Drug Resistance Genes Research Articles

Hierarchy and interconnected networks in the WhiB7 mediated transcriptional response to antibiotic stress in Mycobacterium abscessus.

Mycobacterium abscessus is intrinsically resistant to antibiotics effective against other pathogenic mycobacteria largely due to the drug-induced expression of genes that confer resistance. WhiB7 is a major hub controlling the induction of resistance to ribosome-targeting antibiotics. It activates the expression of >100 genes, 7 of which are known determinants of drug resistance; the function of most genes within the regulon is however unknown, but some conceivably encode additional mechanisms of resistance. Furthermore, the hierarchy of gene expression within the regulon, if any, is poorly understood. In the present work we have identified 56 WhiB7 binding sites using chromatin immunoprecipitation sequencing (CHIP-Seq) which accounts for the WhiB7-dependent upregulation of 72 genes, and find that M. abscessus WhiB7 functions exclusively as a transcriptional activator at promoters recognized by σA/σB. We have investigated the role of 18 WhiB7 regulated genes in drug resistance. Our results suggest that while some genes within the regulon (eg. erm41, hflX, eis2 and the ABCFs) play a major role in resistance, others make smaller contributions (eg. MAB_4324c and MAB_1409c) and the observed hypersensitivity ΔMabwhiB7 is a cumulative effect of these individual contributions. Moreover, our CHIP-Seq data implicate additional roles of WhiB7 induced genes beyond antibiotic resistance. Finally, we identify a σH-dependent network in aminoglycoside and tigecycline resistance which is induced upon drug exposure and is further activated by WhiB7 demonstrating the existence of a crosstalk between components of the WhiB7-dependent and -independent circuits.

Emerging and comparative genomic analysis of a novel plasmid carrying blaKPC-2 in Citrobacter freundii

Objective: To explore the drug resistance mechanism and gene structure characteristics of a carbapenemase-producing novel incompatibility group plasmid pNY2385-KPC from Citrobacter freundii. Methods: A multi-drug resistant strain was obtained from urine samples of patients with fever in the emergency ward of Li Huili Hospital, Ningbo Medical Center. Bacterial species was preliminary identified and finally confirmed by 16S rRNA gene amplification and the average nucleotide identity alignment, respectively. The minimum inhibitory concentrations of the antimicrobial agents were determined by VITEK 2 Compact System. The complete genome sequence was obtained by "third-generation" sequencing methods, and then detailed annotation of gene function and comparative genomic analysis of plasmid structure were carried out by BLASTP/BLASTN, RefSeq, ConservedDomains, ResFinder, Isfinder, etc. Results: The pNY2385-KPC carried by citrobacter freundii NY2385 belonged a novel incompatibility group, and contained blaKPC-2 and conjugative transfer (type Ⅳ secretory system, T4SS) genes, which could induce conjugative transfer. A total of 15 plasmids of the same type as pNY2385-KPC were retrieved by NCBI, which were from Citrobacter freundii, and the rest were from Serratia marcescens, Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae, Raoultella planticola and other bacteria, and were broad-host-range plasmids. The sequence comparative analysis of all 6 of the novel plasmid from Citrobacter freundii showed that the structure of the novel plasmid had certain conserved property, with Tn6296 variant structure carrying blaKPC-2, and plasmid pCF1807-3 had both repApNY2385-KPC and repAIncX8. Conclusion: The pNY2385-KPC type plasmids in Citrobacter freundii carried blaKPC-2 resistance gene, which were divided into two subtypes: repApNY2385-KPC single replicator and repApNY2385-KPC/repAIncX8 complex replicator, belonging to broad-host-range plasmids. And as a mobile genetic element, the plasmids promote the spread of blaKPC-2.

Core–Shell Silica Nanoparticle-Based Barcodes Combined with a Hybridization Chain Reaction for Multiplex Quantitative Detection of Bacterial Drug-Resistance Genes

Core–Shell Silica Nanoparticle-Based Barcodes Combined with a Hybridization Chain Reaction for Multiplex Quantitative Detection of Bacterial Drug-Resistance Genes

Targeted elimination of Vancomycin resistance gene vanA by CRISPR-Cas9 system

ObjectiveThe purpose of this study is to reduce the spread of the vanA gene by curing the vanA-harboring plasmid of vancomycin-resistant using the CRISPR-Cas9 system.MethodsTwo specific spacer sequence (sgRNAs) specific was designed to target the vanA gene and cloned into plasmid CRISPR-Cas9. The role of the CRISPR-Cas system in the plasmid elimination of drug-resistance genes was verified by chemically transformation and conjugation delivery methods. Moreover, the elimination efficiency in strains was evaluated by plate counting, PCR, and quantitative real-time PCR (qPCR). Susceptibility testing was performed by broth microdilution assay and by Etest strips (bioMérieux, France) to detect changes in bacterial drug resistance phenotype after drug resistance plasmid clearance.ResultsIn the study, we constructed a specific prokaryotic CRISPR-Cas9 system plasmid targeting cleavage of the vanA gene. PCR and qPCR results indicated that recombinant pCas9-sgRNA plasmid can efficiently clear vanA-harboring plasmids. There was no significant correlation between sgRNA lengths and curing efficiency. In addition, the drug susceptibility test results showed that the bacterial resistance to vancomycin was significantly reduced after the vanA-containing drug-resistant plasmid was specifically cleaved by the CRISPR-Cas system. The CRISPR-Cas9 system can block the horizontal transfer of the conjugated plasmid pUC19-vanA.ConclusionIn conclusion, our study demonstrated that CRISPR-Cas9 achieved plasmid clearance and reduced antimicrobial resistance. The CRISPR-Cas9 system could block the horizontal transfer of plasmid carrying vanA. This strategy provided a great potential to counteract the ever-worsening spread of the vanA gene among bacterial pathogens and laid the foundation for subsequent research using the CRISPR-Cas9 system as adjuvant antibiotic therapy.

Сравнительный биоинформатический анализ состава генов антимикробной устойчивости в геномах представителей рода Corynebacterium

Currently, multidrug resistance of bacterial infectious agents poses a serious threat to the global public health. The following Corynebacterium strains are of special importance for infections, including hospital-acquired ones: C. amycolatum, C. urealyticum, C. striatum, C. jeikeium, C. aurimucosum, C. genitalium that are resistant to the broad spectrum of antimicrobial drugs. The study was aimed to conduct bioinformatics analysis of the pool of antimicrobial resistance genes in the published genomes of some members of the genus Corynebacterium. The data on the whole genome nucleotide sequences of 22 Corynebacterium isolates readily available from NCBI GenBank were assessed. Bioinformatics analysis of the whole genome sequences conducted in order to search for antimicrobial resistance genes in the specified genomes was performed using the PATRIC online resource. It was found that the genomes provided comprised various combinations of 25 antimicrobial drug resistance genes. Amino acid substitutions in GyrA (positions 87, 88 and 91) were revealed in some Corynebacterium strains, through which quinolone/fluoroquinolone resistance could be realized.

The MAGMA pipeline for comprehensive genomic analyses of clinical Mycobacterium tuberculosis samples.

Whole genome sequencing (WGS) holds great potential for the management and control of tuberculosis. Accurate analysis of samples with low mycobacterial burden, which are characterized by low (<20x) coverage and high (>40%) levels of contamination, is challenging. We created the MAGMA (Maximum Accessible Genome for Mtb Analysis) bioinformatics pipeline for analysis of clinical Mtb samples. High accuracy variant calling is achieved by using a long seedlength during read mapping to filter out contaminants, variant quality score recalibration with machine learning to identify genuine genomic variants, and joint variant calling for low Mtb coverage genomes. MAGMA automatically generates a standardized and comprehensive output of drug resistance information and resistance classification based on the WHO catalogue of Mtb mutations. MAGMA automatically generates phylogenetic trees with drug resistance annotations and trees that visualize the presence of clusters. Drug resistance and phylogeny outputs from sequencing data of 79 primary liquid cultures were compared between the MAGMA and MTBseq pipelines. The MTBseq pipeline reported only a proportion of the variants in candidate drug resistance genes that were reported by MAGMA. Notable differences were in structural variants, variants in highly conserved rrs and rrl genes, and variants in candidate resistance genes for bedaquiline, clofazmine, and delamanid. Phylogeny results were similar between pipelines but only MAGMA visualized clusters. The MAGMA pipeline could facilitate the integration of WGS into clinical care as it generates clinically relevant data on drug resistance and phylogeny in an automated, standardized, and reproducible manner.

Correlation analysis between Raman spectral signature and transcriptomic features of carbapenem-resistant Klebsiella pneumoniae

The Raman microspectroscopy technology has been successfully applied to evaluate the molecular composition of living cells for identifying cell types and states, but the rationale behind it was not well investigated. In this study, we acquired single-cell Raman spectra (SCRS) of three Klebsiella pneumoniae (K. pneumoniae) strains with different Carbapenem resistant mechanisms and analyzed them with machine learning algorithm. Two carbapenem resistant Klebsiella pneumoniae (CRKP) strains can be successfully distinguished from susceptible strain and CRKP with KPC or IMP carbapenemases can be classified with an overall accuracy achieving 100 %. Furthermore, we performed a correlation analysis between transcriptome and Raman spectra, and found that Raman shifts such as 752 and 1039 cm−1 highly correlated with drug resistance genes expression and could be regarded as Raman biomarkers for CRKP with different mechanisms. The findings of the study provide a theoretical basis for identifying the relationship between Raman spectra and transcriptome of bacteria, as well as a novel method for rapid identification of CRKP and their carbapenemases types.

CRISPR/Cas9 Screen Identifies CPX-351 and 7+3 Regimens Response Modulators with Distinct Sensitive and Resistant Profiles

CPX-351 (dual-drug liposomal encapsulation of daunorubicin and cytarabine in a synergistic 1:5 molar ratio) is approved for newly diagnosed high-risk and secondary AML as an alternative to the widely used cytarabine and daunorubicin (7+3) regimen. Despite newly approved drugs, many AML patients still developed treatment resistance and relapse, leading to poor survival. Amid mounting research into the molecular mechanisms of AML therapies, treatment resistance remains a major concern. To overcome treatment resistance, it is necessary to examine the molecular mechanisms underlying the CPX-351 and 7+3 treatment response. We conducted custom CRISPR/Cas9 synthetic-lethal screens targeting 2440 genes (AML-relevant genes, cytarabine and daunorubicin pharmacology, and druggable genes) in six AML cell lines, profiled drug-sensitive/-resistant modulators, and identified novel targets for overcoming drug resistance in CPX-351 and 7+3 regimen. Six AML cell lines were transduced with a custom library and puromycin was selected before pool transduced cells were treated with DMSO, the IC30-IC50 of cytarabine, daunorubicin, or CPX-351 for 7 doubling-times at 500x coverage. All samples were processed with genomic DNA, sgRNAs were amplified by PCR, and Illumina NovaSeq 6000 SP 100SR sequencing was performed. The abundance of sgRNA was analyzed using MAGeCK-RRA to estimate the RRA drug response score (Drug vs. Control conditions at 7-doubling times) or RRA essential score (Control at 7-doubling time vs. Control at Day 0). Genes with an average RRA score of &amp;lt; -1 across six AML cell lines were defined as drug-resistant or essential genes (negative selection), whereas genes with an average RRA score of &amp;gt; 1 were categorized as sensitive or suppressor genes (positive selection). Next, comparisons of functional pathways with over-representation analysis were performed on resistant and sensitive genes across three drugs to determine differences in their functions. At the average RRA response score cut-off, 61, 163, and 112 were identified as drug-resistant genes, while 46, 93, and 86 were sensitive genes to cytarabine, daunorubicin, or CPX-351 (provided by Jazz Pharmaceuticals), respectively. Fig 1A showed a Venn diagram of all significant resistant and sensitive genes across all three drugs with 14 common significant genes and 73, 174, and 118 unique genes to cytarabine, daunorubicin and CPX-351, respectively, along with their essential score. Among these significant common response genes, some notable genes included ABCC1, SAMHD1 and TP53, where CRISPR knocking out screens led to depletion or enrichment of sgRNA when treated with drugs across all AML cell lines. ABCC1 is a drug-resistant gene that encodes the ATP Binding Cassette C1-mediated drug efflux transporter; therefore, knocking out the efflux transporter causing drug resistance, would increase the lethality of AML cells . Our result also showed ABCC1 isa tumor suppressor gene; because knocking out the ABCC1 gene improves cell survival and growth . SAMHD1 is another notable gene that encodes for deoxynucleoside triphosphate (dNTP) tri-phosphohydrolase that cleaves physiological dNTPs into deoxyribonucleosides and inorganic triphosphate. SAMHD1 has been shown to cause resistance in cytarabine, but there is limited evidence of its response to other drugs. In this study, we showed that SAMHD1 is a resistant gene for CPX-351 and a sensitive gene for daunorubicin, and it is a tumor suppressor gene. Lastly, TP53 is one of the significant common sensitive genes to cytarabine, daunorubicin, and CPX-351. TP53 encodes for the well-known tumor suppressor gene tumor protein 53, and mutations in TP53 have been shown to affect drug response. In this study, we demonstrated that knocking out TP53 in all AML cell lines contributed to cell survival and growth as a tumor suppressor gene and a drug-sensitive gene across the three drugs. Comparing significant resistant and sensitive genes across CPX-351, cytarabine, and daunorubicin, as shown in Fig 1B, revealed that resistant genes shared some common involvement in the cell cycle and cellular response to stress, but resistant genes for each drug displayed more distinct functional pathways. Moreover, each drug's sensitive genes revealed distinct functional enrichment pathways. Our current research is focused on identifying novel drug targets to combat drug resistance in the CPX-351 and 7+3 regimens.

Leveraging Custom CRISPR/Cas9 Screens to Identify AraC-Daunorubicin-Etoposide (ADE), Gemtuzumab Ozogamicin (GO), and Bortezomib Response Modulators Association with Clinical Outcomes in Pediatric AML

Cytarabine, daunorubicin, and etoposide (ADE) remain the standard backbone chemotherapy for pediatric AML. In addition to ADE, gemtuzumab ozogamicin (GO) and bortezomib have been evaluated in the COG's AAML0531 and AAML1031 clinical trials, respectively. Nonetheless, when these experimental agents were combined with ADE, ADE+GO had shown to improve EFS but not OS, while ADE+bortezomib displayed no additional benefit over ADE alone. Thus, treatment options for pediatric AML are still limited, and development of resistance is a major concern, resulting in poor clinical outcomes. To overcome treatment resistance, it is crucial to understand the underlying molecular mechanisms of ADE±GO or bortezomib. Herein, we conducted custom CRISPR/Cas9 synthetic-lethal screens targeting 2440 genes (including AML-relevant genes, pharmacologically relevant genes, and druggable genes), in six AML cell lines with exposure to ara-C, daunorubicin, etoposide, GO and bortezomib followed by integration of the results with outcome data from multiple clinical trials where induction treatment was either standard ADE chemotherapy, ADE+GO or ADE+bortezomib. Six AML cell lines were transduced with a custom library and puromycin selected. Then pool transduced cell populations were then treated with DMSO, the IC30-IC50 levels of cytarabine, daunorubicin, etoposide, GO, or bortezomib for 7-doubling-times at 500x coverage. All samples were processed with genomic DNA extraction, sgRNAs were amplified by PCR, and were subjected to Illumina NovaSeq 6000 SP 100SR sequencing. The abundance of sgRNA was analyzed using MAGeCK-RRA to estimate the RRA drug response score (drug vs. control conditions at 7-doubling times). Genes with an average RRA score of &amp;lt; -1 across six AML cell lines were defined as drug-resistant genes (negative selection), whereas genes with an average RRA score of &amp;gt; 1 were categorized as sensitive genes (positive selection). Next, we investigated leukemic cell gene expression levels of the drug-resistant and drug-sensitive genes with clinical outcomes. At the average RRA score cutoff from CRISPR screens, 459 genes met the significance as resistant or sensitive genes to at least one drug from ADE's screens as ADE genes, 244 genes were significant from ADE genes and GO's screen as ADE+GO genes, and 166 genes were significant from ADE genes and bortezomib'screen as ADE+bortezomib genes ( Fig 1a). ADE genes were evaluated with clinical outcomes with patients treated with ADE AML02 (n=163), ADE arm of AAML0531 (n=201) and AAML1031 (n=411); while ADE+GO genes were evaluated in ADE+GO arm of AAML0531 (n=205) and ADE+bortezomib genes were evaluated in ADE+bortezomib arm of AAML1031 (n=436). Genes showing drug resistance in CRISPR screens and associated with detrimental outcomes (EFS, OS, or MRD1) at high expression were deemed clinically and biologically important drug genes and vice versa. Our preliminary results showed 21 met the criteria as clinically and biologically important drug-resistant or drug-sensitive genes, where genes were significant in multiple CRISPR screens and multiple pediatric AML cohorts ( Fig 1b). Six genes were determined to resist multiple drugs, with higher expression associated with worse outcomes across multiple cohorts. Particularly, ABCC1 encodes the ATP Binding Cassette C1-mediated drug efflux transporter; therefore, knocking out the efflux transporter causing drug resistance, would increase the lethality of AML cells, resulting in poor outcomes in multiple cohorts in patients treated with ADE alone and ADE with GO or bortezomib. Five genesshowed significant as sensitive genes for multiple drugs in our CRISPR screens with high expression association with favorable outcomes in multiple cohorts. Interestingly, two genes were identified as bortezomib-sensitive genes with high expression in patients treated with ADE+bortezemib in AAML1031 cohort had better outcomes. Other genes were revealed to be sensitive to one drug but resistant to another, indicating that antagonistic pleiotropy effects between genes and drugs may affect clinical outcomes. Current research focuses on developing a pipeline to predict different drug combination outcomes for prioritizing drug treatments and identifying novel drug targets to overcome drug resistance to ADE+GO or bortezomib.

Exceptional Response to BTK Inhibitors in Aggressive Lymphomas Linked to Chronic Selective Autophagy

Diffuse large B cell lymphoma (DLBCL) is an aggressive cancer that is profoundly heterogeneous, both molecularly and phenotypically, presenting a challenge for precision medicine. Inhibitors of Bruton tyrosine kinase (BTK) block B cell receptor (BCR)-dependent NF-κB signaling and are particularly effective in DLBCL with mutations in the BCR subunit CD79B and MYD88 (MCD DLBCL). MCD tumors are enriched for a multiprotein supercomplex, termed the My-T-BCR, that is nucleated by MYD88 L265P, TLR9 and the BCR, and serves as a central hub of NF-κB signaling. The integrity of the My-T-BCR complex is rapidly compromised following BTK inhibition, but the molecular mechanisms responsible for the dissolution of this molecular machine have not been elucidated. To investigate the underlying mechanisms regulating the My-T-BCR, we used genome wide CRISPR-Cas9 screens in MCD DLBCL cell line models treated with the BTK inhibitors (BTKi) and compared the results to screens with inhibitors of SYK, IKK, mTOR, and BRD4. We identified several drug resistance genes encoding known negative regulators of BCR, NF-κB, and PI3 kinase signaling that were recurrently inactivated by mutation and/or deletion in DLBCL biopsies. Unexpectedly, we identified multiple autophagy-related genes involved autophagosome formation ( ATG9A, ATG101, ATG13, RB1CC1 and ATG14) and autophagosome membrane expansion ( ATG2A, WIPI2, WDR45) that strongly counteracted the toxicity of BTKi (≥3 SD). Notably, these autophagy genes did not promote resistance to the mTOR inhibitors, which induces classical autophagy. To gain further insight, we generated BTKi-resistant cell lines deficient in ATG9A or ATG101 (ATG KO) and performed genome-wide CRISPR screens and RNA-seq with or without BTKi. In doing so, we observed 11 ATG genes that displayed epistatic interactions, no longer conferring BTKi resistance in ATG KO MCD cells (p≤0.05). We also observed the buffering of many NF-κB negative regulators (p≤0.001) and the increased sensitivity to loss of NF-κB positive regulators (p≤0.05). Furthermore, gene expression studies showed a marked decrease of BCR, MYD88 and NF-κB signatures in control BTKi-treated cells, whereas ATG KOs demonstrated a rescue of these same gene signatures and displayed higher levels of nuclear NF-κB localization upon BTKi treatment. MCD patients display the highest levels of NF-κB gene expression compared to other genetic subtypes. Interestingly, MCD patients also displayed the lowest gene expression profile for a subset of the ATG genes (p≤2.7x10 -6), suggesting that this pathway may be counter-selected during MCD pathogenesis. As autophagy promotes the lysosomal degradation of proteins, and disruption of autophagy promoted NF-κB signaling in MCD cell lines, we assessed global protein levels and localization of proteins to the My-T-BCR in ATG KO cells using mass spectrometry. Selective autophagy receptors TAX1BP1, NBR1 and p62 were significantly upregulated in ATG KOs and displayed significant enrichment of proximity to MYD88 L265Pin ATG KOs (p≤0.01), suggesting MYD88 L265P is targeted for degradation by selective autophagy. To test this, we engineered MCD cell lines with dual fluorescent autophagy reporters (GFP-RFP fusions) for TAX1BP1, NBR1, p62, or MYD88 L265P. Each reporter displayed an accumulation of GFP relative to RFP in ATG KOs or upon treatment with the autophagy blocking v-ATPase inhibitor bafilomycin. To identify genes that modulate selective autophagy of MYD88 L265P, we performed a CRISPR screen in MCD cells stably expressing a MYD88 L265P-RFP-GFP reporter. We observed deletion of the same epistatic interactors of ATG9A in BTKi survival screens also blocked MYD88 L265P autophagic degradation. Conversely, deletion of BTK, mTORC1-related genes and IRF4 were all among the top-ranked genes that promoted MYD88 L265P autophagic degradation. We validated these findings using chemical inhibitors of each gene alone or in combination and observed synergy for the promotion of MYD88 L265P autophagic degradation. Collectively, we identified a non-canonical form of selective autophagy that chronically degrades MYD88 L265P, is counter-selected in MCD tumors, and is promoted by BTK inhibitors. Our findings help to elucidate the exceptional benefit of BTK-targeted therapies in the MCD DLBCL subtype and offer a rationally designed combination therapy regimen to specifically degrade this mutant allele of MYD88.

Phenotypic and genomic insights into the pathogenicity and antimicrobial resistance of an Enterobacter roggenkampii strain isolated from diseased silver arowana (Osteoglossum bicirrhosum).

Enterobacter roggenkampii is an opportunistic pathogen that causes infections in a wide range of hosts. A bacterial strain named EOBSR_19 was isolated from diseased silver arowana, Osteoglossum bicirrhosum. This bacterium was identified as E. roggenkampii based on the phenotypic characteristics and sequence analysis of the16S rDNA and gyrB genes. Average nucleotide identity and phylogenetic analysis based on the whole genome sequence further confirmed the bacterial taxonomy of EOBSR_19. Artificial experimental infection indicated that EOBSR_19 was pathogenic to fish. Antimicrobial susceptibility test showed it was multi-drug resistant. The EOBSR_19 was found to be resistant to 18 antibiotics belonging to quinolones, macrolides, sulfonamides, aminoglycosides, and β-lactams classes. The whole genome sequencing analysis showed that EOBSR_19 carried 730 virulence genes that were annotated for different functional modules, such as adhesion and invasion, secretion system, siderophore transport system and bacterial toxin. Among them, the virulence genes related to adhesion and invasion were the most abundant. In addition, drug resistance genes involved in multiple mechanisms of antimicrobial resistance were identified in its genomics, including multidrug resistance efflux pumps, antibiotic inactivating enzymes, and antibiotic binding site mutations. Its genomic analysis via whole-genome sequencing provided insights into the pathogenicity and antimicrobial resistance.

Phenotypic identification of Candida species and relative expression of candida drug resistance (CDR1) gene in fluconazole resistant and sensitive Candida albicans

ABSTRACT:&#x0D; Background and objective:&#x0D; Candida albicans belongs to the genus Candida. It is a commensal yeast in healthy individuals and can cause wide variety of infections in immune-compromised patients. The aim of the present study was to observe the frequency of Candida species, resistant profile of C. albicans against fluconazole and over-expression of CDR1 gene in both fluconazole resistant and sensitive C. albicans.&#x0D; Methodology:&#x0D; A total number of 206 Candida species were collected on Sabouraud Dextrose agar from Jinnah hospital Lahore. The isolates were re-confirmed by using various microbiological techniques like wet preparation, Gram stain and Germ tube test. Identification upto the species level was done by APIC 20AUX. Antifungal susceptibility profile of C. albicans against fluconazole was determined by broth microdilutiom method. Real Time PCR was performed on both the sensitive and resistant C. albicans to observe the role of CDR1 gene against fluconazole resistance. Results:&#x0D; &#x0D; tropicals was the most common species with the frequency of 66 (32.0%) followed by C. albicans 41(19.9), C. glabrata 24 (11.7%), C. lusitaniae 14 (6.8%) and other less common species. The majority of infected patients were females (59.2%) than males (41%). Antifungal susceptibility testing showed that 80% C. albicans were sensitive to fluconazole. Results of gene expression showed that CDR1 gene play a major role towards fluconazole resistance in C.albicans.Conclusion: Fluconazole was found to be effective in treating infections caused by C. albicans. &#x0D; &#x0D; Keywords: Candida albicans, Candida Drug Resistant Gene 1, Polymerase Chain Reaction, Candida Tropicalis, Candida glabrata, and Candida lusitaniae

Analysis of changes in the genome of Vibrio cholerae O1 El Tor genovariants during the current period of the cholera pandemic

Introduction. The genome variability of genetic variants of El Tor cholera agent has led to the emergence of strains carrying mutations in various genes associated with epidemically important pathogen properties. This situation requires an assessment of the trends in these changes in order to predict the pathogenic potential of previously unknown variants and promptly develop new tools for their diagnostics and prevention.&#x0D; The purpose of this work was to analyze the dynamic changes in pathogenicity and drug resistance genes of V. cholerae El Tor genetic variants from endemic countries and Russia.&#x0D; Materials and methods. We analyzed complete genome nucleotide sequences of 104 V. cholerae El Tor strains from the NCBI Gen Bank and European Nucleotide Archive databases, as well as those obtained by us. The nucleotide sequences were analyzed using the UGEN v. 45.1 software. The dendrogram was constructed using maximum parsimony algorithm in BioNumerics v.7.6 software package based on the multiple alignment generated using the Snippy 4.6.0 program.&#x0D; Results. Genomic sequences of 103 strain genovariants isolated on the territory of nine endemic countries of Asia and Africa, as well as in Russia in 1991-2022, have been compared. It is shown that the process of genovariant genome changing was multistage and occurred due to the continuous accumulation of point mutations in key (ctxB and tcpA) and additional (rtxA) genes of pathogenicity and core genes of antibiotic resistance (gyrA, parC and carR), as well as a deletion in SXT element. The most important was the change in the ctxB gene and the emergence of new genovariants with the ctxB7 allele, which replaced the previously prevalent strains. Analysis of altered genome regions of 83 strains from endemic regions has revealed eight genotypes, while the strains (21 isolates) imported to Russia belonged to only five of them including highly virulent strains with the ctxB7 allele and lost PolR biovar-specific feature due to carR gene mutation. The established close phylogenetic relatedness of genovariants from Russia with strains from endemic Asian countries confirms their importation from this region.&#x0D; Conclusion. The sequential occurrence and accumulation of mutations in the pathogenicity and drug resistance genes in the genome of genovariants in endemic regions have been shown, which leads to a change in their epidemically important features. The importation of new highly virulent genovariants into Russia has been established, which indicates the need for an ongoing assessment of changes in the genome of this pathogen for the timely development of adequate means of gene diagnostics and prevention.

Isolation and Characterization of New Bacteriophages against Staphylococcal Clinical Isolates from Diabetic Foot Ulcers.

Staphylococcus sp. is the most common bacterial genus in infections related to diabetic foot ulcers (DFUs). The emergence of multidrug-resistant bacteria places a serious burden on public health systems. Phage therapy is an alternative treatment to antibiotics, overcoming the issue of antibiotic resistance. In this study, six phages (SAVM01 to SAVM06) were isolated from effluents and were used against a panel of staphylococcal clinical samples isolated from DFUs. A genomic analysis revealed that the phages belonged to the Herelleviridae family, with sequences similar to those of the Kayvirus genus. No lysogeny-associated genes, known virulence or drug resistance genes were identified in the phage genomes. The phages displayed a strong lytic and antibiofilm activity against DFU clinical isolates, as well as against opportunistic pathogenic coagulase-negative staphylococci. The results presented here suggest that these phages could be effective biocontrol agents against staphylococcal clinical isolates from DFUs.

Revitalizing pond culture system: Harnessing the power of composite nanopeptide C[sbnd]I20, 1,3-1,6-β-glucan, and anthocyanidin biotherapy as an antibiotic substitute for efficient management of bacterial diseases in bullfrogs

Bacterial diseases in aquaculture ponds have significant negative effects on sustainable development. Antibiotic resistance in aquatic pathogens has reduced the effectiveness of traditional treatments. To address this, composite additives that boost the natural defense system are being explored. In this study, we focused on four common symptoms in bullfrog ponds: amyosthenia, black body color, crooked head, and pythiosis. We also observed overlapping symptoms such as amyosthenia + crooked head and black body color + pythiosis. The overall incidence rate of these symptoms was 85.6%, with Aeromonas hydrophila and Elizabethkingia miricola identified as the responsible pathogens. To treat bullfrog bacterial diseases, we used two treatments: florfenicol (FF) at 20 mg/Kg and a composite I20 (CI) containing nanopeptide CI20 (2 g/Kg), β-glucan (1 g/Kg), and anthocyanidin (1 g/Kg). We implemented a staged therapy approach, comprising initial assessment, self-cure intervention, and continuous treatment stages, along with continuous monitoring to assess the effectiveness and usage protocol of CI. In the initial therapy stage, oral CI or florfenicol improved body color and rapidly reduced morbidity rate and daily mortality rate. CI biological agent exerted anti-bacterial infection by improving mucosal barrier, antioxidant enzyme activity (T-AOC and TSOD), and innate immunity index (LZM activity, leukocyte number). The CI treatment demonstrated significant reductions in tissue injury (intestine, liver, spleen) and tissue bacterial load. Following the stage therapy, the CI group exhibited lower morbidity and daily mortality rates compared to the FF group. Additionally, the total protection rate of the CI group surpassed that of the FF and control groups during the stage therapy. The antibiotic resistance and drug resistance genes (FIoR and FexA) and virulence genes (HxlR, ClpP, OmpA, and FlaA) in A. hydrophila and E. mirabilis were markedly induced by florfenicol treatment, respectively. In continuing therapy stage (42 days), the morbidity, protection rate, and daily mortality of the CI group were better than that of the FF group. In conclusion, composite I20 biotherapy effectively treats the bacterial disease of bullfrogs in pond system, providing a prospectively novel strategy for controlling bacterial disease in aquaculture.

The effect of paclitaxel- and fisetin-loaded PBM nanoparticles on apoptosis and reversal of drug resistance gene ABCG2 in ovarian cancer

BackgroundHigh-grade serous ovarian cancer (OvCa) is the most common type of epithelial OvCa. It is usually diagnosed in advanced stages, leaving a woman’s chance of survival below 50%. Despite traditional chemotherapeutic therapies, there is often a high recurrence rate following initial treatments. Hence, a targeted drug delivery system is needed to attack the cancer cells and induce apoptosis, overcome acquired drug resistance, and protect normal cells from cytotoxicity. The present study shows that targeting folate receptor alpha (FRα) through planetary ball milling (PBM) nanoparticles (NPs) induces apoptosis in OvCa cells.ResultsHuman tissue microarrays (TMAs) show overexpression of FRα in Stage IV OvCa tissues compared to matched normal tissues. They provide a focus for a targeted delivery system. We formulated PBM nanoparticles encapsulated with paclitaxel (PTX) or fisetin (Fis) and conjugated with folic acid (FA). The cytotoxic effect of these PBM NPs reduced the concentration of the toxic chemotherapy drug PTX by five-fold. The combined treatment of PTX-FA NPs and Fis-FA NPs inhibited cell proliferation and induced apoptosis more extensively than the individual drugs alone. Apoptosis of OvCa cells, determined by flow cytometry, showed an increase from 14.4 to 80.4% (OVCAR3 cells) and from 2.69 to 90.0% (CAOV3 cells) in the number of apoptotic cells. Also, expressions of the pro-apoptotic markers, BAK and active caspase-3, were increased after PTX-FA + Fis-FA PBM NP treatment. In addition to looking at targeted treatment effects on apoptosis, drug resistance was investigated. Drug resistance in OvCa cells was reversed by ABCG2, an ABC-transporter marker.ConclusionsOur study shows that PTX-FA and Fis-FA PBM NPs directly target platinum-resistant OvCa cells, induce cytotoxic/apoptotic effects, and reverse multi-drug resistance (MDR). These findings allow us to create new clinical applications using PTX-FA and Fis-FA combination nanoparticles to treat drug-resistant cancers.

YTHDF1 enhances stemness and chemoresistance in triple-negative breast cancer cells by upregulating SIAH2.

Triple-negative breast cancer (TNBC) is the most lethal and aggressive subtype of breast cancer, and chemoresistance is the major determinant of TNBC treatment failure. This study explores the molecular mechanism of TNBC chemoresistance.The Cancer Genome Atlas, breast cancer integrative platform, and GEPIA databases were used to analyze the expression and correlation of YTHDF1 and seven in absentia homology 2 (SIAH2) in breast cancer. Knockdown of YTHDF1 and SIAH2, or overexpression of SIAH2 in vitro and in vivo, was conducted to evaluate the impact of changes in YTHDF1 and SIAH2 expression on TNBC cell proliferation, apoptosis, stemness, drug resistance, and Hippo pathway gene expression.YTHDF1 and SIAH2 were highly expressed in breast cancer patients and TNBC cells. Knockdown of YTHDF1 and SIAH2 significantly inhibited proliferation and stemness and promoted apoptosis and chemosensitivity of TNBC cells. Mechanistically, the knockdown of YTHDF1 inhibited the expression of SIAH2, thereby downregulating the Hippo pathway, which inhibited proliferation and stemness and promoted apoptosis and chemosensitivity of TNBC cells.The current findings revealed the regulatory mechanism of YTHDF1 in TNBC and clarified the role of the YTHDF1/SIAH2 axis in TNBC drug resistance and stemness. This could provide new insights into the vital role of targeting YTHDF1/SIAH2 to suppress drug resistance and stemness in TNBC cells.

Polymorphism of Drug Resistance Genes dhfr and dhps in Plasmodium falciparum Isolates among Chinese Migrant Workers Who Returned from Ghana in 2013.

In 2013, an epidemic of falciparum malaria involving over 820 persons unexpectedly broke out in Shanglin County, Guangxi Zhuang Autonomous Region, China, after a large number of migrant workers returned from Ghana, where they worked as gold miners. Herein, we selected 146 isolates randomly collected from these patients to investigate the resistance characteristics of the parasite to sulfadoxine-pyrimethamine (SP) by screening mutations in the dhfr and dhps genes. All 146 isolates were successfully genotyped for dhps, and only 137 samples were successfully genotyped for dhfr. In the dhfr gene, point mutations occurred at three codons: 51 (83.2%, 114/137), 59 (94.9%, 130/137), and 108 (96.4%, 132/137). In the dhps gene, mutations occurred at four codons: 436 (36.3%, 53/146 for S436A, 0.7%, 1/146 for S436Y), 437 (95.2%, 139/146), 540 (3.4%, 5/146), and 613 (2.7%, 4/146). All 146 isolates had mutations in at least one codon, either within dhfr or dhps. Quadruple mutation I51R59N108/G437 (41.1%, 60/146) of partial or low resistance level was the most prevalent haplotype combination. Quintuple I51R59N108/G437E540 accounted for 2.1% (3/146). Sextuple I51R59N108/A436G437S613 was also found and accounted for 1.4% (2/146). A chronological assay incorporating two sets of resistance data from the studies of Duah and Amenga-Etego provided an overview of the resistance trend from 2003 to 2018. During this period, the results we obtained generally coincided with the total development tendency of SP resistance. It can be concluded that Plasmodium falciparum samples collected from Chinese migrant workers from Ghana presented prevalent but relatively partial or low resistance to SP. A chronological assay incorporating two sets of data around 2013 indicates that our results possibly reflect the SP resistance level of Ghana in 2013 and that the possibility of increased resistance exists. Therefore, reasonable drug use and management should be strengthened while also maintaining a continuous screening of resistance to SP. These findings also underscore the need to strengthen the prevention of malaria importation from overseas and focus on preventing its reintroduction and transmission in China.

Engineering a metastatic stroma directs the osteosarcoma tumour transcriptome in a spatially specific manner

The biological behaviour of the cancer cell is in part controlled by its microenvironment and in part by underlying genetics. The interaction of cancer cells with the surrounding stroma directs cancer progression. The most common primary malignant tumour of the bone, osteosarcoma, is a highly complex disease in terms of its progression, invasion, and metastasis. To better understand disease characteristics and develop accurate therapies for osteosarcoma, novel tissue models have been developed to systematically understand how facets of the tumour microenvironment (TME) can influence cancer cell behaviour. This study aims to understand how osteosarcoma invasion and metastasis is influenced by different types of stromal cells in the TME. The engineering of complex tumouroids, which segregate the tumour and stromal cells in specific 3D compartments, allows for the study of tumour-stroma interaction and invasion of cells between different compartments. Complex tumouroids comprised a central osteosarcoma tumour mass with different engineered stromal compartments, representative of the primary bone or metastatic lung. Spatial Transcriptomics (ST) unveiled that osteosarcoma cells upregulated metastatic genes such as IFI27 in the presence of a lung stromal compartment, whereas a bone stromal compartment resulted in the upregulated of oncogenes and drug resistance genes such as DUSP1 and HIF1A within the osteosarcoma compartment. Engineering a primary stromal cell compartment compared to a metastatic stromal cell compartment unveiled how the regulation of cell proliferation, invasion, extracellular matrix (ECM) remodelling, metastasis markers and tumour suppressor markers was altered in the osteosarcoma transcriptome.

The F pilus serves as a conduit for the DNA during conjugation between physically distant bacteria.

Horizontal transfer of F-like plasmids by bacterial conjugation is responsible for disseminating antibiotic resistance and virulence determinants among pathogenic Enterobacteriaceae species, a growing health concern worldwide. Central to this process is the conjugative F pilus, a long extracellular filamentous polymer that extends from the surface of plasmid donor cells, allowing it to probe the environment and make contact with the recipient cell. It is well established that the F pilus can retract to bring mating pair cells in tight contact before DNA transfer. However, whether DNA transfer can occur through the extended pilus has been a subject of active debate. In this study, we use live-cell microscopy to show that while most transfer events occur between cells in direct contact, the F pilus can indeed serve as a conduit for the DNA during transfer between physically distant cells. Our findings enable us to propose a unique model for conjugation that revises our understanding of the DNA transfer mechanism and the dissemination of drug resistance and virulence genes within complex bacterial communities.