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The paper delves into an in-depth analysis of the intrinsic resistance of the inverted pendulum system which causes the modeling of the system to differ from the actual system. Our primary objective revolves around the implementation and subsequent optimization of fuzzy logic controllers (FLC), drawing inspiration from human perceptual assessments. The processing comprises comprehensive mathematical system modeling, intrinsic resistance examination, and improved fuzzy logic control with detailed membership function and rule design. In addition, we conduct a comparative analysis with the widely recognized linear quadratic regulator (LQR) algorithm, which is considered the conventional control algorithm. The result demonstrates that the improved FLC outperforms the conventional LQR algorithm overshoot mitigation, thereby underscoring its superior efficacy and optimality. [ABSTRACT FROM AUTHOR]

Definitive chemoradiotherapy (CRT) is a cornerstone of treatment for locoregionally advanced head and neck cancer (HNC). Research is ongoing on how to improve the tumor response to treatment and limit normal tissue toxicity. A major limitation in that regard is the growing occurrence of intrinsic or acquired treatment resistance in advanced cases. In this review, we will discuss how overexpression of efflux pumps, perturbation of apoptosis-related factors, increased expression of antioxidants, glucose metabolism, metallotheionein expression, increased DNA repair, cancer stem cells, epithelial-mesenchymal transition, non-coding RNA and the tumour microenvironment contribute towards resistance of HNC to chemotherapy and/or radiotherapy. These mechanisms have been investigated for years and been exploited for therapeutic gain in resistant patients, paving the way to the development of new promising drugs. Since in vitro studies on resistance requires a suitable model, we will also summarize published techniques and treatment schedules that have been shown to generate acquired resistance to chemo- and/or radiotherapy that most closely mimics the clinical scenario. [ABSTRACT FROM AUTHOR]

In the absence of data on the reporting of L. monocytogenes resistance to antibiotics, we sought to determine which clonal complexes (CCs)/sequence types (STs) circulate in the food chain in Kosovo and to determine their antibiogram profiles to a panel of 18 antibiotics. From a total of 114 isolates, 21 different typical STs were identified by multilocus sequence typing (MLST). Each isolate derived from the food categories was subjected to tests to verify its susceptibility to the selected antibiotics according to the designed Sensititre GPN3F panel. Among the different STs that were identified, CC9-ST9 was more abundant in meat products (38.75%) while CC29-ST29 was more abundant (24.0%) in dairy products. Moreover, these isolates showed marked resistance against levofloxacin (22.8%), gentamicin and rifampicin (17.5%), quinupristin/dalfopristin (14.9%), erythromycin (11.4%), penicillin (7.89%), tetracycline (1.75%), and streptomycin (0.88%). A total of 27 multiple antibiotic resistance (MAR) phenotypes were observed amongst the isolates, which ranged from 3 to 12. The ARI of the food category including meat and meat products (MMP, 0.22) and fish meat products (FMP, 0.26) were >0.2, the permissible Krumperman threshold. The number of strains with MAR values >0.2 was 34, (29.8%). The identification of typical multidrug-resistant STs among L. monocytogenes isolates in Kosovo constitutes a potential threat to food safety and public health, which requires a continuous and expanded surveillance system to prevent the further spread of antimicrobial resistant (AMR) isolates. [ABSTRACT FROM AUTHOR]

Abstract Background With poor prognosis and high mortality, pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies. Standard of care therapies for PDAC have included gemcitabine for the past three decades, although resistance often develops within weeks of chemotherapy initiation through an array of possible mechanisms. Methods We reanalyzed publicly available RNA-seq gene expression profiles of 28 PDAC patient-derived xenograft (PDX) models before and after a 21-day gemcitabine treatment using our validated analysis pipeline to identify molecular markers of intrinsic and acquired resistance. Results Using normalized RNA-seq quantification measurements, we first identified oxidative phosphorylation and interferon alpha pathways as the two most enriched cancer hallmark gene sets in the baseline gene expression profile associated with intrinsic gemcitabine resistance and sensitivity, respectively. Furthermore, we discovered strong correlations between drug-induced expression changes in glycolysis and oxidative phosphorylation genes and response to gemcitabine, which suggests that these pathways may be associated with acquired gemcitabine resistance mechanisms. Thus, we developed prediction models using baseline gene expression profiles in those pathways and validated them in another dataset of 12 PDAC models from Novartis. We also developed prediction models based on drug-induced expression changes in genes from the Molecular Signatures Database (MSigDB)’s curated 50 cancer hallmark gene sets. Finally, pathogenic TP53 mutations correlated with treatment resistance. Conclusion Our results demonstrate that concurrent upregulation of both glycolysis and oxidative phosphorylation pathways occurs in vivo in PDAC PDXs following gemcitabine treatment and that pathogenic TP53 status had association with gemcitabine resistance in these models. Our findings may elucidate the molecular basis for gemcitabine resistance and provide insights for effective drug combination in PDAC chemotherapy.

Simple Summary: Lung adenocarcinoma, the most prevalent type of lung cancer, presents a significant treatment challenge owing to drug resistance. This study aimed to investigate the role of long non-coding RNAs (lncRNAs) in promoting intrinsic resistance in three lung adenocarcinoma cell lines from the onset of treatment. Drug-tolerant persister (DTP) cells, a subset of cancer cells with the ability to survive and proliferate after exposure to therapeutic drugs, were generated. RNA sequencing was used to investigate the differential expression of lncRNAs, and the clinical relevance of lncRNAs was assessed in a cohort of lung adenocarcinoma patients from The Cancer Genome Atlas. Knockdown of these lncRNAs increases the sensitivity of the analyzed cell lines to therapeutic drugs. This study provides an opportunity to investigate the role of lncRNAs in the genetic and epigenetic mechanisms underlying intrinsic resistance. Lung adenocarcinoma is the most prevalent form of lung cancer, and drug resistance poses a significant obstacle in its treatment. This study aimed to investigate the overexpression of long non-coding RNAs (lncRNAs) as a mechanism that promotes intrinsic resistance in tumor cells from the onset of treatment. Drug-tolerant persister (DTP) cells are a subset of cancer cells that survive and proliferate after exposure to therapeutic drugs, making them an essential object of study in cancer treatment. The molecular mechanisms underlying DTP cell survival are not fully understood; however, long non-coding RNAs (lncRNAs) have been proposed to play a crucial role. DTP cells from lung adenocarcinoma cell lines were obtained after single exposure to tyrosine kinase inhibitors (TKIs; erlotinib or osimertinib). After establishing DTP cells, RNA sequencing was performed to investigate the differential expression of the lncRNAs. Some lncRNAs and one mRNA were overexpressed in DTP cells. The clinical relevance of lncRNAs was evaluated in a cohort of patients with lung adenocarcinoma from The Cancer Genome Atlas (TCGA). RT–qPCR validated the overexpression of lncRNAs and mRNA in the residual DTP cells and LUAD biopsies. Knockdown of these lncRNAs increases the sensitivity of DTP cells to therapeutic drugs. This study provides an opportunity to investigate the involvement of lncRNAs in the genetic and epigenetic mechanisms that underlie intrinsic resistance. The identified lncRNAs and CD74 mRNA may serve as potential prognostic markers or therapeutic targets to improve the overall survival (OS) of patients with lung cancer. [ABSTRACT FROM AUTHOR]

Abstract Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) is crucial for patients with lung cancer harboring EGFR mutations. However, almost all patients experience disease progression, regardless of their response to the targeted therapy, necessitating the development of additional treatment options. Two patients with lung cancer harboring EGFR‐L858R mutations in exon 21 were treated by surgical resection during successful osimertinib treatment. Because the pathological diagnosis was suspected to be pleural metastasis, osimertinib treatment was continued until disease progression. We analyzed the evolution of genomic alterations and the levels of AXL using tumor specimens obtained by repeated biopsies during the course of treatment: initial diagnosis, operation, and disease progression. Genetic alterations detected at the three time points were dramatically changed and showed reductions in numbers, while EGFR‐L858R mutations were detected in all samples tested in both patients. Immunohistochemical expression of AXL remained positive from the beginning of analysis to disease progression. Clonal evolution under oncogenesis is related to gradual accumulation of genomic alterations during tumor growth. However, our case series revealed that volume reduction procedures may cause this phenomenon. Therefore, identification of intrinsic drug‐resistant cells in tumors may be as important as detection of acquired genetic alterations.

In the absence of data on the reporting of L. monocytogenes resistance to antibiotics, we sought to determine which clonal complexes (CCs)/sequence types (STs) circulate in the food chain in Kosovo and to determine their antibiogram profiles to a panel of 18 antibiotics. From a total of 114 isolates, 21 different typical STs were identified by multilocus sequence typing (MLST). Each isolate derived from the food categories was subjected to tests to verify its susceptibility to the selected antibiotics according to the designed Sensititre GPN3F panel. Among the different STs that were identified, CC9-ST9 was more abundant in meat products (38.75%) while CC29-ST29 was more abundant (24.0%) in dairy products. Moreover, these isolates showed marked resistance against levofloxacin (22.8%), gentamicin and rifampicin (17.5%), quinupristin/dalfopristin (14.9%), erythromycin (11.4%), penicillin (7.89%), tetracycline (1.75%), and streptomycin (0.88%). A total of 27 multiple antibiotic resistance (MAR) phenotypes were observed amongst the isolates, which ranged from 3 to 12. The ARI of the food category including meat and meat products (MMP, 0.22) and fish meat products (FMP, 0.26) were >0.2, the permissible Krumperman threshold. The number of strains with MAR values >0.2 was 34, (29.8%). The identification of typical multidrug-resistant STs among L. monocytogenes isolates in Kosovo constitutes a potential threat to food safety and public health, which requires a continuous and expanded surveillance system to prevent the further spread of antimicrobial resistant (AMR) isolates.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) is crucial for patients with lung cancer harboring EGFR mutations. However, almost all patients experience disease progression, regardless of their response to the targeted therapy, necessitating the development of additional treatment options. Two patients with lung cancer harboring EGFR‐L858R mutations in exon 21 were treated by surgical resection during successful osimertinib treatment. Because the pathological diagnosis was suspected to be pleural metastasis, osimertinib treatment was continued until disease progression. We analyzed the evolution of genomic alterations and the levels of AXL using tumor specimens obtained by repeated biopsies during the course of treatment: initial diagnosis, operation, and disease progression. Genetic alterations detected at the three time points were dramatically changed and showed reductions in numbers, while EGFR‐L858R mutations were detected in all samples tested in both patients. Immunohistochemical expression of AXL remained positive from the beginning of analysis to disease progression. Clonal evolution under oncogenesis is related to gradual accumulation of genomic alterations during tumor growth. However, our case series revealed that volume reduction procedures may cause this phenomenon. Therefore, identification of intrinsic drug‐resistant cells in tumors may be as important as detection of acquired genetic alterations. [ABSTRACT FROM AUTHOR]

Simple Summary: The BRAF V600E mutation is frequently found in cancer. It activates the MAPK pathway and promotes cancer cell proliferation, making BRAF an excellent target for anti-cancer therapy. While BRAF-targeted therapy is highly effective for melanoma, it is often ineffective against other cancers. This study uses a proteolysis targeting chimera (PROTAC) to probe the role of BRAF V600E in colorectal and triple-negative breast cancer cells. The study reveals a diverse set of biochemical and proliferative responses to BRAF V600E degradation: some cancer cells are killed by BRAF degradation, while others utilize additional oncogenic drivers, such as Src kinase phosphatidylinositol 3-kinase, to resist the effect of BRAF degradation. These responses provide a mechanistic explanation for the efficacy of BRAF-targeted therapy for some cancers and the intrinsic resistance in others. The BRAF V600E mutation is frequently found in cancer. It activates the MAPK pathway and promotes cancer cell proliferation, making BRAF an excellent target for anti-cancer therapy. While BRAF-targeted therapy is highly effective for melanoma, it is often ineffective against other cancers harboring the BRAF mutation. In this study, we evaluate the effectiveness of a proteolysis targeting chimera (PROTAC), SJF-0628, in directing the degradation of mutated BRAF across a diverse panel of cancer cells and determine how these cells respond to the degradation. SJF-0628 treatment results in the degradation of BRAF V600E and a decrease in Mek activation in all cell lines tested, but the effects of the treatment on cell signaling and cell proliferation are cell-line-specific. First, BRAF degradation killed DU-4475 and Colo-205 cells via apoptosis but only partially inhibited the proliferation of other cancer cell lines. Second, SJF-0628 treatment resulted in co-degradation of MEK in Colo-205 cells but did not have the same effect in other cell lines. Finally, cell lines partially inhibited by BRAF degradation also contain other oncogenic drivers, making them multi-driver cancer cells. These results demonstrate the utility of a PROTAC to direct BRAF degradation and reveal that multi-driver oncogenesis renders some colorectal cancer cells resistant to BRAF-targeted treatment. [ABSTRACT FROM AUTHOR]

Resistance to cisplatin is the main cause of treatment failure in lung adenocarcinoma. Drug-tolerant-persister (DTP) cells are responsible for intrinsic resistance, since they survive the initial cycles of treatment, representing a reservoir for the emergence of clones that display acquired resistance. Although the molecular mechanisms of DTP cells have been described, few studies have investigated the earliest molecular alterations of DTP cells in intrinsic resistance to cisplatin. In this work, we report a gene expression signature associated with the emergence of cisplatin-DTP cells in lung adenocarcinoma cell lines. After a single exposure to cisplatin,we sequenced the transcriptome of cisplatin-DTPs to identify differentially expressed genes. Bioinformatic analysis revealed that early cisplatin- DTP cells deregulate metabolic and proliferative pathways to survive the drug insult. Interaction network analysis identified three highly connected submodules in which SOCS1 had a significant participation in controlling the proliferation of cisplatin-DTP cells. Expression of the candidate genes and their corresponding protein was validated in lung adenocarcinoma cell lines. Importantly, the expression level of SOCS1 was different between CDDP-susceptible and CDDPresistant lung adenocarcinoma cell lines. Moreover, knockdown of SOCS1 in the CDDP-resistant cell line partially promoted its susceptibility to CDDP. Finally, the clinical relevance of the candidate genes was analyzed in silico, according to the overall survival of cisplatin-treated patients from The Cancer Genome Atlas. Survival analysis showed that downregulation or upregulation of the selected genes was associated with overall survival. The results obtained indicate that these genes could be employed as predictive biomarkers or potential targets to improve the effectiveness of CDDP treatment in lung cancer patients. [ABSTRACT FROM AUTHOR]

The qualified presumption of safety (QPS) approach was developed to provide a regularly updated generic pre‐evaluation of the safety of microorganisms intended for use in the food or feed chains. Safety concerns identified for a taxonomic unit (TU) are, where possible, confirmed at the species/strain or product level and reflected by 'qualifications' which should be assessed at strain and/or product level by EFSA's Scientific Panels. The generic qualification 'the strains should not harbour any acquired antimicrobial resistance (AMR) genes to clinically relevant antimicrobials' applies to all QPS bacterial TUs. The different EFSA risk assessment areas use the same approach to assess the qualification related to AMR genes. In this statement, the terms 'intrinsic' and 'acquired' AMR genes were defined for the purpose of EFSA's risk assessments, and they apply to bacteria used in the food and feed chains. A bioinformatic approach is proposed for demonstrating the 'intrinsic'/'acquired' nature of an AMR gene. All AMR genes that confer resistance towards 'critically important', 'highly important' and 'important' antimicrobials, as defined by the World Health Organisation (WHO), found as hits, need to be considered as hazards (for humans, animals and environment) and need further assessment. Genes identified as responsible for 'intrinsic' resistance could be considered as being of no concern in the frame of the EFSA risk assessment. 'Acquired' AMR genes resulting in a resistant phenotype should be considered as a concern. If the presence of the 'acquired' AMR gene is not leading to phenotypic resistance, further case‐by‐case assessment is necessary. [ABSTRACT FROM AUTHOR]

Objective: Antibiotics resistance in bacteria is an important public health problem recently. Resistance rates vary from country to country, from region to region, and even from hospital to hospital. Recently, tigecycline and colistin have been widely used due to increasing multi-resistance in Gram-negative bacteria. The aim of the study was to investigate the changes in colistin and tigecycline intrinsic-resistant bacteria ratio and the effects of these drugs on the hospital flora throughout the hospital due to the widespread use of these drugs. Materials and Methods: In our microbiology laboratory, clinical samples were processed according to the general microbiology procedure. Bacterial identification and antibiogram susceptibility tests were performed with VITEK®2 Compact, MALDI-TOF mass spectrometry (bioMérieux, France). Results were evaluated according to CLSI and EUCAST criteria. The data were scanned retrospectively between the years 2012 and 2019. Chi-square test was used in statistical analysis. Significance was evaluated at the p<0.05 level. Results: While the number of Escherichia coli isolates decreased over the years, an increase was detected in Klebsiella spp. tigecycline intrinsic-resistant bacteria, Proteus spp., Providencia spp., and Pseudomonas spp. When evaluated with the general number of bacteria isolated over the years, Pseudomonas spp., a three-fold increase was found in 2018 and 2019 and in colistin intrinsic-resistant bacteria, Morganella spp., Proteus spp., Providencia spp., Yersinia spp., Burkholderia spp., and Eliseabethkingea spp. No statistically significant increase was detected. A significant increase was detected in Serratia spp. Conclusion: Changes were detected in intrinsic-resistant bacteria and in antibiotic distribution. The antibiotic drugs used affect the entire flora, including the microbiota. The importance of patient care and antibiotic management should be emphasized, targeted therapies treatments should be made that the microbiota will not change, or fecal transplantation methods, which are new methods to replace microbiota, should be investigated. [ABSTRACT FROM AUTHOR]

Abstract Background RNA 5-methylcytosine (m5C) modification plays critical roles in the pathogenesis of various tumors. However, the function and molecular mechanism of RNA m5C modification in tumor drug resistance remain unclear. Methods The correlation between RNA m5C methylation, m5C writer NOP2/Sun RNA methyltransferase family member 2 (NSUN2) and EGFR-TKIs resistance was determined in non-small-cell lung cancer (NSCLC) cell lines and patient samples. The effects of NSUN2 on EGFR-TKIs resistance were investigated by gain- and loss-of-function assays in vitro and in vivo. RNA-sequencing (RNA-seq), RNA bisulfite sequencing (RNA-BisSeq) and m5C methylated RNA immunoprecipitation-qPCR (MeRIP-qPCR) were performed to identify the target gene of NSUN2 involved in EGFR-TKIs resistance. Furthermore, the regulatory mechanism of NSUN2 modulating the target gene expression was investigated by functional rescue and puromycin incorporation assays. Results RNA m5C hypermethylation and NSUN2 were significantly correlated with intrinsic resistance to EGFR-TKIs. Overexpression of NSUN2 resulted in gefitinib resistance and tumor recurrence, while genetic inhibition of NSUN2 led to tumor regression and overcame intrinsic resistance to gefitinib in vitro and in vivo. Integrated RNA-seq and m5C-BisSeq analyses identified quiescin sulfhydryl oxidase 1 (QSOX1) as a potential target of aberrant m5C modification. NSUN2 methylated QSOX1 coding sequence region, leading to enhanced QSOX1 translation through m5C reader Y-box binding protein 1 (YBX1). Conclusions Our study reveals a critical function of aberrant RNA m5C modification via the NSUN2-YBX1-QSOX1 axis in mediating intrinsic resistance to gefitinib in EGFR-mutant NSCLC.

Metallo-β-lactamases (MBLs) encoding carbapenem resistance in wastewater are a well-known serious threat to human health. Twelve Pseudomonas otitidis isolates obtained from a municipal wastewater treatment plant (WWTP) in Hawaii were found to possess a subclass B3 MBL – POM (P. otitidis MBL), with a minimum inhibition concentration (MIC) range of 8–16 mg/L. The unrooted neighbor-joining phylogenetic tree showed that these blaPOM genes isolated in wastewater samples (n = 12) were distinctly different from other reference genes isolated from clinical, freshwater, animal, and soil samples except for isolates MR7, MR8, and MR11. MR7, MR8, and MR11 were found to have 4, 3, and 3 amino acid substitutions when compared to the type strain MC10330T and were closely clustered to the clinical reference genes. The meropenem hydrolysis experiment showed that isolates with multiple amino acid substitutions completely hydrolyzed 64 mg/L of meropenem in 7 h. The emergence of the opportunistic pathogen P. otitidis chromosomally encoding blaPOM in the treated municipal wastewater is an alarming call for the spread of this MBL in the environment. Further studies are required to understand the mechanism and regulation of this carbapenem-resistant β-lactamase in order to fill in the knowledge gap. HIGHLIGHTS Wastewater surveillance detected numerous carbapenem-resistant Pseudomonas otitidis isolates that showed minimum inhibition concentrations up to 16 mg/L of meropenem.; The blaPOM genes in the wastewater P. otitidis isolates possess mutations not observed previously in clinical and environmental sources.; Some wastewater P. otitidis isolates with multiple amino acid substitutions in the blaPOM gene catalyzed fast hydrolysis of carbapenem.;

ABSTRACT The polymorphism at amino acid 17 of quinolone resistance-determining region of GyrA has been stated with a potential role in fluoroquinolone susceptibility in different mycobacterial species. However, no study has provided dependable evidence so far. Here, we verified that gene-edited Mycobacterium abscessus mutants bearing Ser/Gly at this position were more susceptible to fluoroquinolones than their parent strain and the revertant that supports mycobacteria containing Ser/Gly at this position were more susceptible to fluoroquinolones than those containing Ala. IMPORTANCE Fluoroquinolones (FQs) play a key role in the treatment regimens against tuberculosis and non-tuberculous mycobacterial infections. However, there are significant differences in the sensitivities of different mycobacteria to FQs. In this study, we proved that this is associated with the polymorphism at amino acid 17 of quinolone resistance-determining region of Gyrase A by gene editing. This is the first study using CRISPR-associated recombination for gene editing in Mycobacterium abscessus to underscore the contribution of the amino acid substitutions in GyrA to FQ susceptibilities in mycobacteria.

In recent years, with the increases in microorganisms that express a multitude of antimicrobial resistance (AMR) mechanisms, the threat of antimicrobial resistance in the global population has reached critical levels. The introduction of the COVID-19 pandemic has further contributed to the influx of infections caused by multidrug-resistant organisms (MDROs), which has placed significant pressure on healthcare systems. For over a century, the potential for light-based approaches targeted at combatting both cancer and infectious diseases has been proposed. They offer effective killing of microbial pathogens, regardless of AMR status, and have not typically been associated with high propensities of resistance development. To that end, the goal of this review is to describe the different mechanisms that drive AMR, including intrinsic, phenotypic, and acquired resistance mechanisms. Additionally, the different light-based approaches, including antimicrobial photodynamic therapy (aPDT), antimicrobial blue light (aBL), and ultraviolet (UV) light, will be discussed as potential alternatives or adjunct therapies with conventional antimicrobials. Lastly, we will evaluate the feasibility and requirements associated with integration of light-based approaches into the clinical pipeline. [ABSTRACT FROM AUTHOR]

Resistance to cisplatin is the main cause of treatment failure in lung adenocarcinoma. Drug-tolerant-persister (DTP) cells are responsible for intrinsic resistance, since they survive the initial cycles of treatment, representing a reservoir for the emergence of clones that display acquired resistance. Although the molecular mechanisms of DTP cells have been described, few studies have investigated the earliest molecular alterations of DTP cells in intrinsic resistance to cisplatin. In this work, we report a gene expression signature associated with the emergence of cisplatin-DTP cells in lung adenocarcinoma cell lines. After a single exposure to cisplatin, we sequenced the transcriptome of cisplatin-DTPs to identify differentially expressed genes. Bioinformatic analysis revealed that early cisplatin-DTP cells deregulate metabolic and proliferative pathways to survive the drug insult. Interaction network analysis identified three highly connected submodules in which SOCS1 had a significant participation in controlling the proliferation of cisplatin-DTP cells. Expression of the candidate genes and their corresponding protein was validated in lung adenocarcinoma cell lines. Importantly, the expression level of SOCS1 was different between CDDP-susceptible and CDDP-resistant lung adenocarcinoma cell lines. Moreover, knockdown of SOCS1 in the CDDP-resistant cell line partially promoted its susceptibility to CDDP. Finally, the clinical relevance of the candidate genes was analyzed in silico, according to the overall survival of cisplatin-treated patients from The Cancer Genome Atlas. Survival analysis showed that downregulation or upregulation of the selected genes was associated with overall survival. The results obtained indicate that these genes could be employed as predictive biomarkers or potential targets to improve the effectiveness of CDDP treatment in lung cancer patients.

Background: RNA 5-methylcytosine (m5C) modification plays critical roles in the pathogenesis of various tumors. However, the function and molecular mechanism of RNA m5C modification in tumor drug resistance remain unclear. Methods: The correlation between RNA m5C methylation, m5C writer NOP2/Sun RNA methyltransferase family member 2 (NSUN2) and EGFR-TKIs resistance was determined in non-small-cell lung cancer (NSCLC) cell lines and patient samples. The effects of NSUN2 on EGFR-TKIs resistance were investigated by gain- and loss-of-function assays in vitro and in vivo. RNA-sequencing (RNA-seq), RNA bisulfite sequencing (RNA-BisSeq) and m5C methylated RNA immunoprecipitation-qPCR (MeRIP-qPCR) were performed to identify the target gene of NSUN2 involved in EGFR-TKIs resistance. Furthermore, the regulatory mechanism of NSUN2 modulating the target gene expression was investigated by functional rescue and puromycin incorporation assays. Results: RNA m5C hypermethylation and NSUN2 were significantly correlated with intrinsic resistance to EGFR-TKIs. Overexpression of NSUN2 resulted in gefitinib resistance and tumor recurrence, while genetic inhibition of NSUN2 led to tumor regression and overcame intrinsic resistance to gefitinib in vitro and in vivo. Integrated RNA-seq and m5C-BisSeq analyses identified quiescin sulfhydryl oxidase 1 (QSOX1) as a potential target of aberrant m5C modification. NSUN2 methylated QSOX1 coding sequence region, leading to enhanced QSOX1 translation through m5C reader Y-box binding protein 1 (YBX1). Conclusions: Our study reveals a critical function of aberrant RNA m5C modification via the NSUN2-YBX1-QSOX1 axis in mediating intrinsic resistance to gefitinib in EGFR-mutant NSCLC. [ABSTRACT FROM AUTHOR]

Metallo-β-lactamases (MBLs) encoding carbapenem resistance in wastewater are a well-known serious threat to human health. Twelve Pseudomonas otitidis isolates obtained from a municipal wastewater treatment plant (WWTP) in Hawaii were found to possess a subclass B3 MBL – POM (P. otitidis MBL), with a minimum inhibition concentration (MIC) range of 8–16 mg/L. The unrooted neighbor-joining phylogenetic tree showed that these blaPOM genes isolated in wastewater samples (n=12) were distinctly different from other reference genes isolated from clinical, freshwater, animal, and soil samples except for isolates MR7, MR8, and MR11. MR7, MR8, and MR11 were found to have 4, 3, and 3 amino acid substitutions when compared to the type strain MC10330T and were closely clustered to the clinical reference genes. The meropenem hydrolysis experiment showed that isolates with multiple amino acid substitutions completely hydrolyzed 64 mg/L of meropenem in 7 h. The emergence of the opportunistic pathogen P. otitidis chromosomally encoding blaPOM in the treated municipal wastewater is an alarming call for the spread of this MBL in the environment. Further studies are required to understand the mechanism and regulation of this carbapenem-resistant β-lactamase in order to fill in the knowledge gap. [ABSTRACT FROM AUTHOR]

Pulmonary infections caused by Mycobacterium abscessus (MAB) have been increasing in incidence in recent years, leading to chronic and many times fatal infections due to MAB's natural resistance to most available antimicrobials. The use of bacteriophages (phages) in clinics is emerging as a novel treatment strategy to save the lives of patients suffering from drug-resistant, chronic, and disseminated infections. The substantial research indicates that phage–antibiotic combination therapy can display synergy and be clinically more effective than phage therapy alone. However, there is limited knowledge in the understanding of the molecular mechanisms in phage–mycobacteria interaction and the synergism of phage–antibiotic combinations. We generated the lytic mycobacteriophage library and studied phage specificity and the host range in MAB clinical isolates and characterized the phage's ability to lyse the pathogen under various environmental and mammalian host stress conditions. Our results indicate that phage lytic efficiency is altered by environmental conditions, especially in conditions of biofilm and intracellular states of MAB. By utilizing the MAB gene knockout mutants of the MAB_0937c/MmpL10 drug efflux pump and MAB_0939/pks polyketide synthase enzyme, we discovered the surface glycolipid diacyltrehalose/polyacyltrehalose (DAT/PAT) as one of the major primary phage receptors in mycobacteria. We also established a set of phages that alter the MmpL10 multidrug efflux pump function in MAB through an evolutionary trade-off mechanism. The combination of these phages with antibiotics significantly decreases the number of viable bacteria when compared to phage or antibiotic-alone treatments. This study deepens our understanding of phage–mycobacteria interaction mechanisms and identifies therapeutic phages that can lower bacterial fitness by impairing an antibiotic efflux function and attenuating the MAB intrinsic resistance mechanism via targeted therapy. [ABSTRACT FROM AUTHOR]

Simple Summary: Cell culture models of cancer typically favor proliferative but therapy-sensitive cells because body-like selection pressures are absent. To address this limitation, we previously described a function-based selection strategy to model deep intrinsic resistance in cultures of triple-negative breast cancer cells. Our methods were designed to reveal therapy-resistant, adaptable cancer cells that opportunistically switch between quiescence and proliferation. To determine the validity of this approach in identifying noncytotoxic drugs that could inhibit highly resistant breast cancer cells, we used our novel cell culture model to evaluate a well-known BET bromodomain inhibitor, JQ1, which modulates the cancer epigenome. JQ1 has been found to inhibit resistant cancer cells in several cancer types, including breast cancer. Low-dose JQ1 inhibited the growth of highly adaptable/resistant breast cancer cells in our cell culture model. Our results support the validity of a cell culture-based approach for modeling cancer. We treated highly metabolically adaptable (SUM149-MA) triple-negative inflammatory breast cancer cells and their control parental SUM149-Luc cell line with JQ1 for long periods to determine its efficacy at inhibiting therapy-resistant cells. After 20 days of treatment with 1–2 µM of JQ1, which killed majority of cells in the parental cell line, a large number of SUM149-MA cells survived, consistent with their pan-resistant nature. Interestingly, though, the JQ1 treatment sensitized resistant cancer cells in both the SUM149-MA and SUM149-Luc cell lines to subsequent treatment with doxorubicin and paclitaxel. To measure JQ1-mediated sensitization of resistant cancer cells, we first eradicated approximately 99% of relatively chemotherapy-sensitive cancer cells in culture dishes by long treatments with doxorubicin or paclitaxel, and then analyzed the remaining resistant cells for survival and growth into colonies. In addition, combination, rather than sequential, treatment with JQ1 and doxorubicin was also effective in overcoming resistance. Notably, Western blotting showed that JQ1-treated cancer cells had significantly lower levels of PD-L1 protein than did untreated cells, indicating that JQ1 treatment may reduce tumor-mediated immune suppression and improve the response to immunotherapy targeting PD-L1. Finally, JQ1 treatment with a low 62.5 nM dose sensitized another resistant cell line, FC-IBC02-MA, to treatment with doxorubicin and paclitaxel. [ABSTRACT FROM AUTHOR]

Zhijian Zhang,1,&ast; Wei Wang,2,&ast; Yufeng Wang,3 Zhongtan Xue,3 Shanshan Li,2 Yu Pang2 1Department of Respiratory and Critical Care Medicine, the Second Medical Center of Chinese PLA General Hospital, Beijing, 100036, People’s Republic of China; 2Department of Bacteriology and Immunology, Beijing Key Laboratory on Drug-Resistant Tuberculosis Research, Beijing Tuberculosis and Thoracic Tumor Research Institute/Beijing Chest Hospital, Capital Medical University, Beijing, 101149, People’s Republic of China; 3Innovation Alliance on Tuberculosis Diagnosis and Treatment, Beijing, 101149, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Shanshan Li; Yu Pang, Department of Bacteriology and Immunology, Beijing Key Laboratory on Drug-Resistant Tuberculosis Research, Beijing Tuberculosis and Thoracic Tumor Research Institute/Beijing Chest Hospital, Capital Medical University, Beijing, 101149, People’s Republic of China, Tel +86-010-89509368 ; +86-010-89509162, Email lss9011@126.com; pangyupound@163.comAbstract: We aimed to determine the prevalence of amikacin (AMK) resistance of clinical Mycobacterium abscessus (MAB) isolates and to investigate if AMK resistance was induced by AMK exposure. A total of 75 MAB isolates underwent susceptibility testing for AMK after 3 and 14 days of incubation, respectively. The partial fragment of the rrs gene conferring AMK resistance was sequenced. The MIC values for AMK ranged from 0.5 to 128 μg/mL, with MIC50 and MIC90 values of 2 and 32 μg/mL, respectively. In addition, 9.3% of isolates (7/75) were resistant to AMK, all of which harbored a mutation within the rrs locus, including six with A1408G mutation and one with a C1409T mutation. Of note, the MICs of three isolates were significantly increased from 2 μg/mL to 64 μg/mL (one isolate) and 2 μg/mL to 128 μg/mL (two isolates), suggesting that three of the MAB isolates had inducible resistance to AMK. In conclusion, our data demonstrate that approximately one-tenth of clinical MAB isolates in Beijing harbored AMK resistance due to the acquisition of rrs mutations. Additionally, we firstly identified that intrinsic AMK resistance is inducible in MAB isolates, highlighting the urgent need to establish a proper method for the in vitro detection of AMK susceptibility in MAB.Keywords: amikacin, intrinsic resistance, inducible, Mycobacterium abscessus

IntroductionIn this work, 170 strains covering 13 species from the Lactobacillaceae family were analyzed to determine minimal inhibitory concentration (MIC) distributions to nine antimicrobial agents, and genes potentially conferring resistance. This allows a proposal of tentative Epidemiological Cut-Offs (ECOFFs) that follows the phylogeny for interpretation of resistance in the 13 species.MethodsThe 170 strains originated from different sources, geographical areas, and time periods. MICs for nine antibiotics were determined according to the ISO 10932 standard for lactobacillia and by a modified CLSI-method for Leuconostoc and Pediococcus which ensured sufficient growth. The strains were whole genome sequenced, subtyped by core genome analysis, and assessed for the presence of antibiotic resistance genes using the ResFinder and NCBI AMRFinder databases.Results and discussionThe data provide evidence that antimicrobial susceptibility follows phylogeny instead of fermentation pattern and accordingly, tentative ECOFFs were defined. For some species the tentative ECOFFs for specific antibiotics are above the cut-off values set by the European Food Safety Authority (EFSA) which are primarily defined according to fermentation pattern or at genus level. The increased tolerance for specific antibiotics observed for some species was evaluated to be innate, as only for one strain phenotypic resistance was found to be related to an acquired resistance gene. In general, more data are needed to define ECOFFs and since the number of isolates available for industrial relevant bacterial species are often limited compared to clinically relevant species, it is important; 1) that strains are unambiguously defined at species level and subtyped through core genome analysis, 2) MIC determination are performed by use of a standardized method to define species-specific MIC distributions and 3) that known antimicrobial resistance genes are determined in whole genome sequences to support the MIC determinations.

Background: Cutaneous melanoma is a lethal form of skin cancer with morbidity and mortality rates highest amongst European, North American and Australasian populations. The developments of targeted therapies (TTs) directed at the oncogene BRAF and its downstream mediator MEK, and immune checkpoint inhibitors (ICI), have revolutionized the treatment of metastatic melanoma, improving patient outcomes. However, both TT and ICI have their limitations. Although TTs are associated with high initial response rates, these are typically short‐lived due to resistance. Conversely, although ICIs provide more durable responses, they have lower initial response rates. Due to these distinct yet complementary response profiles, it has been proposed that sequencing ICI with TT could lead to a high frequency of durable responses whilst circumventing the toxicity associated with combined ICI + TT treatment. However, several questions remain unanswered, including the mechanisms underpinning this synergy and the optimal sequencing strategy. The key to determining this is to uncover the biology of each phase of the therapeutic response. Aims and methods: In this review, we show that melanoma responds to TT and ICI in three phases: early response, minimal residual disease (MRD) and disease progression. We explore the effects of ICI and TT on melanoma cells and the tumour immune microenvironment, with a particular focus on MRD which is predicted to underpin the development of acquired resistance in the third phase of response. Conclusion: In doing so, we provide a new framework which may inform novel therapeutic approaches for melanoma, including optimal sequencing strategies and agents that target MRD, thereby ultimately improving clinical outcomes for patients. [ABSTRACT FROM AUTHOR]

Serratia marcescens is a ubiquitous bacterium from order Enterobacterales displaying a high genetic plasticity that allows it to adapt and persist in multiple niches including soil, water, plants, and nosocomial environments. Recently, S. marcescens has gained attention as an emerging pathogen worldwide, provoking infections and outbreaks in debilitated individuals, particularly newborns and patients in intensive care units. S. marcescens isolates recovered from clinical settings are frequently described as multidrug resistant. High levels of antibiotic resistance across Serratia species are a consequence of the combined activity of intrinsic, acquired, and adaptive resistance elements. In this review, we will discuss recent advances in the understanding of mechanisms guiding resistance in this opportunistic pathogen. [ABSTRACT FROM AUTHOR]

Serratia marcescens is a ubiquitous bacterium from order Enterobacterales displaying a high genetic plasticity that allows it to adapt and persist in multiple niches including soil, water, plants, and nosocomial environments. Recently, S. marcescens has gained attention as an emerging pathogen worldwide, provoking infections and outbreaks in debilitated individuals, particularly newborns and patients in intensive care units. S. marcescens isolates recovered from clinical settings are frequently described as multidrug resistant. High levels of antibiotic resistance across Serratia species are a consequence of the combined activity of intrinsic, acquired, and adaptive resistance elements. In this review, we will discuss recent advances in the understanding of mechanisms guiding resistance in this opportunistic pathogen.

Tuberculosis (TB) is among the most difficult infections to treat, requiring several months of multidrug therapy to produce a durable cure. The reasons necessitating long treatment times are complex and multifactorial. However, one major difficulty of treating TB is the resistance of the infecting bacterium, Mycobacterium tuberculosis (Mtb), to many distinct classes of antimicrobials. This review will focus on the major gaps in our understanding of intrinsic drug resistance in Mtb and how functional and chemical-genetics can help close those gaps. A better understanding of intrinsic drug resistance will help lay the foundation for strategies to disarm and circumvent these mechanisms to develop more potent antitubercular therapies. [ABSTRACT FROM AUTHOR]

Antimicrobial resistance has not been a new phenomenon. Still, the number of resistant organisms, the geographic areas affected by emerging drug resistance, and the magnitude of resistance in a single organism are enormous and mounting. Disease and disease-causing agents formerly thought to be contained by antibiotics are now returning in new forms resistant to existing therapies. Antimicrobial resistance is one of the most severe and complicated health issues globally, driven by interrelated dynamics in humans, animals, and environmental health sectors. Coupled with various epidemiological factors and a limited pipeline for new antimicrobials, all these misappropriations allow the transmission of drug-resistant organisms. The problem is likely to worsen soon. Antimicrobial resistance in general and antibiotic resistance in particular is a shared global problem. Actions taken by any single country can adversely or positively affect the other country. Targeted coordination and prevention strategies are critical in stopping the spread of antibiotic-resistant organisms and hence its overall management. This article has provided in-depth knowledge about various methods that can help mitigate the emergence and spread of antimicrobial resistance globally. Key points: • Overview of antimicrobial resistance as a global challenge and explain various reasons for its rapid progression. • Brief about the intrinsic and acquired resistance to antimicrobials and development of antibiotic resistance in bacteria. • Systematically organized information is provided on different strategies for tackling antimicrobial resistance for the welfare of human health. [ABSTRACT FROM AUTHOR]

Tuberculosis (TB) is among the most difficult infections to treat, requiring several months of multidrug therapy to produce a durable cure. The reasons necessitating long treatment times are complex and multifactorial. However, one major difficulty of treating TB is the resistance of the infecting bacterium, Mycobacterium tuberculosis (Mtb), to many distinct classes of antimicrobials. This review will focus on the major gaps in our understanding of intrinsic drug resistance in Mtb and how functional and chemical-genetics can help close those gaps. A better understanding of intrinsic drug resistance will help lay the foundation for strategies to disarm and circumvent these mechanisms to develop more potent antitubercular therapies.

In recent years, with the increases in microorganisms that express a multitude of antimicrobial resistance (AMR) mechanisms, the threat of antimicrobial resistance in the global population has reached critical levels. The introduction of the COVID-19 pandemic has further contributed to the influx of infections caused by multidrug-resistant organisms (MDROs), which has placed significant pressure on healthcare systems. For over a century, the potential for light-based approaches targeted at combatting both cancer and infectious diseases has been proposed. They offer effective killing of microbial pathogens, regardless of AMR status, and have not typically been associated with high propensities of resistance development. To that end, the goal of this review is to describe the different mechanisms that drive AMR, including intrinsic, phenotypic, and acquired resistance mechanisms. Additionally, the different light-based approaches, including antimicrobial photodynamic therapy (aPDT), antimicrobial blue light (aBL), and ultraviolet (UV) light, will be discussed as potential alternatives or adjunct therapies with conventional antimicrobials. Lastly, we will evaluate the feasibility and requirements associated with integration of light-based approaches into the clinical pipeline.

Esophageal cancer (EC) is a common malignant gastrointestinal (GI) cancer in adults. Although surgical technology combined with neoadjuvant chemoradiotherapy has advanced rapidly, patients with EC are often diagnosed at an advanced stage and the five-year survival rate remains unsatisfactory. The poor prognosis and high mortality in patients with EC indicate that effective and validated therapy is of great necessity. Recently, immunotherapy has been successfully used in the clinic as a novel therapy for treating solid tumors, bringing new hope to cancer patients. Several immunotherapies, such as immune checkpoint inhibitors (ICIs), chimeric antigen receptor T-cell therapy, and tumor vaccines, have achieved significant breakthroughs in EC treatment. However, the overall response rate (ORR) of immunotherapy in patients with EC is lower than 30%, and most patients initially treated with immunotherapy are likely to develop acquired resistance (AR) over time. Immunosuppression greatly weakens the durability and efficiency of immunotherapy. Because of the heterogeneity within the immune microenvironment and the highly disparate oncological characteristics in different EC individuals, the exact mechanism of immunotherapy resistance in EC remains elusive. In this review, we provide an overview of immunotherapy resistance in EC, mainly focusing on current immunotherapies and potential molecular mechanisms underlying immunosuppression and drug resistance in immunotherapy. Additionally, we discuss prospective biomarkers and novel methods for enhancing the effect of immunotherapy to provide a clear insight into EC immunotherapy. [ABSTRACT FROM AUTHOR]

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Resistance to spectinomycin emerged after widely used for treatment of gonorrhea. Previous studies revealed that Lysobacter enzymogenes strain C3 (LeC3) exhibited elevated level of intrinsic resistance to spectinomycin. In this study, we screened a Tn5 transposon mutant library of LeC3 to elucidate the underlying molecular mechanisms of spectinomycin resistance. Insertion sites in 15 out of 19 mutants recovered with decreased spectinomycin resistance were located on two ribosomal RNA operons at different loci, indicating the pivotal role of ribosomal RNAs in conferring spectinomycin resistance in L. enzymogenes. The other mutants harbored mutations in the tuf, rpoD, mltB, and purB genes. Among them, the tuf and rpoD genes, respectively, encode a translation elongation factor Tu and an RNA polymerase primary sigma factor. They both contribute to protein biosynthesis, where ribosomal RNAs play essential roles. The mltB gene, whose product is involved in cell-wall recycling, was not only associated with resistance against spectinomycin, but also conferred resistance to osmotic stress and ampicillin. In addition, mutation of the purB gene, for which its product is involved in the biosynthesis of inosine and adenosine monophosphates, led to decreased spectinomycin resistance. Addition of exogenous adenine at lower concentration in medium restored the growth deficiency in the purB mutant and increased bacterial resistance to spectinomycin. These results suggest that while cell-wall recycling and purine biosynthesis might contribute to spectinomycin resistance, target rRNAs play critical role in spectinomycin resistance in L. enzymogenes. [ABSTRACT FROM AUTHOR]

Annotations of non-pathogenic bacterial genomes commonly reveal putative antibiotic resistance genes and the potential risks associated with such genes is challenging to assess. We have examined a putative tetracycline tet(L) gene (conferring low level tetracycline resistance), present in the majority of all publicly available genomes of the industrially important operational group Bacillus amyloliquefaciens including the species B. amyloliquefaciens, Bacillus siamensis and Bacillus velezensis. The aim was to examine the risk of transfer of the putative tet(L) in operational group B. amyloliquefaciens through phylogenetic and genomic position analysis. These analyses furthermore included tet(L) genes encoded by transferable plasmids and other Gram-positive and -negative bacteria, including Bacillus subtilis. Through phylogenetic analysis, we could group chromosomally and plasmid-encoded tet(L) genes into four phylogenetic clades. The chromosomally encoded putative tet(L) from operational group B. amyloliquefaciens formed a separate phylogenetic clade; was positioned in the same genomic region in the three species; was not flanked by mobile genetic elements and was not found in any other bacterial species suggesting that the gene has been present in a common ancestor before species differentiation and is intrinsic. Therefore the gene is not considered a safety concern, and the risk of transfer to and expression of resistance in other non-related species is considered negligible. We suggest a subgrouping of the tet(L) class into four groups (tet(L)1.1, tet(L)1.2 and tet(L)2.1, tet(L)2.2), corresponding with the phylogenetic grouping and tet(L) from operational group B. amyloliquefaciens referred to as tet(L)2.2. Phylogenetic analysis is a useful tool to correctly differentiate between intrinsic and acquired antibiotic resistance genes. [ABSTRACT FROM AUTHOR]

Pseudomonas spp. are ubiquitous microorganisms that exhibit intrinsic and acquired resistance to many antimicrobial agents. Pseudomonas aeruginosa is the most studied species of this genus due to its clinical importance. In contrast, the Pseudomonas fluorescens complex consists of environmental and, in some cases, pathogenic opportunistic microorganisms. The records of antimicrobial-resistant P. fluorescens are quite scattered, which hinders the recognition of patterns. This review compiles published data on antimicrobial resistance in species belonging to the P. fluorescens complex, which were identified through phylogenomic analyses. Additionally, we explored the occurrence of clinically relevant antimicrobial resistance genes in the genomes of the respective species available in the NCBI database. Isolates were organized into two categories: strains isolated from pristine sites and strains isolated from human-impacted or metal-polluted sites. Our review revealed that many reported resistant phenotypes in this complex might be related to intrinsic features, whereas some of them might be ascribed to adaptive mechanisms such as colistin resistance. Moreover, a few studies reported antimicrobial resistance genes (ARGs), mainly β-lactamases. In-silico analysis corroborated the low occurrence of transferable resistance mechanisms in this Pseudomonas complex. Both phenotypic and genotypic assays are necessary to gain insights into the evolutionary aspects of antimicrobial resistance in the P. fluorescens complex and the possible role of these ubiquitous species as reservoirs of clinically important and transmissible ARGs. [ABSTRACT FROM AUTHOR]

Simple Summary: While the PI3K/AKT/mTORC1 pathway is highly active in breast cancer, mTORC1-targeting drugs are not effective in all breast cancer subtypes. To identify potential resistance mechanisms, we utilized a mouse model of breast cancer that continues to grow in the presence of the mTORC1 inhibitor, rapamycin. This treatment caused changes in the activity of genes that control the environment surrounding the tumor cells (the extracellular matrix or ECM). To determine if the ECM can modulate mTORC1 inhibitor effectiveness, we targeted focal adhesion kinase (FAK), an integral protein that mediates signaling from the ECM into the cell. In models that are relatively resistant to mTORC1 inhibitors, blocking FAK improved the ability of mTORC1 inhibitors to suppress tumor growth. However, in models that are sensitive to mTORC1 inhibitors, FAK suppression had no effect. These results provide preclinical evidence that the dual targeting of FAK and mTORC1 may improve therapeutic impact in cancers that are resistant to mTORC1 inhibitors. The PI3K/AKT/mTORC1 pathway is a major therapeutic target for many cancers, particularly breast cancer. Everolimus is an mTORC1 inhibitor used in metastatic estrogen receptor-positive (ER+) and epidermal growth factor receptor 2-negative (HER2-) breast cancer. However, mTORC1 inhibitors have limited efficacy in other breast cancer subtypes. We sought to discover collateral sensitivities to mTORC1 inhibition that could be exploited to improve therapeutic response. Using a mouse model of breast cancer that is intrinsically resistant to mTORC1 inhibition, we found that rapamycin alters the expression of numerous extracellular matrix genes, suggesting a potential role for integrins/FAK in controlling mTORC1-inhibitor efficacy. FAK activation was also inversely correlated with rapamycin response in breast cancer cell lines. Supporting its potential utility in patients, FAK activation was observed in >50% of human breast cancers. While blocking FAK in mouse models of breast cancer that are highly responsive to rapamycin had no impact on tumor growth, FAK inhibition sensitized rapamycin-resistant tumors to mTORC1 inhibition. These data reveal an innate dependency on FAK when mTORC1 signaling is lost in tumors that are resistant to mTORC1 inhibitors. They also suggest a precision medicine approach to improving mTORC1 inhibitor efficacy in resistant cancers by suppressing FAK signaling. [ABSTRACT FROM AUTHOR]

Abstract Background Enterococcus casseliflavus is rarely isolated from human specimens. To the best of our knowledge, there are no reports on its detailed treatment course and prognosis. Here, we present the first known case of E. casseliflavus endocarditis with a detailed treatment course. Case presentation An 86-year-old Japanese woman was transferred to the emergency department with dyspnoea, wheezing, and lumbago. Her medical history included hypertension, chronic kidney disease, idiopathic interstitial pneumonia, and rectal carcinoma. Physical examination revealed expiratory wheezes and a diastolic murmur (Levine 2/6) at the 4th right sternal border. Chest radiography revealed bilateral interstitial opacities and slight cardiac dilatation. Transthoracic echocardiography demonstrated the presence of mobile vegetation with perforation, prolapse, and regurgitation of the aortic valve. With a suspicion of infective endocarditis, we started administering intravenous ampicillin/sulbactam. Thereafter, blood cultures identified E. casseliflavus through matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. The antimicrobial treatment was then switched to ampicillin plus gentamicin. The patient underwent aortic valve replacement on the thirteenth hospital day. She was administered intravenous ampicillin and gentamicin for 6 weeks. The patient was discharged 8 weeks after admission. Conclusions Our case demonstrated that E. casseliflavus could cause infective endocarditis, which can be successfully treated with a 6-week regimen of ampicillin and gentamicin in combination with proper surgical treatment.

ABSTRACT Mycobacterium abscessus is an emerging human pathogen leading to significant morbidity and even mortality, intrinsically resistant to almost all the antibiotics available and so can be a nightmare. Mechanisms of its intrinsic resistance remain not fully understood. Here, we selected and confirmed an M. abscessus transposon mutant that is hypersensitive to multiple drugs including rifampin, rifabutin, vancomycin, clofazimine, linezolid, imipenem, levofloxacin, cefoxitin, and clarithromycin. The gene MAB_0189c encoding a putative arabinosyltransferase C was found to be disrupted, using a newly developed highly-efficient strategy combining next-generation sequencing and multiple PCR. Furthermore, selectable marker-free deletion of MAB_0189c recapitulated the hypersensitive phenotype. Disruption of MAB_0189c resulted in an inability to synthesize lipoarabinomannan and markedly enhanced its cell envelope permeability. Complementing MAB_0189c or M. tuberculosis embC restored the resistance phenotype. Importantly, treatment of M. abscessus with ethambutol, a first-line antituberculosis drug targeting arabinosyltransferases of M. tuberculosis, largely sensitized M. abscessus to multiple antibiotics in vitro. We finally tested activities of six selected drugs using a murine model of sustained M. abscessus infection and found that linezolid, rifabutin, and imipenem were active against the MAB_0189c deletion strain. These results identified MAB_0189 as a crucial determinant of intrinsic resistance of M. abscessus, and optimizing inhibitors targeting MAB_0189 might be a strategy to disarm the intrinsic multiple antibiotic resistance of M. abscessus. IMPORTANCE Mycobacterium abscessus is intrinsically resistant to most antibiotics, and treatment of its infections is highly challenging. The mechanisms of its intrinsic resistance remain not fully understood. Here we found a transposon mutant hypersensitive to a variety of drugs and identified the transposon inserted into the MAB_0189c (orthologous embC coding arabinosyltransferase, EmbC) gene by using a newly developed rapid and efficient approach. We further verified that the MAB_0189c gene played a significant role in its intrinsic resistance by decreasing the cell envelope permeability through affecting the production of lipoarabinomannan in its cell envelope. Lastly, we found the arabinosyltransferases inhibitor, ethambutol, increased activities of nine selected drugs in vitro. Knockout of MAB_0189c made M. abscessus become susceptible to 3 drugs in mice. These findings indicated that potential powerful M. abscessus EmbC inhibitor might be used to reverse the intrinsic resistance of M. abscessus to multiple drugs.

Annotations of non-pathogenic bacterial genomes commonly reveal putative antibiotic resistance genes and the potential risks associated with such genes is challenging to assess. We have examined a putative tetracycline tet(L) gene (conferring low level tetracycline resistance), present in the majority of all publicly available genomes of the industrially important operational group Bacillus amyloliquefaciens including the species B. amyloliquefaciens, Bacillus siamensis and Bacillus velezensis. The aim was to examine the risk of transfer of the putative tet(L) in operational group B. amyloliquefaciens through phylogenetic and genomic position analysis. These analyses furthermore included tet(L) genes encoded by transferable plasmids and other Gram-positive and -negative bacteria, including Bacillus subtilis. Through phylogenetic analysis, we could group chromosomally and plasmid-encoded tet(L) genes into four phylogenetic clades. The chromosomally encoded putative tet(L) from operational group B. amyloliquefaciens formed a separate phylogenetic clade; was positioned in the same genomic region in the three species; was not flanked by mobile genetic elements and was not found in any other bacterial species suggesting that the gene has been present in a common ancestor before species differentiation and is intrinsic. Therefore the gene is not considered a safety concern, and the risk of transfer to and expression of resistance in other non-related species is considered negligible. We suggest a subgrouping of the tet(L) class into four groups (tet(L)1.1, tet(L)1.2 and tet(L)2.1, tet(L)2.2), corresponding with the phylogenetic grouping and tet(L) from operational group B. amyloliquefaciens referred to as tet(L)2.2. Phylogenetic analysis is a useful tool to correctly differentiate between intrinsic and acquired antibiotic resistance genes.

In this review, we analyse the impact of oncogenic Ras mutations in mediating cancer drug resistance and the progress made in the abrogation of this resistance, through pharmacological targeting. At a physiological level, Ras is implicated in many cellular proliferation and survival pathways. However, mutations within this small GTPase can be responsible for the initiation of cancer, therapeutic resistance and failure, and ultimately disease relapse. Often termed "undruggable," Ras is notoriously difficult to target directly, due to its structure and intrinsic activity. Thus, Ras‐mediated drug resistance remains a considerable pharmacological problem. However, with advances in both analytical techniques and novel drug classes, the therapeutic landscape against Ras is changing. Allele‐specific, direct Ras‐targeting agents have reached clinical trials for the first time, indicating there may, at last, be hope of targeting such an elusive but significant protein for better more effective cancer therapy. LINKED ARTICLES: This article is part of a themed issue on New avenues in cancer prevention and treatment (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.12/issuetoc [ABSTRACT FROM AUTHOR]

It is generally accepted that fosfomycin activity is higher in the presence of glucose-6-phosphate, since its inducible transporter UhpT is one of the gates for fosfomycin entry. Accordingly, fosfomycin susceptibility tests are performed in the presence of this sugar; however, since Stenotrophomonas maltophilia lacks UhpT, it is doubtful that glucose-6-phosphate might be a fosfomycin adjuvant in this microorganism. The aim of the work was to determine whether glucose-6-phosphate or other metabolites may alter the activity of fosfomycin against S. maltophilia. To that goal, checkerboard assays were performed to analyze the synergy and antagonism of compounds, such as glucose-6-phosphate, fructose, phosphoenolpyruvate, and glyceraldehyde-3-phosphate, among others, with fosfomycin. Besides, minimal inhibitory concentrations of fosfomycin against a set of clinical S. maltophilia isolates presenting different levels of expression of the SmeDEF efflux pump were determined in the presence and absence of said compounds. Finally, intracellular fosfomycin concentrations were determined using a bioassay. Our results show that, opposite to what has been described for other bacteria, glucose-6-phosphate does not increase fosfomycin activity against S. maltophilia ; it is a fosfomycin antagonist. However, other metabolites such as fructose, phosphoenolpyruvate and glyceraldehyde-3-phosphate, increase fosfomycin activity. Consistent with these results, glucose-6-phosphate decreases fosfomycin internalization (a feature against current ideas in the field), while the other three compounds increase the intracellular concentration of this antibiotic. These results support that current standard fosfomycin susceptibility tests made in the presence of glucose-6-phosphate do not account for the actual susceptibility to this antibiotic of some bacteria, such as S. maltophilia. Finally, the innocuous metabolites that increase S. maltophilia susceptibility to fosfomycin found in this work are potential adjuvants, which might be included in fosfomycin formulations used for treating infections by this resistant pathogen. [ABSTRACT FROM AUTHOR]

It is generally accepted that fosfomycin activity is higher in the presence of glucose-6-phosphate, since its inducible transporter UhpT is one of the gates for fosfomycin entry. Accordingly, fosfomycin susceptibility tests are performed in the presence of this sugar; however, since Stenotrophomonas maltophilia lacks UhpT, it is doubtful that glucose-6-phosphate might be a fosfomycin adjuvant in this microorganism. The aim of the work was to determine whether glucose-6-phosphate or other metabolites may alter the activity of fosfomycin against S. maltophilia. To that goal, checkerboard assays were performed to analyze the synergy and antagonism of compounds, such as glucose-6-phosphate, fructose, phosphoenolpyruvate, and glyceraldehyde-3-phosphate, among others, with fosfomycin. Besides, minimal inhibitory concentrations of fosfomycin against a set of clinical S. maltophilia isolates presenting different levels of expression of the SmeDEF efflux pump were determined in the presence and absence of said compounds. Finally, intracellular fosfomycin concentrations were determined using a bioassay. Our results show that, opposite to what has been described for other bacteria, glucose-6-phosphate does not increase fosfomycin activity against S. maltophilia; it is a fosfomycin antagonist. However, other metabolites such as fructose, phosphoenolpyruvate and glyceraldehyde-3-phosphate, increase fosfomycin activity. Consistent with these results, glucose-6-phosphate decreases fosfomycin internalization (a feature against current ideas in the field), while the other three compounds increase the intracellular concentration of this antibiotic. These results support that current standard fosfomycin susceptibility tests made in the presence of glucose-6-phosphate do not account for the actual susceptibility to this antibiotic of some bacteria, such as S. maltophilia. Finally, the innocuous metabolites that increase S. maltophilia susceptibility to fosfomycin found in this work are potential adjuvants, which might be included in fosfomycin formulations used for treating infections by this resistant pathogen.

Pulmonary infections caused by Mycobacterium abscessus (MAB) have been increasing in incidence in recent years, leading to chronic and many times fatal infections due to MAB’s natural resistance to most available antimicrobials. The use of bacteriophages (phages) in clinics is emerging as a novel treatment strategy to save the lives of patients suffering from drug-resistant, chronic, and disseminated infections. The substantial research indicates that phage–antibiotic combination therapy can display synergy and be clinically more effective than phage therapy alone. However, there is limited knowledge in the understanding of the molecular mechanisms in phage–mycobacteria interaction and the synergism of phage–antibiotic combinations. We generated the lytic mycobacteriophage library and studied phage specificity and the host range in MAB clinical isolates and characterized the phage’s ability to lyse the pathogen under various environmental and mammalian host stress conditions. Our results indicate that phage lytic efficiency is altered by environmental conditions, especially in conditions of biofilm and intracellular states of MAB. By utilizing the MAB gene knockout mutants of the MAB_0937c/MmpL10 drug efflux pump and MAB_0939/pks polyketide synthase enzyme, we discovered the surface glycolipid diacyltrehalose/polyacyltrehalose (DAT/PAT) as one of the major primary phage receptors in mycobacteria. We also established a set of phages that alter the MmpL10 multidrug efflux pump function in MAB through an evolutionary trade-off mechanism. The combination of these phages with antibiotics significantly decreases the number of viable bacteria when compared to phage or antibiotic-alone treatments. This study deepens our understanding of phage–mycobacteria interaction mechanisms and identifies therapeutic phages that can lower bacterial fitness by impairing an antibiotic efflux function and attenuating the MAB intrinsic resistance mechanism via targeted therapy.

Polymyxin is the “last resort” of antibiotics. The self-induced resistance to polymyxin in Gram-negative bacteria could be mediated by lipopolysaccharide (LPS) modification, which is regulated by the two-component system, PhoP/PhoQ. Yersinia enterocolitica is a common foodborne pathogen. However, PhoP/PhoQ has not been thoroughly studied in Y. enterocolitica. In this study, the functions of PhoP/PhoQ in Y. enterocolitica intrinsic resistance were investigated. The resistance of Y. enterocolitica was found to decrease with the deletion of PhoP/PhoQ. Further, PhoP/PhoQ was found to play an important role in maintaining membrane permeability, intercellular metabolism, and reducing membrane depolarization. Based on subsequent studies, the binding ability of polymyxin to Y. enterocolitica was decreased by the modification of LPS with structures, such as L-Ara4N and palmitate. Analysis of the gene transcription levels revealed that the LPS modification genes, pagP and arn operon, were downregulated with the deletion of PhoP/PhoQ in Y. enterocolitica during exposure to polymyxin. In addition, pmrA, pmrB, and eptA were downregulated in the mutants compared with the wild-type strain. Such findings demonstrate that PhoP/PhoQ contributes to the intrinsic resistance of Y. enterocolitica toward polymyxins. LPS modification with L-Ara4N or palmitate is mainly responsible for the resistance of Y. enterocolitica to polymyxins. The transcription of genes related to LPS modification and PmrA/PmrB can be both affected by PhoP/PhoQ in Y. enterocolitica. This study adds to current knowledge regarding the role of PhoP/PhoQ in intrinsic resistance of Y. enterocolitica to polymyxin.

Several factors affect drug delivery from dry powder inhalers (DPIs). Some are related to patient's physiological characteristics, while others depend on DPIs' technical aspects. The patient's inspiratory airflow rate (IAR) affects the pressure drop and the turbulence needed to disaggregate the powder inside a DPI. The present study investigated whether lung function limitations occurring in asthmatic adolescents affect their IAR when inhaling through a DPI simulator. Eighteen consecutive adolescents with asthma were recruited, and IAR was randomly assessed at low-, mid-, and high-resistance regimens. A multiple logistic model was developed to evaluate the association of patients' lung function characteristics and devices' resistance with the probability to achieve the expected IAR (E-IAR). The mean value of E-IAR achieved seemed to be sex- and age-independent. Low- and high-resistance regimens were less likely to consent the E-IAR level (odds ratio [OR] = 0.035 and OR = 0.004, respectively). Only the basal residual volume and the inspiratory resistance, but not the Forced Expiratory Volume in 1 s (FEV1), seemed to affect the extent of IAR in asthmatic adolescents (OR = 1.131 and OR = 0.290, respectively). The results suggest that the assessment of current lung function is crucial for choosing the proper DPI for asthmatic adolescents. [ABSTRACT FROM AUTHOR]