral heterogeneity and promote refractory potential. However, the mastermind of the cells which is critical for the treatment resistance remains unknown. Here, we aimed to identify the mechanisms of the treatment resistance of the CRC by focusing on typical stem cells. We selected several stem cell-related genes such as pluripotency related genes, based on the expression profile analysis of CRC patients along with drug susceptibility assays using patient-derived organoids. Despite the small number of cancer stem cells, the single cell produced heterogeneous cell populations in vitro and in vivo, expressing various differentiation and stem biomarkers, and confirmed by the knockdown analysis. Single-cell trajectory analysis suggested that heterogeneous populations including cancer stem and some persister cells were derived from the stem cells following administration of anti-cancer drugs. Furthermore, the stem cells formed heterogeneous liver metastases at a high rate, and these unique cells were observed in the bloodstream of CRC patients. This present study unveils a mechanism of CRC treatment resistance and highlights the potential of stem marker-expressing cells as CTCs. Citation Format: Norikatsu Miyoshi, Shiki Fujino, Yuki Sekido, Tsuyoshi Hata, Takayuki Ogino, Hidekazu Takahashi, Mamoru Uemura, HIrofumi Yamamoto, Yuichiro Doki, Hidetoshi Eguchi. Heterogeneity inducible tumor stem cells relate distant metastasis and the treatment resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2630.