Resistance In Clinical Settings Research Articles

Photoactivated riboflavin inhibits planktonic and biofilm growth of Candida albicans and non-albicans Candida species

Fungal infections caused by Candida species pose a serious threat to humankind. Antibiotics abuse and the ability of Candida species to form biofilm have escalated the emergence of drug resistance in clinical settings and hence, rendered it more difficult to treat Candida-related diseases. Lethal effects of Candida infection are often due to inefficacy of antimicrobial treatments and failure of host immune response to clear infections. Previous studies have shown that a combination of riboflavin with UVA (riboflavin/UVA) light demonstrate candidacidal activity albeit its mechanism of actions remain elusive. Thus, this study sought to investigate antifungal and antibiofilm properties by combining riboflavin with UVA against Candida albicans and non-albicans Candida species. The MIC20 for the fluconazole and riboflavin/UVA against the Candida species tested was within the range of 0.125–2 μg/mL while the SMIC50 was 32 μg/mL. Present findings indicate that the inhibitory activities exerted by riboflavin/UVA towards planktonic cells are slightly less effective as compared to controls. However, the efficacy of the combination towards Candida species biofilms showed otherwise. Inhibitory effects exerted by riboflavin/UVA towards most of the tested Candida species biofilms points towards a variation in mode of action that could make it an ideal alternative therapeutic for biofilm-related infections.

Abstract LB388: Super potent and efficacious ALK/ROS1/TRK inhibitor that overcomes multiple drug-resistant mutations

Abstract Abnormal activation of ALK, ROS1 and TRK is implicated in tumorigenesis, progression, invasion, and metastasis of malignancies through various mechanisms. The first generation TRK inhibitors including larotrectinib and entrectinib have limited applications due to acquired resistance in clinical settings. Multiple on-target mutations occur at the solvent-front, gatekeeper, and xDFG motif inside the ATP binding pocket of the TRK kinase domain. While some candidates under current clinical development aim to address the solvent-front mutation, they are ineffective against the xDFG mutations (e.g.NTRK1.G667X). There is strong unmet medical needs for next generation TRK inhibitors that can address resistance mutations. We employ the synergistic advantages of domain expertise and modern technology to accelerate early stage drug discovery. On our deep learning (DL) centric AI platform, extensive scientific computing supports large scale virtual screening, generative design, drug property prediction and multi-objective lead optimization. Only the optimized candidate compounds are passed along for synthesis and testing to save time and cost. In Ba/F3-TPM3 cellular assays, LB-2159 showed superior potencies that are hundreds to thousands of fold higher than larotrectinib and/or entrectinib, with IC50 values of 0.02 nM for NTRK1.WT, 0.3 nM for NTRK1.G595R and 0.88 nM for NTRK1.G667C. Similar cellular potencies were demonstrated for ALK and ROS1. In xenograft models LB-2159 at 10 mg/kg bid resulted in complete tumor regression in Ba/F3-TPM3-NTRK1-WT tumors, 96% TGI in Ba/F3-TPM3-NTRK1-G595R tumors, 84% TGI and 100% survival in mice bearing Ba/F3-TPM3-NTRK1-G667C tumors. The pharmacokinetic profiles of LB-2159 are overall favorable, demonstrating extended stability and half life. In vitro safety screens yielded clean profiles without any DDI signal or hERG liability. Preclinical safety evaluations exhibited good tolerance in rats. In multiple dose escalation studies LB-2159 was well tolerated at 50 mg/kg/day and beyond. Empowered by our proprietary AI platform, we have discovered the novel ALK/ROS1/TRK inhibitors that overcome clinically known resistance mutations. Our drug candidate exhibited good tolerance in animals in preclinical safety evaluations. In xenograft models that are resistant to current treatments the compound demonstrates strong tumor suppression efficacy. LB-2159 is advancing through IND enabling studies, and its preclinical profiles support the potential for best-in-class treatment for NTRK fusion-positive solid tumors. Citation Format: Li Xing, Jingbing Wang, Wei Zhu. Super potent and efficacious ALK/ROS1/TRK inhibitor that overcomes multiple drug-resistant mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB388.

EGFR signaling and pharmacology in oncology revealed with innovative BRET-based biosensors

Mutations of receptor tyrosine kinases (RTKs) are associated with the development of many cancers by modifying receptor signaling and contributing to drug resistance in clinical settings. We present enhanced bystander bioluminescence resonance energy transfer-based biosensors providing new insights into RTK biology and pharmacology critical for the development of more effective RTK-targeting drugs. Distinct SH2-specific effector biosensors allow for real-time and spatiotemporal monitoring of signal transduction pathways engaged upon RTK activation. Using EGFR as a model, we demonstrate the capacity of these biosensors to differentiate unique signaling signatures, with EGF and Epiregulin ligands displaying differences in efficacy, potency, and responses within different cellular compartments. We further demonstrate that EGFR single point mutations found in Glioblastoma or non-small cell lung cancer, impact the constitutive activity of EGFR and response to tyrosine kinase inhibitor. The BRET-based biosensors are compatible with microscopy, and more importantly characterize the next generation of therapeutics directed against RTKs.

DCAF1-based PROTACs with activity against clinically validated targets overcoming intrinsic- and acquired-degrader resistance

Targeted protein degradation (TPD) mediates protein level through small molecule induced redirection of E3 ligases to ubiquitinate neo-substrates and mark them for proteasomal degradation. TPD has recently emerged as a key modality in drug discovery. So far only a few ligases have been utilized for TPD. Interestingly, the workhorse ligase CRBN has been observed to be downregulated in settings of resistance to immunomodulatory inhibitory drugs (IMiDs). Here we show that the essential E3 ligase receptor DCAF1 can be harnessed for TPD utilizing a selective, non-covalent DCAF1 binder. We confirm that this binder can be functionalized into an efficient DCAF1-BRD9 PROTAC. Chemical and genetic rescue experiments validate specific degradation via the CRL4DCAF1 E3 ligase. Additionally, a dasatinib-based DCAF1 PROTAC successfully degrades cytosolic and membrane-bound tyrosine kinases. A potent and selective DCAF1-BTK-PROTAC (DBt-10) degrades BTK in cells with acquired resistance to CRBN-BTK-PROTACs while the DCAF1-BRD9 PROTAC (DBr-1) provides an alternative strategy to tackle intrinsic resistance to VHL-degrader, highlighting DCAF1-PROTACS as a promising strategy to overcome ligase mediated resistance in clinical settings.

Towards a High-Affinity Peptidomimetic Targeting Proliferating Cell Nuclear Antigen from Aspergillus fumigatus.

Invasive fungal infections (IFIs) are prevalent in immunocompromised patients. Due to alarming levels of increasing resistance in clinical settings, new drugs targeting the major fungal pathogen Aspergillus fumigatus are required. Attractive drug targets are those involved in essential processes like DNA replication, such as proliferating cell nuclear antigens (PCNAs). PCNA has been previously studied in cancer research and presents a viable target for antifungals. Human PCNA interacts with the p21 protein, outcompeting binding proteins to halt DNA replication. The affinity of p21 for hPCNA has been shown to outcompete other associating proteins, presenting an attractive scaffold for peptidomimetic design. p21 has no A. fumigatus homolog to our knowledge, yet our group has previously demonstrated that human p21 can interact with A. fumigatus PCNA (afumPCNA). This suggests that a p21-based inhibitor could be designed to outcompete the native binding partners of afumPCNA to inhibit fungal growth. Here, we present an investigation of extensive structure-activity relationships between designed p21-based peptides and afumPCNA and the first crystal structure of a p21 peptide bound to afumPCNA, demonstrating that the A. fumigatus replication model uses a PIP-box sequence as the method for binding to afumPCNA. These results inform the new optimized secondary structure design of a potential peptidomimetic inhibitor of afumPCNA.

Acquired blaVIM and blaGES Carbapenemase-Encoding Genes in Pseudomonas aeruginosa: A Seven-Year Survey Highlighting an Increasing Epidemiological Threat.

(1) Background: Pseudomonas aeruginosa is a Gram-negative bacterium with several intrinsic and acquired antimicrobial resistance mechanisms. The spread of carbapenemase-encoding genes, an acquired mechanism, enables carbapenem resistance in clinical settings. Detection of the carbapenemase-producer strains is urgent. Therefore, we aimed to characterize carbapenemase production in the clinical strains of P. aeruginosa at a tertiary-care center. (2) Methods: We included clinical strains of P. aeruginosa (from August 2011 to December 2018) with resistance towards at least one carbapenem. Strains were isolated in a tertiary-care center in Mexico City. Antimicrobial susceptibility profiles were determined by broth microdilution. Screening for carbapenemase-encoding genes was performed in all strains. Phenotypic assays (CarbaNP and mCIM) were conducted. Additional modifications to mCIM were also tested. (3) Results: One-hundred seventy-one P. aeruginosa strains out of 192 included in this study were resistant towards at least one of the carbapenems tested. Forty-seven of these strains harbored a carbapenemase-encoding gene. VIM (59.6%) and GES (23.4%) were the most frequently found carbapenemases in our study, followed by IMP (14.9%). (4) Among the most frequent carbapenemase genes identified, metallo-ß-lactamases were the most prevalent, which impair new treatment options. Searching for carbapenemase genes should be performed in resistant isolates to stop transmission and guide antimicrobial treatment.

Deep mutational scanning of influenza A virus neuraminidase facilitates the identification of drug resistance mutations in vivo.

NA is a crucial surface antigen and drug target of influenza A virus. A comprehensive understanding of NA's mutational effect and drug resistance profiles in vivo is essential for comprehending the evolutionary constraints and making informed choices regarding drug selection to combat resistance in clinical settings. In the current study, we established an efficient deep mutational screening system in mouse lung tissues and systematically evaluated the fitness effect and drug resistance to three neuraminidase inhibitors of NA single-nucleotide mutations. The fitness of NA mutants is generally correlated with a natural mutation in the database. The fitness of NA mutants is influenced by biophysical factors such as protein stability, complex formation, and the immune response triggered by viral infection. In addition to confirming previously reported drug-resistant mutations, novel mutations were identified. Interestingly, we identified an allosteric drug-resistance mutation that is not located within the drug-binding pocket but potentially affects drug binding by interfering with NA tetramerization. The dual assessments performed in this study provide a more accurate assessment of the evolutionary potential of drug-resistant mutations and offer guidance for the rational selection of antiviral drugs.

Developing Phage Therapy That Overcomes the Evolution of Bacterial Resistance.

The global rise of antibiotic resistance in bacterial pathogens and the waning efficacy of antibiotics urge consideration of alternative antimicrobial strategies. Phage therapy is a classic approach where bacteriophages (bacteria-specific viruses) are used against bacterial infections, with many recent successes in personalized medicine treatment of intractable infections. However, a perpetual challenge for developing generalized phage therapy is the expectation that viruses will exert selection for target bacteria to deploy defenses against virus attack, causing evolution of phage resistance during patient treatment. Here we review the two main complementary strategies for mitigating bacterial resistance in phage therapy: minimizing the ability for bacterial populations to evolve phage resistance and driving (steering) evolution of phage-resistant bacteria toward clinically favorable outcomes. We discuss future research directions that might further address the phage-resistance problem, to foster widespread development and deployment of therapeutic phage strategies that outsmart evolved bacterial resistance in clinical settings.

Environmental integrons: the dark side of the integron world.

Integrons are bacterial genetic elements notorious for their role in spreading antibiotic resistance in clinical settings. In the natural environment, integrons present a wide and hidden diversity, raising questions as to their broader role in bacterial adaptation. From the One Health perspective, they must be considered a threatening pool of resistance determinants.

Cohort Profile: A European Multidisciplinary Network for the Fight against HIV Drug Resistance (EuResist Network).

The EuResist cohort was established in 2006 with the purpose of developing a clinical decision-support tool predicting the most effective antiretroviral therapy (ART) for persons living with HIV (PLWH), based on their clinical and virological data. Further to continuous extensive data collection from several European countries, the EuResist cohort later widened its activity to the more general area of antiretroviral treatment resistance with a focus on virus evolution. The EuResist cohort has retrospectively enrolled PLWH, both treatment-naïve and treatment-experienced, under clinical follow-up from 1998, in nine national cohorts across Europe and beyond, and this article is an overview of its achievement. A clinically oriented treatment-response prediction system was released and made available online in 2008. Clinical and virological data have been collected from more than one hundred thousand PLWH, allowing for a number of studies on the response to treatment, selection and spread of resistance-associated mutations and the circulation of viral subtypes. Drawing from its interdisciplinary vocation, EuResist will continue to investigate clinical response to antiretroviral treatment against HIV and monitor the development and circulation of HIV drug resistance in clinical settings, along with the development of novel drugs and the introduction of new treatment strategies. The support of artificial intelligence in these activities is essential.

Clinical Aspects of Bacterial Distribution and Antibiotic Resistance in the Reproductive System of Equids.

Antibiotic administration is a standard therapeutic practice for the treatment of reproductive disorders of equids. This might lead to undesirable microbial imbalance and could favour the acquisition of antibiotic resistance. Therefore, it is imperative for clinicians to understand patterns of antibiotic resistance when considering and developing treatment regimes. Continued engagement of clinicians with novel alternative approaches to treat reproductive infections would be essential in order to address this rising threat within the One Health perspective. The objectives of the present review were to present the bacterial infections in the reproductive system of equids (horses, donkeys), to upraise the literature related to the issue of antibiotic resistance of bacteria causing these infections and to discuss the topic from a clinical perspective. Initially, the review summarised the various infections of the reproductive system of equids (genital system of females, genital system of males, mammary glands) and the causal bacteria, providing relevant information about horses and donkeys. Subsequently, the clinical therapeutics of these infections were presented, taking into account the significance of antibiotic resistance of bacteria as a limiting factor in treating the infections. Finally, approaches to circumvent antibiotic resistance in clinical settings were summarized. It was concluded that awareness regarding antibiotic resistance in equine reproductive medicine would increase, as we would recognise the multifaceted problem of resistance. Actions and initiatives within the One Health approach, minimizing the potential dissemination of resistant strains to humans and to the environment, with specific applications in medicine of equids should be appropriately instituted internationally.

Newly Discovered Mechanisms of Antibiotic Self-Resistance with Multiple Enzymes Acting at Different Locations and Stages.

Self-resistance determinants are essential for the biosynthesis of bioactive natural products and are closely related to drug resistance in clinical settings. The study of self-resistance mechanisms has long moved forward on the discovery of new resistance genes and the characterization of enzymatic reactions catalyzed by these proteins. However, as more examples of self-resistance have been reported, it has been revealed that the enzymatic reactions contribute to self-protection are not confined to the cellular location where the final toxic compounds are present. In this review, we summarize representative examples of self-resistance mechanisms for bioactive natural products functional at different cell locations to explore the models of resistance strategies involved. Moreover, we also highlight those resistance determinants that are widespread in nature and describe the applications of self-resistance genes in natural product mining to interrogate the landscape of self-resistance genes in drug resistance-related new drug discovery.

Molecular Dynamics Investigation of MFS Efflux Pump MdfA Reveals an Intermediate State between Its Inward and Outward Conformations.

Multidrug resistance poses a major challenge to antibiotic therapy. A principal cause of antibiotic resistance is through active export by efflux pumps embedded in the bacterial membrane. Major facilitator superfamily (MFS) efflux pumps constitute a major group of transporters, which are often related to quinolone resistance in clinical settings. Although a rocker-switch model is proposed for description of their conformational transitions, detailed changes in this process remain poorly understood. Here we used MdfA from E. coli as a representative MFS efflux pump to investigate factors that can affect its conformational transition in silico. Molecular dynamics (MD) simulations of MdfA's inward and outward conformations revealed an intermediate state between these two conformations. By comparison of the subtle differences between the intermediate state and the average state, we indicated that conformational transition from outward to inward was initiated by protonation of the periplasmic side. Subsequently, hydrophilic interaction of the periplasmic side with water was promoted and the regional structure of helix 1 was altered to favor this process. As the hydrophobic interaction between MdfA and membrane was also increased, energy was concentrated and stored for the opposite transition. In parallel, salt bridges at the cytoplasmic side were altered to lower probabilities to facilitate the entrance of substrate. In summary, we described the total and local changes during MdfA's conformational transition, providing insights for the development of potential inhibitors.

SUMOylation of ERp44 enhances Ero1α ER retention contributing to the pathogenesis of obesity and insulin resistance

ObjectiveAs the only E2 conjugating enzyme for the SUMO system, Ubc9-mediated SUMOylation has been recognized to regulate diverse biological processes, but its impact on adipocytes relevant to obesity and insulin resistance is yet to be elucidated. MethodsWe established adipocyte-specific Ubc9 deficient mice to explore the effects of Ubc9 on obesity and metabolic disorders induced by high-fat diet (HFD) in adult mice. The molecular targets of SUMOylation were explored by liquid chromatography-mass spectrometry, and the regulatory mechanism of SUMOylation in T2D was analyzed. ResultsAdipocyte-specific depletion of Ubc9 (AdipoQ-Cre-Ubc9fl/fl, Ubc9AKO) protected mice from HFD-induced obesity, insulin resistance, and hepatosteatosis. The Ubc9AKO mice were featured by the reduced HFD-induced endoplasmic reticulum (ER) stress and inflammatory response. Mechanically, over nutrition rendered adipocytes to undergo a SUMOylation turnover characterized by the change of SUMOylation levels and substrates. ERp44 displayed the highest change in terms of SUMOylation levels of substrates involved in ER-related functions. The lack of ERp44 SUMOylation at lysine 76 (K76) located within the thioredoxin (TRX)-like domain by Ubc9 deficiency enhanced its degradation and suppressed its covalent binding to Ero1α, an oxidase that exists in the ER but lacks ER retention motif, thereby alleviating endoplasmic reticulum stress by promoting Ero1α secretion. ConclusionsOur data suggest that modulation of ERp44 SUMOylation in adipocytes could be a feasible strategy against obesity and insulin resistance in clinical settings.

Lectin-Fortified Cationic Copper Sulfide Nanoparticles Gain Dual Targeting Capabilities to Treat Carbapenem-Resistant Acinetobacter baumannii Infection.

Targeted drug delivery maximizes the chance to combat infection caused by drug-resistant pathogens. Herein, lectin-fortified cationic copper sulfide (cCuS) nanoparticles were suggested for targeted adhesion to bacterial membranes and to enforce bacterial death. Jacalin, a lectin from jackfruit seed, was conjugated to fluorescein isothiocyanate (FITC), and its ability to recognize bacterial cell surface glycans was demonstrated. Jacalin formed a noncovalent complex with cCuS, which was investigated by fluorescence quenching measurements. The data revealed that jacalin-cCuS (JcCuS) had a good affinity with an association constant K a of 2.27 (± 0.28) × 104 M-1. The resultant JcCuS complex displayed excellent anti-infective activity against carbapenem-resistant Acinetobacter baumannii (CRAB). The minimum inhibitory concentration (MIC) of cCuS was 62.5 μM, which was 2-fold lower than that of the broad-spectrum antibiotic ciprofloxacin. Interestingly, the MIC of JcCuS was reduced to 15.63 μM, which was attributed to jacalin fortification. The mechanistic study unveiled that JcCuS affected the membrane integrity, depolarized the inner membrane, and produced excess reactive oxygen species to combat CRAB at a lower concentration compared to cCuS. A. baumannii formed a biofilm more readily, which played a critical role in pathogenesis and resistance in clinical settings. JcCuS (3.91 μM) displayed stronger antibiofilm activity without affecting the metabolic viability of CRAB. Microscopy analyses confirmed the inhibition of biofilm formation and disruption of the mature biofilm upon treatment with JcCuS. Furthermore, JcCuS hindered pellicle formation and inhibited the biofilm-associated virulence factor of CRAB such as exopolysaccharide, cell surface hydrophobicity, swarming, and twitching mobility. The anti-infective potential of JcCuS was demonstrated by rescuing CRAB-infected zebrafish. The reduction in pathogen proliferation in muscle tissues was observed in the treated group, and the fish recovered from the infection and was restored to normal life within 12 h. The findings illustrate that lectin fortification offers a unique advantage in enhancing the therapeutic potential of antimicrobials against human pathogens of critical priority worldwide.

A Nonclassical Mechanism of β-Lactam Resistance in Methicillin-Resistant Staphylococcus aureus and Its Effect on Virulence.

Methicillin-resistant Staphylococcus aureus (MRSA) is a group of pathogenic bacteria that are infamously resistant to β-lactam antibiotics, a property attributed to the mecA gene. Recent studies have reported that mutations associated with the promoter region of pbp4 demonstrated high levels of β-lactam resistance, suggesting the role of PBP4 as an important non-mecA mediator of β-lactam resistance. The pbp4-promoter-associated mutations have been detected in strains with or without mecA. Our previous studies that were carried out in strains devoid of mecA described that pbp4-promoter-associated mutations lead to PBP4 overexpression and β-lactam resistance. In this study, by introducing various pbp4-promoter-associated mutations in the genome of a MRSA strain, we demonstrate that PBP4 overexpression can supplement mecA-associated resistance in S. aureus and can lead to increased β-lactam resistance. The promoter and regulatory region of pbp4 is shared with a divergently transcribed gene, abcA, which encodes a multidrug exporter. We demonstrate that the promoter mutations caused an upregulation of pbp4 and downregulation of abcA, confirming that the resistant phenotype is associated with PBP4 overexpression. PBP4 has also been associated with staphylococcal pathogenesis, however, its exact role remains unclear. Using a Caenorhabditis elegans model, we demonstrate that strains having increased PBP4 expression are less virulent than wild-type strains, suggesting that β-lactam resistance mediated via PBP4 likely comes at the cost of virulence. IMPORTANCE Our study demonstrates the ability of PBP4 to be an important mediator of β-lactam resistance in not only methicillin-susceptible Staphylococcus aureus (MSSA) background strains as previously demonstrated but also in MRSA strains. When present together, PBP2a and PBP4 overexpression can produce increased levels of β-lactam resistance, causing complications in treatment. Thus, this study suggests the importance of monitoring PBP4-associated resistance in clinical settings, as well as understanding the mechanistic basis of associated resistance, so that treatments targeting PBP4 may be developed. This study also demonstrates that S. aureus strains with increased PBP4 expression are less pathogenic, providing important hints about the role of PBP4 in S. aureus resistance and pathogenesis.

A novel of quinoxaline derivatives tagged with pyrrolidinyl scaffold as a new class of antimicrobial agents: Design, synthesis, antimicrobial activity, and molecular docking simulation

Globally, infectious diseases are becoming harder to treat due to antibiotic resistance, which has reached high levels. Additionally, developing new therapeutic options to combat the growing antimicrobial resistance in clinical settings is necessary. The new 3-(pyrrolidin-1-yl)quinoxaline derivatives 4–10 conjugated with a different substituent at C2 through ether or amine linkage were synthesized via nucleophilic substitution reaction. The structure of quinoxaline derivatives was confirmed by IR, 1H NMR, and 13C NMR spectra. The antimicrobial activity of quinoxaline derivatives 4–10 varied from good to potency against the tested strains. The quinoxaline derivatives 4, 6, and 7 exhibited excellent activity, especially against B. pumilis and E. cloacae, with MIC values of (7.8, 15.6, and 3.91 µg/mL) and (15.6, 7.8, and 15.6 µg/mL) compared with Ciprofloxacin (7.8 and 15.6 µg/mL). Further, hybrid quinoxaline with different methanimine moieties at C2 showed moderate to good antimicrobial activity, except methanimine 9 and 10 that had MIC values equipotent to Ciprofloxacin 15.6 and 31.25 µg/mL against E. coli and S. faecalis, respectively. On the other hand, all these derivatives revealed good to weak antifungal activity with MIC (31.25–500 µg/mL) and MFC (62.5–1000 µg/mL) against A. niger and C. albicans. Moreover, most of the quinoxaline derivatives exhibited bactericidal and fungicidal activity, except for quinoxaline derivatives 9 and 10, which displayed bacteriostatic against E. coli. The molecular docking simulation showed lower binding energy with different types of interaction at the active site of DNA gyrase pocket indicating that these compounds could inhibit the enzyme and cause promising antimicrobial effects.

Bibliometric trends of scientific publications on antimicrobial resistance in Escherichia coli in Peru from 2009-2019.

Antibiotic resistance is a growing problem worldwide. While the most visible aspect is the direct effect of antimicrobial resistance in clinical settings, this problem affects all microorganisms in all environments. Nevertheless, most of the data available on antibiotic resistance outside clinical settings are from high-income countries, with the current situation in most low- and middle-income countries remaining under-reported. Escherichia coli is considered a good marker of “antibiotic pressure” due to its cosmopolitan distribution. In this scenario, we selected E. coli as an appropriate microorganism to perform an initial bibliometric analysis of publications focused on antimicrobial resistance outside disease-causing microorganisms. Thus, the present manuscript performs an analysis of the studies reporting measurable levels of antibiotic resistance of E. coli not causing human disease in Peru published during the period from 2009-2019. Following a search in PubMed and Google, 35 documents were selected as reporting measurable data of antibiotic resistance. Of these, 8 (22.8%) were reported in thesis format. The mean delay between sampling and publishing in article format was 3.7 years, with one study having been published 18 years after sampling. The number of publications developed in Peru describing antimicrobial resistance levels in E. coli out of hospital settings in the period 2009-2019 is scarce. Of these, a relevant number is of unpublished thesis. These findings showing the gap of knowledge and lack of accessible data about antimicrobial resistance out of hospital environments. Efforts and strategies must be developed to fill this knowledge gap.

Nanopore Sequencing for Characterization of HIV-1 Recombinant Forms.

ABSTRACTHigh genetic diversity, including the emergence of recombinant forms (RFs), is one of the most prominent features of human immunodeficiency virus type 1 (HIV-1). Conventional detection of HIV-1 RFs requires pretreatments, i.e., cloning or single-genome amplification, to distinguish them from dual- or multiple-infection variants. However, these processes are time-consuming and labor-intensive. Here, we constructed a new nanopore sequencing-based platform that enables us to obtain distinctive genetic information for intersubtype RFs and dual-infection HIV-1 variants by using amplicons of HIV-1 near-full-length genomes or two overlapping half-length genome fragments. Repeated benchmark tests of HIV-1 proviral DNA revealed consensus sequence inference with a reduced error rate, allowing us to obtain sufficiently accurate sequence data. In addition, we applied the platform for sequence analyses of 9 clinical samples with suspected HIV-1 RF infection or dual infection according to Sanger sequencing-based genotyping tests for HIV-1 drug resistance. For each RF infection case, replicated analyses involving our nanopore sequencing-based platform consistently produced long consecutive analogous consensus sequences with mosaic genomic structures consisting of two different subtypes. In contrast, we detected multiple heterologous sequences in each dual-infection case. These results demonstrate that our new nanopore sequencing platform is applicable to identify the full-length HIV-1 genome structure of intersubtype RFs as well as dual-infection heterologous HIV-1. Since the genetic diversity of HIV-1 continues to gradually increase, this system will help accelerate full-length genome analysis and molecular epidemiological surveillance for HIV-1.IMPORTANCE HIV-1 is characterized by large genetic differences, including HIV-1 recombinant forms (RFs). Conventional genetic analyses require time-consuming pretreatments, i.e., cloning or single-genome amplification, to distinguish RFs from dual- or multiple-infection cases. In this study, we developed a new analytical system for HIV-1 sequence data obtained by nanopore sequencing. The error rate of this method was reduced to ~0.06%. We applied this system for sequence analyses of 9 clinical samples with suspected HIV-1 RF infection or dual infection, which were extracted from 373 cases of HIV patients based on our retrospective analysis of HIV-1 drug resistance genotyping test results. We found that our new nanopore sequencing platform is applicable to identify the full-length HIV-1 genome structure of intersubtype RFs as well as dual-infection heterologous HIV-1. Our protocol will be useful for epidemiological surveillance to examine HIV-1 transmission as well as for genotypic tests of HIV-1 drug resistance in clinical settings.

Colistin Resistance and Molecular Characterization of the Genomes of mcr-1-Positive Escherichia coli Clinical Isolates.

Research on resistance against polymyxins induced by the mcr-1 gene is gaining interest. In this study, using agar dilution method, polymerase chain reaction, and comparative genomic analysis, we investigated the colistin resistance mechanism of clinical E. coli isolates. The minimum inhibitory concentration (MIC) analysis results revealed that of the 515 isolates tested, bacteria with significantly increased MIC levels against colistin were isolated in 2019. Approximately one-fifth (17.14% to 19.65%) of the isolates showed MIC values ≥1 mg/L against colistin in 2015, 2016, and 2017. However, in 2019, up to three-quarters (74.11%, 146/197) of the isolates showed MIC values ≥1 mg/L against colistin indicating an increase in colistin resistance. Six isolates (EC7518, EC4968, EC3769, EC16, EC117, EC195, 1.13%, 6/515) were found to carry the mcr-1 gene and a novel mcr-1 variant with Met2Ile mutation was identified in EC3769. All six strains showed higher MIC levels (MIC=4 mg/L) than any mcr-1-negative strains (MIC ≤ 2 mg/L). Whole-genome sequencing of the six mcr-1-positive isolates revealed that EC195 carried the highest number of resistance genes (n = 28), nearly a half more than those of the following EC117 (n = 19). Thus, EC195 showed a wider resistance spectrum and higher MIC levels against the antimicrobials tested than the other five isolates. Multi-locus sequence typing demonstrated that these mcr-1-positive strains belonged to six different sequence types. The six mcr-1 genes were located in three different incompatibility group plasmids (IncI2, IncHI2 and IncX4). The genetic context of mcr-1 was related to a sequence derived from Tn6330 (ISApl1-mcr-1-pap2-ISApl1). Investigations into the colistin resistance mechanism and characterization of the molecular background of the mcr genes may help trace the development and spread of colistin resistance in clinical settings.