Methods in molecular biology (Clifton, N.J.)Min Zhang, Po Hu, Hailing Jin
Antimicrobial peptides (AMPs) represent the first line of host immune defense against a broad spectrum of microorganisms. Whether naturally occurring or synthetically designed, these small peptides can potently inhibit microbial growth and remain stable under diverse conditions. Most AMPs act by directly disrupting fungal or bacterial membranes. In this study, we identified a glycine-rich peptide with strong antifungal activity against Botrytis cinerea. We further investigated its mode of action (MOA) and demonstrated its preventive antifungal activity in plants. Our work focuses primarily on the in vitro characterization of AMP features in the context of plant-Botrytis interactions.
Methods in molecular biology (Clifton, N.J.)H M Suraj, André Fleissner, Jan A L van Kan
Botrytis cinerea, a major fungal pathogen of crops, has been extensively studied for its resistance to antifungal plant metabolites and agrochemicals. Evaluating the sensitivity of B. cinerea to these compounds is crucial for understanding its pathogenicity and for developing effective plant protection strategies. This study describes two methods for antifungal sensitivity assessment: the spot assay, which visualizes fungal growth on agar medium supplemented with antifungal compounds, and the resazurin assay, which quantifies fungal metabolic activity in liquid culture. Using saponins as antifungal compounds, we demonstrate the utility of these methods on B. cinerea. These methods provide simple, reliable, and reproducible protocols to assess the sensitivity of antifungal compounds on B. cinerea.
Methods in molecular biology (Clifton, N.J.)Alexander W E Franz
Chikungunya virus (CHIKV) is a human pathogenic, mosquito-borne virus (arbovirus), which is causing epidemic outbreaks among human populations in Africa, Asia, Europe, and South- and Central America including the Caribbean. The development of novel approaches to prevent mosquito transmission of the virus in the field requires a detailed study of CHIKV interactions with its mosquito vectors in the laboratory. In this chapter, we describe how to prepare Aedes aegypti mosquitoes for the infection with freshly cultivated CHIKV. We also describe quantitative and qualitative viral detection assays in mosquitoes based on plaque assays and the amplification of saliva samples in cell culture.
Methods in molecular biology (Clifton, N.J.)Akino Emi, Youichi Suzuki
The cytopathic effect (CPE) is a key to understanding the pathogenicity of the chikungunya virus (CHIKV) and is useful for evaluating viral infectivity in vitro. The plaque assay, based on the CPE induced by viral infection, provides an infectious virus titer for test samples. Additionally, the assay is simple, quantitative, and cost-effective. Thus, the plaque assay remains the gold standard for determining the infectivity of CHIKV. However, manually counting of virus-induced plaques with the naked eye or under the microscope is often time-consuming and labor-intensive. Therefore, automated plaque-counting software would improve the consistency and efficiency of plaque counting. We recently developed plaQuest, a stand-alone Windows software that enables rapid, reliable plaque counting for CHIKV. In this chapter, we describe the basic procedure for detecting and counting the CHIKV-formed plaques in a 24-well plate.
Methods in molecular biology (Clifton, N.J.)Sebastiano Volpe, Claudia Filomatori
The plaque-forming assay is the gold-standard method for measuring infectious viral particles. In this technique, lytic viruses infect and destroy host cells, but their spread is restricted by a viscous overlay medium. As a result, new viral particles can only infect neighboring cells, leading to localized clear areas known as plaques, which become visible after staining the remaining living cells. The number of plaques reflects the number of infectious viral particles initially present in the sample and is reported as plaque-forming units (PFU) per sample volume. In this protocol, we describe step by step how to perform a plaque-forming assay to determine the concentration of chikungunya virus in a cell culture supernatant.
Methods in molecular biology (Clifton, N.J.)Aakriti Singh, Barbara S Sixt
The recent application of experimental and computational drug discovery workflows has identified hundreds of new molecules that display potent, and in some cases highly selective, activity against bacteria of the genus Chlamydia. The full value of these novel antichlamydial molecules, however, depends critically on our ability to determine their molecular targets and modes of action. A particularly powerful and widely applied strategy for identifying candidate targets involves selecting for bacterial mutants that have acquired resistance to a compound's inhibitory activity, followed by determining the mutations responsible for this resistance. Such mutations often pinpoint the compound's direct target or reveal cellular pathways that modulate target engagement. Here, we present a detailed, step-by-step protocol for applying this approach to the clinically important, human-pathogenic species Chlamydia trachomatis. The workflow includes: (1) determining compound potency by quantitative dose-response analysis, (2) generating a resistant C. trachomatis mutant through serial passaging under progressively increasing selective pressure, (3) confirming the resistance phenotype, and (4) isolating bacterial genomic DNA for whole-genome sequencing to identify resistance-associated mutations and facilitate downstream target discovery.
We adapted the plaque reduction neutralization test (PRNT) to detect chikungunya virus (CHIKV) neutralizing antibodies in liver homogenates instead of serum. This approach enables serological investigation using tissues collected from animal carcasses as nonhuman primate ones, during arbovirus surveillance, expanding opportunities for CHIKV monitoring and strengthening epidemiological surveillance efforts.
Veterinary medicine and scienceZineb Soubai, Nadia Ziyate, Rim Rais, Hamza Elhrech, Oumayma Aguerd, Abdelhakim Bouyahya, Taoufiq Benali, Mohamed Akhazzane, Hiba Mahir, Siham Fellahi, Waleed …
BACKGROUND: Campylobacter is one of the leading causes of gastroenteritis, and the increasing prevalence of antimicrobial resistance calls for new treatment alternatives, such as essential oils (EOs). OBJECTIVES: This study aims to investigate the prevalence of antimicrobial-resistant Campylobacter strains recovered from poultry in Morocco, and to evaluate the antibacterial effects of the commercial EOs from cinnamon (CIEO), clove (CLEO) and oregano (OREO) alone and in combination with ciprofloxacin on Campylobacter coli and Campylobacter jejuni using in vitro and in silico approaches. METHODS: A total of 643 caecal samples were collected from poultry across four regions of Morocco, and C. coli and C. jejuni strains were isolated and tested for antibiotic susceptibility, following EUCAST guidelines. The antibacterial activity of selected EOs was assessed individually and in combination with antibiotics through broth microdilution and checkerboard assays. Additionally, molecular docking was conducted between the major EOs chemotypes and resistance-associated protein targets (RNApα, CmeB, CmeR, DHPS, Erm(B), GyrA, GyrB, PBP2 and TetR). RESULTS: The prevalence of Campylobacter spp. among the collected samples was 15.52% (100/643). Many isolates exhibited strong resistance to nalidixic acid (90%), ciprofloxacin (85%) and tetracycline (78%). The studied EOs demonstrated a high antibacterial activity, with the MIC value ranging from 0.0312% to 0.0625% (v/v). A high level of synergism was achieved with OREO, while the interactions with CIEO and CLEO were considered antagonistic. In silico molecular docking revealed that caryophyllene exhibited strong binding affinities towards key resistance-associated proteins, including GyrB (binding energy: -7.4 kcal/mol), CmeB (-7.5 kcal/mol) and PBP2 (-6.4 kcal/mol), suggesting potential interference with DNA gyrase activity, multidrug efflux and cell wall synthesis, respectively. CONCLUSIONS: These findings suggest that selected EOs, particularly OREO, warrant further investigation as potential adjuncts to conventional antibiotics against multidrug-resistant Campylobacter.
Journal of peptide science : an official publication of the European Peptide SocietyMei Tao, Runze Huang, Fangfang Hu, Xuejun Zhao, Nan Cen, Cenying Liu, Wei Li, Zhenhua Luo
Candida parapsilosis (C. parapsilosis) has caused more infections in recent years, and resistance to conventional antifungal agents is also rising. This study evaluated the antifungal potential of a newly designed MAF-1A-derived peptide against C. parapsilosis. Four derivatives were chemically synthesized and structurally characterized. Our hybrid method included the use of computational tools for hydrophobic optimization, amphipathic α-helix stabilization, and peptide-membrane interaction prediction together with experimental validations. We synthesized four designed derivatives (8KGF7, 9GIF6, 10KLF6, and 10GIF6) by Fmoc-based solid-phase chemistry. Its antifungal activity against C. parapsilosis was assessed using the broth microdilution assay. Trypsin stability was evaluated by incubating 10KLF6 with trypsin followed by measurement of residual antifungal activity. Hemolytic safety was assessed using human red blood cells via hemolytic activity assays. The results demonstrated that 10KLF6 exhibited up to 15-fold higher antifungal activity against C. parapsilosis compared to MAF-1A. Additionally, 10KLF6 retained more than 20% of its antifungal activity after 100 min of trypsin digestion and showed low hemolytic toxicity. Collectively, 10KLF6 possesses potent antifungal activity as well as favorable stability and safety profiles against C. parapsilosis, which supports its further exploration as a promising candidate for the development of potent and stable antifungal peptide agents.
Journal of peptide science : an official publication of the European Peptide SocietyHoan Tran Khanh, Trang-Hoang Ngoc-Ngan, Hai Bui Thi Phuong, Anh Luong The, Nguyen Ba Nhiet, Le Minh Bui, Huy Luong Xuan, Duc Nguyen Minh
The global rise of multidrug-resistant (MDR) pathogens has created an urgent need for alternative antimicrobial agents. In this study, we evaluated the antibacterial activity of LTX-315, a synthetic cationic 9-mer peptide originally developed as an oncolytic agent, against six ATCC reference strains and six clinical isolates, including methicillin-resistant Staphylococcus aureus (MRSA), extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae, MDR Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Burkholderia cepacia. LTX-315 exhibited broad-spectrum antibacterial activity, with MICs of 8-32 μM against reference strains and as low as 4 μM against several MDR clinical isolates, whereas chloramphenicol and kanamycin were ineffective. Double-disk diffusion assays indicated synergistic interactions with aminoglycosides, chloramphenicol, and vancomycin, and checkerboard assays confirmed synergy-to-additivity interactions with gentamicin and vancomycin against S. aureus (FICI 0.38-0.75), yielding fourfold to 16-fold reductions in antibiotic dose. These findings demonstrate that LTX-315 retains potent activity against clinically relevant MDR pathogens and may serve as both a standalone antimicrobial peptide and a potential antibiotic adjuvant.
Reviews in medical virologyBlanca Taboada, Selene Zárate, Lorena Díaz-González, Juan Manuel Hurtado
Dengue remains a major public health concern in Mexico, with incidence and clinical burden increasing substantially in recent years. Using national surveillance data from 2020 to 2024, this study provides a comprehensive overview of the changing epidemiological landscape, with an emphasis on geographic distribution, temporal trends, serotype dynamics, and disease severity. Over this period, Mexico saw an almost fivefold increase in dengue cases, reaching over 123,000 confirmed infections and 426 deaths in 2024, the highest annual number recorded. Seasonal patterns shifted, with sustained transmission extending into unusual winter months from late 2023. Serotyping data revealed a shift in predominant virus serotypes, with DENV2 predominating until 2022, DENV1 peaking in 2021, and DENV3 rising sharply from 2022 onward, becoming dominant in 2023 and almost exclusive by 2024. This shift coincided with a sustained rise in dengue with warning signs (from 16.9% in 2020 to > 42% in 2023-2024) and higher hospitalisation rates, while severe dengue remained below 5%. Although DENV3 dominated nationally, DENV1 and DENV2 persisted in specific states, and DENV4 circulated sporadically, particularly in southern and northern regions. Several traditionally low-prevalence states experienced atypical outbreaks, underscoring the potential for sudden epidemic expansion. Climatic analysis showed a consistent positive association between temperature and DENV3 prevalence, with precipitation effects varying by year. Odds ratio analysis suggested serotype-specific differences in severity across years. These findings underscore the heterogeneous and dynamic nature of dengue transmission in Mexico and highlight the need for regional surveillance, prevention, and clinical preparedness strategies.
Pakistan journal of pharmaceutical sciencesFakhra Batool, Muhammad Wajid, Hayat Ali Alzahrani, Tahir Ali Chohan, Ahd A Mansour, Mohd Imran, Abida Khan, Darakhshan Masroor
BACKGROUND: Plant-derived bioactive compounds have significant pharmaceutical applications. OBJECTIVES: This study focused on the methanol extract of Astragalus tephrosoides (A. tephrosoides) to evaluate its phytochemical composition, biological activities and in-silico properties. METHOD: Phytochemical screening, GC-MS profiling, antioxidant (DPPH), antimicrobial and phytotoxic assays were performed. Molecular docking against bacterial proteins (DNA gyrase, lanosterol-14-α-demethylase, PBP2A and chitin synthase) and density functional theory (DFT) analyses were conducted for the bioactive compounds identified by GC-MS. RESULTS: Phytochemical analysis confirmed the presence of alkaloids, steroids, terpenoids, saponins, phenolics, volatile oils and coumarins in the extract. GC-MS analysis revealed the presence of 17 bioactive compounds. The extract exhibited notable antioxidant activity (IC50 = 0.66 mg/mL), comparable to that of ascorbic acid (IC50 = 0.58 mg/mL). Antibacterial effects were observed with net absorbance values of 0.860 (E. coli, P. aeruginosa), 0.770 (B. subtilis), 0.656 (S. aureus) and 0.550 (S. typhi) and MICs of 1.0-0.12 µg/mL. The extract exhibited antifungal activity comparable to that of miconazole and moderate phytotoxicity (66.6%) at 1000 μg/mL. Docking revealed moderate binding affinities, while DFT analysis revealed significant HOMO/LUMO gaps, indicating their thermodynamic stability. CONCLUSION: A. tephrosoides contains bioactive compounds with promising antioxidant, antimicrobial and moderate phytotoxic activities, as supported by computational studies.
Sexual healthDaniela Eassey, Jason J Ong, Ken Koh, David Nolan, David Baker, Mark O'Reilly, Beng Eu, Jillian Lau, Fiona Bisshop, Jessica Kent, Andrés Doblado-Maldonado, Dan…
BACKGROUND: Although clinical trials and real-world studies demonstrate the efficacy and tolerability of dolutegravir and lamivudine (DTG + 3TC), Australian real-world evidence remains limited despite differences in access to health care and prescribing context. Here, we present the motivations for switching to DTG/3TC (a fixed-dose, single-tablet regimen) and treatment outcomes. METHODS: We performed a retrospective, observational analysis of individuals with HIV with an undetectable viral load (VL; <50 copies/mL) who switched to DTG/3TC during a 24-month inclusion window from 1 December 2020 to 1 December 2022, in nine Australian clinics. Healthcare providers completed an electronic survey using baseline demographics of people with HIV and other clinical information gathered from participant medical records. Data were collected after December 2023 to allow a 12-month follow-up. Primary endpoints were baseline demographics, clinical characteristics, and motivations for switching to DTG/3TC. Secondary endpoints included virologic outcomes and rates and reasons for DTG/3TC discontinuation. RESULTS: Overall, 276 individuals with HIV who switched to DTG/3TC were included. Most were male (97%) and White (76%), with a median (interquartile range) age of 54 (45-61) years. The most common antiretroviral therapy before DTG/3TC switch was abacavir/dolutegravir/lamivudine (54%). The most common reason for switching to DTG/3TC was clinician preference for two-drug regimen (43%). Most individuals (98%) maintained virologic suppression (VL <50 copies/mL), and none experienced virologic failure. Through Month 12, one (<1%) individual discontinued DTG/3TC. CONCLUSIONS: These real-world data support the use of DTG/3TC as a viable treatment strategy in this Australian population of individuals with HIV.
Sexual healthRebecca Houghton, Anik Ray, Alison Mahony, Lisa McCann, Brent Mackie, Joubert van der Merwe, Rick Varma, Arthur Wong
BACKGROUND: Overseas-born gay, bisexual, and other men who have sex with men (GBMSM) experience higher HIV notification rates compared with Australian-born GBMSM. This disparity is perpetuated by delayed access to HIV testing and biomedical prevention driven by a range of factors including Medicare eligibility, language and cultural barriers, and unfamiliarity with the health system. Community-based services offer an alternative pathway to HIV testing that may better serve migrant GBMSM facing challenges accessing mainstream healthcare settings. METHODS: We conducted a cross-sectional study comparing settings of first HIV test within our service, either peer-led community-based testing or traditional clinic-based testing for overseas-born GBMSM who arrived in Australia between January 2022 and January 2024. Participants were grouped by time of arrival to first HIV test (either less than 6 months or more than 6 months). Comparative analysis and multivariable logistic regression were used to identify characteristics associated with attendance at the community-based setting. RESULTS: A total of 1204 GBMSM were included with 424 (35.2%) attending community-based setting for their first HIV test after arrival. Those who attended community-based testing were more likely to have arrived in Australia more than 6 months ago (66.8% vs 26.9%) and never previously tested for HIV (37.5% vs 17.2%). These factors were also associated with higher likelihood of community-based testing in the multivariable model (aOR 5.27, 95% CI 3.98-6.97 and aOR 2.13, 95% CI 1.57-2.91 respectively). CONCLUSION: Community-based, peer-led HIV testing services play an important role in engaging overseas-born GBMSM who may be under-served by traditional clinic-based models, particularly those with longer residence in Australia and no prior HIV testing.
Sexual healthRongyan Li, Feifei Li, Yi Zhou, Ye Zhang, Dan Wu, Xinyuan Zhang, Fengshi Jing, Jie Fan, Xi He, Joseph D Tucker, Weiming Tang
BACKGROUND: Secondary distribution of HIV self-testing (HIVST) can extend testing through social and sexual networks of men who have sex with men, but the peer subgroups reached by this approach remain insufficiently understood. We aimed to identify latent profiles of peers who returned HIVST results, and assess index-peer factors associated with peer class membership. METHODS: We conducted a secondary analysis of pooled HIVST secondary distribution studies and program phases among men who have sex with men in Zhuhai, China, from October 2019 to December 2023. Peers received HIVST kits from indexes and returned valid results online. Latent class analysis used four indicators, with assignment by highest posterior probability: previous HIV testing, two or more recent male partners, recent condomless anal intercourse (CAI) and disclosure of sexual orientation or same-sex behavior. Exploratory multinomial logistic regression with cluster-robust standard errors accounting for clustering by index examined index-peer associations. RESULTS: Among 611 unique indexes, 259 successfully distributed kits to 584 peers. Three peer classes were identified: recent-CAI, lower-disclosure peers (17.0%), no-recent-CAI, lower-testing peers (64.2%) and previously tested, higher-disclosure peers (18.8%). Compared with the previously tested, higher-disclosure class, index disclosure was inversely associated with membership in the other two classes. Index CAI was positively associated with recent-CAI, lower-disclosure membership and inversely associated with no-recent-CAI, lower-testing membership. CONCLUSIONS: Peers who returned HIVST results comprised groups with distinct testing histories, recent sexual behaviors and disclosure patterns. These findings characterize heterogeneity among result-returning peers and may inform future evaluation of differentiated follow-up strategies.
ChemMedChemLe Zhai, Jiazhu Chigan, Yao Zhao, Hua-Ping Xie, Yi-Qiang Wu, Sheng Zhang, Ke-Wu Yang
Due to the lack of metallo-β-lactamase (MβL) inhibitors for clinical purposes, the antibiotic resistance mediated by MβL poses a clear threat to public health. In this work, twelve symmetrical o-methylpyridine substituted tertiary amines P1-P6 and their Dy(III) complexes D1-D6 were synthesized, characterized, and confirmed. Nine compounds displayed inhibitory activity against MβLs from subclasses B1, B2, and B3. Encouragingly, D2 and D6 exhibited dual inhibitory activity targeting both imipenem-hydrolyzing MβL S (ImiS) and MβL L1 (L1). Mechanistic studies uncovered P2 exerted a competitive inhibition mode on New Delhi MβL-1 (NDM-1), while D6 showed a noncompetitive inhibition mode against ImiS and L1. Microbiological assays indicated the obtained inhibitors could restore the antibacterial activity of cefazolin and meropenem against six bacterial strains, including resistant ones, while P4, D1, and D6 alone exhibited bactericidal effect. Cytotoxicity assays showed the tested inhibitors performed a reasonable biological toxicity against L929 cells. Docking simulation reveals a molecular-level rationale for the dual-target inhibitory activity of D2, indicating the introduction of Dy(III) can regulate the inhibitory activity against both ImiS and L1. In short, the study demonstrate o-methylpyridine substituted tertiary amines and their Dy(III) complexes are promising scaffolds for the development of MβLs inhibitors and antibacterial agents.
ChemMedChemRadu-George Bulai, Martina Parolini, Kara Anne Cummins, Sam Purnal, Eveline Lescrinier, Jef Rozenski, Joleen Masschelein, Peter Verwilst
The continuous rise and spread of antimicrobial resistance poses a severe threat to global health and creates an urgent need for the development of new classes of antibiotics. One promising target is FabV, an enoyl-acyl carrier protein reductase that is crucial for the survival of certain pathogenic Gram-negative species such as the opportunistic nosocomial bacterium P. aeruginosa. Previously, our group investigated the structure-activity relationship of diaryl ether-based inhibitors of FabV and uncovered p-N,N-dimethylsulfamoyl phenyl substitution as a privileged motif, exemplified by compounds 1a (IC50 = 0.25 μM) and 1e (IC50 = 0.19 μM). In the present study, we explored skeletal modifications of the diaryl ether scaffold with the aim of improving aqueous solubility of the compounds while maintaining potent activity. Our efforts uncovered compound 6k (IC50 = 0.53 μM), in which the alkyl chain is replaced by a 3-cyanocyclobutyl group and which shows a fivefold increase in kinetic solubility compared to the parent compound 1a. Similarly, benzene-to-pyridine replacement in the A-ring led to significant solubility gains with minimal loss of potency. These results showcase an effective strategy for optimizing solubility in inhibitors targeting enzymes with highly lipophilic natural substrates.
Zhonghua jie he he hu xi za zhi = Zhonghua jiehe he huxi zazhi = Chinese journal of tuberculosis and respiratory diseasesC Z Zhang, S S Lyu, X D Han, H Wang, Y Sun, X D Liu
Objective: To investigate the molecular epidemiological characteristics, antimicrobial resistance mechanisms, and clinical risk factors for infection with carbapenem-resistant Klebsiella pneumoniae (CRKP), and to examine the relationship between antimicrobial resistance and virulence. Methods: A total of 528 Klebsiella pneumoniae (KP) isolates and corresponding clinical data were collected from hospitalized patients at Qingdao Municipal Hospital between November 2016 and October 2023. Based on antimicrobial susceptibility testing results, 80 patients with CRKP infection were assigned to the CRKP group. Patients with carbapenem-susceptible K. pneumoniae (CSKP) isolates recovered within 7 days before or after the CRKP isolation date were selected as matched controls for each patient in the CRKP group. If insufficient eligible controls were available, the matching window was expanded to 30 days before or after the isolation date. Ultimately, 160 matched patients were included in the CSKP group. Whole-genome sequencing was performed on all included isolates to systematically characterize their molecular epidemiological features and resistance and virulence gene profiles. A multivariable logistic regression model was used to identify independent risk factors for CRKP infection. Spearman rank correlation analysis was performed to assess the correlation between resistance and virulence gene scores. Results: Multivariable logistic regression analysis showed that a history of antimicrobial agent use in the 30 days before infection (OR=4.411, 95%CI: 1.749-11.128; Wald χ²=9.882; P<0.01) and invasive abdominal procedures in 30 days before infection (OR=6.846, 95%CI: 3.141-14.922; Wald χ²=23.411; P<0.01) were independent risk factors for CRKP infection. ST11 was the predominant sequence type among CRKP isolates (80.0%), and the blaKPC-2 gene was the predominant resistance determinant (88.8%). The predominant CRKP clone, ST11, underwent a serotype transition from ST11-K25/O5 to ST11-K64/O2a. At the overall population level, the antimicrobial resistance gene score was negatively correlated with the virulence gene score (Spearman ρ=-0.321, P<0.001). However, subgroup analysis showed no significant correlation between the two scores within the same ST11 clone (Spearman ρ=0.020, P=0.872). These findings suggest that the overall negative correlation was a population-level artifact caused by inherent phenotypic differences among distinct clonal groups, rather than an evolutionary trade-off in which the acquisition of antimicrobial resistance within the same clone resulted in reduced virulence. The intensive care unit (ICU) was the central hub for nosocomial clonal transmission of CRKP. The predominant clone may have disseminated from the ICU to general wards through patient transfers, healthcare workers, or medical equipment, thereby forming interdepartmental transmission chains. Conclusions: A history of antimicrobial agent use and invasive abdominal procedures in the 30 days before CRKP infection were independent risk factors for CRKP infection. CRKP isolates frequently carried plasmids coharboring multiple antimicrobial resistance determinants and exhibited extensive antimicrobial resistance.
Sheeppox virus (SPPV) is a highly host-restricted capripoxvirus, and its biological behavior in heterologous cells remains poorly defined. Although heterologous cell lines such as Vero cells are widely used for SPPV propagation and vaccine production, the effects of prolonged adaptation to these systems on viral replication and release dynamics have not been systematically examined. In this study, the SP3-Nur strain of SPPV (PV167793.1) was serially passaged 45 times in Vero cells. The parent virus (P0) was assessed in lamb testicular (LT) cells, while Vero-adapted passage levels (P15, P30, and P45) were evaluated in both Vero and LT cells to examine how passage levels affect viral replication and extracellular virus release. Replication kinetics were quantified at 0, 2, 4, and 6 days post-infection (DPI) using plaque-forming unit (PFU) assays and quantitative PCR (qPCR). In Vero cells, while virus production was increased from 2.54 log10 PFU/ml (P15) to 5.73 log10 PFU/ml (P30) at 6 DPI, accompanied by an increase in mean plaque area from 7,266 μm2 to 42,309 μm2, respectively, while at P45, reduced extracellular release (4.26 log10 PFU/ml at 6 DPI) and smaller plaques (5,319 μm2) were observed. Plaque size differences between P15 and P30 and between P30 and P45 were statistically significant (p < 0.01). The qPCR data in Vero cells revealed that viral genome copies in supernatant declined from 5.73 to 4.72 log10 copies/ml, whereas cell-associated viral genome copies remained stable at 5.45 to 5.13 log₁₀ copies/ml in passages P30 and P45, respectively, at 6 DPI, indicating impaired virus release rather than loss of intracellular replication. A similar trend was observed in LT cells. This study showed biphasic phenotypic adaptation in heterologous cells characterized by altered replication dynamics and impaired viral release. This study provides important insights into SPPV biology in Vero cells, having implications in its virulence, vaccine production, and the development of a suitable animal model.
JMIR research protocolsMarjan Javanbakht, Lisa B Hightow-Weidman, Sean D Young, Nora E Rosenberg, Kathryn E Muessig, Sarah Schoetz Dean, Aimee Rochelle, Warren Scott Comulada, Pamina…
BACKGROUND: Despite advances in HIV treatment, many people living with HIV fail to achieve or maintain viral suppression. Individuals who experience interruptions in HIV care, adherence challenges, and viral nonsuppression are often underrepresented in clinic-based cohort studies. Virtual cohort methodologies offer opportunities to recruit, engage, and retain these populations while integrating longitudinal behavioral, biomarker, and digital engagement data needed to characterize dynamic changes in HIV care engagement and viral suppression. OBJECTIVE: The Exploring, Predicting, and Intervening on Long-Term Viral Suppression Electronically (EPI-LoVE) study was developed to establish a national virtual cohort of people living with HIV with current viral nonsuppression or at an elevated risk of future viral nonsuppression. We describe the study design, recruitment, data collection methods, retention strategies, and baseline cohort characteristics. METHODS: Participants were recruited through digital advertising, a national HIV care provider network, and referrals from previous research studies. Eligible participants were adults living with HIV who were currently virally unsuppressed or considered at elevated risk of future viral nonsuppression based on a history of detectable HIV viral load, recent missed HIV care appointments, or a qualifying mental health diagnosis. Following online screening, participant eligibility was confirmed through telephone verification and supporting documentation. Participants completed online surveys every 3 months and HIV viral load assessments every 6 months over 24 months. Data were collected through a study-specific mobile health platform integrating longitudinal behavioral surveys, remote biomarker collection, app engagement metrics, and frequent "Speak Up" microassessments of time-varying behavioral and psychosocial factors associated with HIV care engagement and viral suppression. RESULTS: The study was funded in May 2023. Participant recruitment occurred between February 2024 and June 2025, and longitudinal data collection is ongoing, with study findings anticipated in spring 2028. A total of 4789 individuals expressed interest in the study, 2739 completed screening, and 2262 met eligibility criteria. A total of 1109 consented, and 1051 enrolled. Among the 2262 eligible participants, 63.4% (n=1435) qualified based on a detectable viral load during the previous 12 months, whereas the remainder qualified based on recent missed HIV care appointments or a qualifying mental health diagnosis. Among the 1051 participants, the cohort was predominantly Black or African American (n=677, 64.4%), with a median age of 36 (IQR 31-43) years; 70.8% (n=744) reported recent substance use, and 63.7% (n=669) screened positive for moderate-to-severe depressive symptoms. Baseline HIV viral load data were obtained from 863 (82.1%) participants. CONCLUSIONS: EPI-LoVE demonstrates the feasibility of recruiting, verifying, and engaging a geographically diverse national virtual cohort of people living with HIV at elevated risk of viral nonsuppression. By integrating longitudinal behavioral assessments, biomarker collection, and digital engagement data within a single platform, EPI-LoVE provides a scalable framework for studying the dynamic determinants of HIV care engagement and informing future personalized digital interventions.
Annals of medicineSuiyuan Lin, Jingming Yao, Qingxian Fu, Yali Wu, Shiqu Deng, Bin Wang
BACKGROUND: Urinary tract infections (UTIs) are common in pediatrics. This study aimed to develop and validate a machine learning model for predicting urine culture positivity in children with suspected UTIs and to characterize regional pathogen and antimicrobial resistance profiles. METHODS: Clinical and laboratory data were collected from children with suspected UTIs. Variables were selected via univariate analysis, LASSO regularization, and recursive feature elimination. Machine learning models were constructed using selected variables, followed by validation and interpretability analysis. A web application was developed to assist in the early prediction of urine culture positivity. RESULTS: Infants aged 0-12 months accounted for 61.10% of enrolled children. The overall positive rate of urine culture was 42.17%, higher in boys. Gram-negative bacteria dominated the pathogen spectrum, with the two major strains exhibiting high rates of resistance to first- to third-generation cephalosporins and susceptibility to β-lactam/β-lactamase inhibitor combinations and carbapenems. Seven variables were used to construct seven machine learning models, among which the random forest (RF) model yielded the best performance, with an AUC of 0.814 (95% CI: 0.742-0.875), and sensitivity of 0.770 (95% CI: 0.662-0.865). A web-based calculator was established for individualized prediction. CONCLUSIONS: We developed an RF-based online tool using seven routine laboratory parameters, demonstrating favorable predictive performance for pediatric urine culture positivity. Furthermore, our mapped local pathogen and antimicrobial resistance profiles help clinicians initiate rational empirical antibiotic therapy.
World journal of microbiology & biotechnologyAyodele Timilehin Adesoji, Vittoria Mattioni Marchetti, Claudia Cortimiglia, Francesca Piscopiello, Ilaria Petrizzi, Aurora Piazza, Emmanuel Dayo Alabi, Pier S…
BACKGROUND: Extended-spectrum β-lactamases (ESBL) and AmpC β-lactamase-producing Enterobacter species are pathogens of public health concern. In this study, we investigated the resistome, virulome, and plasmidome of circulating Enterobacter hormaechei isolated from humans, animals, and ready-to-eat (RTE) food in Dutsin-Ma, northwest Nigeria. MATERIALS AND METHODS: Enterobacter species were isolated from human, and RTE food samples using MacConkey and CHROM agar. Species identification and antibiotics susceptibility testing (AST) were performed using Microscan autoSCAN-4 and MALDI-TOF MS, respectively. Whole-genome sequencing (WGS) and bioinformatics analysis were performed to validate the species identities. WGS also determined sequence types (STs), resistome, plasmidome, and virulome using PubMLST, ResFinder, PlasmidFinder, and Virulence Factors Database (VFDB) via ABRicate, respectively. RESULTS: E. hormaechei subs. xiangfangensis was the dominant species isolated from humans (6/8, 75%) and animals (1/8, 12.5%), while E. hormaechei subs. oharae was exclusively found in RTE food (1/8, 12.5%). The most frequent sequence type (ST) was ST813, found solely in humans, along with ST511, ST454, and the emerging high-risk ST171. ST68 was identified in food, and novel ST (ST3186) were identified in animal and human samples, respectively. All isolates exhibited 100% resistance to ampicillin and amoxicillin-clavulanic acid, with 62.5% resistant to each of aztreonam, ceftazidime, ciprofloxacin, gentamicin, tobramycin and cefotaxime, and 25% to colistin. E. hormaechei (62.5%) isolates carried the blaCTX-M-15 gene, encoding ESBL-production, along with AmpC genes such as blaCMH-3, and also harbored resistance genes for aminoglycosides, fluoroquinolones, sulfonamides, tetracyclines, and trimethoprim. The IncHI2 (50%) was identified as the dominant plasmid type followed by Col (pHAD28) (25%), IncFIB (25%), and IncFII (11.1%). The most (62.5%) prevalence virulence gene among the isolates is ompA encoding adhesion while each of entA, entB and fepD encoding iron acquisition were least (12.5%) occurred. CONCLUSION: This study provides the first genomic insight into Enterobacter species in northwest Nigeria, highlighting their role in the spread of antimicrobial resistance. E. hormaechei subsp. xiangfangensis was identified as the main reservoir of ESBL and AmpC β-lactamases in this study. The discovery of novel STs with diverse virulence factors and plasmid replicon indicates potential horizontal gene transfer across different ecological niches. Our findings underscore the need for robust surveillance and control measures to combat the spread of MDR Enterobacter species across human, animal, and food niches.
BMJ openKlaske J Vliegenthart-Jongbloed, Oana Mihaela Inescu, Filip Fijołek, Isabella Pepe Razzolini, Tristan J Barber, José I Bernardino, Silvia Nozza, Christina K Ps…
INTRODUCTION: Across Europe, many people with HIV are diagnosed late despite repeated contact with hospital services for HIV indicator conditions. These conditions flag a possible underlying HIV infection for which HIV testing is recommended. They provide an opportunity to identify people with HIV, yet implementation of indicator condition-based testing remains insufficient in hospital practice. The #aware.hiv Europe study was developed to address this gap by embedding HIV teams into routine care to normalise HIV testing. METHODS AND ANALYSIS: #aware.hiv Europe is a stepped-wedge cluster randomised trial in 30 hospitals across ten European countries. Five clusters of six hospitals each will sequentially transition from control to implementation periods when local HIV teams led by an infectious diseases specialist will be installed. Intervention activities include hospital-wide peer audit and feedback on missed testing opportunities, targeted education, stigma reduction activities and strengthening of linkage to HIV prevention and care. Patients with predefined HIV indicator conditions are identified using International Classification of Diseases, 10th Revision (ICD-10) diagnosis codes, confirmed through manual review.The primary outcome is the change in HIV testing rate among patients with confirmed HIV indicator conditions. Secondary outcomes include HIV case detection, cascades of diagnosis, care and prevention, variation in testing practices, healthcare professional knowledge and stigma, and implementation outcomes. Analyses will use mixed effects regression models accounting for clustering and time within the stepped-wedge design. ETHICS AND DISSEMINATION: The study has ethical approval in all hospitals to use routinely collected clinical data under exemption from informed consent for patient level data. Results will be disseminated through peer reviewed publications, conferences and collaboration with clinical and community partners with the goal to inform HIV testing policies. TRIAL REGISTRATION NUMBER: NCT06900829.
Host defense peptides (HDPs) possess both direct bacteriocidal and immunomodulatory activities. In this study, chicken cathelicidin-1 (cCATH-1) and chicken cathelicidin-3 (cCATH-3) were selected based on their markedly different minimum inhibitory concentrations (MICs) against avian pathogenic Escherichia coli (APEC) strains, with MICs of cCATH-1 being 4- to 16-fold lower than those of cCATH-3. However, following intraperitoneal challenge with APEC strain E058 (4 × 10⁶ CFU/mouse), 80% of cCATH-3 treated mice survived while 20% of cCATH-1 treated mice survived. Flow cytometry (FC) revealed significantly lower peripheral blood Ly6Chigh monocytes but higher peritoneal Ly6Chigh macrophages in cCATH-3-treated mice at 4 and 8 h post-treatment. Peritoneal CCL3 secretion was significantly upregulated in cCATH-3 treated mice at 8 h post-treatment. Upon knockout of macrophages, the protection of cCATH-3 in mice against APEC E058 challenge was markedly abolished. In vitro, peritoneal macrophages treated with cCATH-3 showed stronger bacteriocidal activity than those treated with cCATH-1. Mouse CCL-3 (mCCL3)-specific antibody neutralize the effect of mCCL3 on macrophages chemotaxis in vitro. mCCL3-specific antibody administration resulted in 0% survival in cCATH-3-treated mice versus 60% survival in cCATH-3-treated mice administered with mock antibody, while cCATH-1-treated mice showed 0% survival administered with either mCCL3-specific antibody or mock antibody after APEC E058 strain challenge. In summary, cCATH-3 provides robust protection against APEC infection compared with cCATH-1 in mice, partially through CCL3-mediated macrophage chemotaxis and enhanced bacteriocidal activity in activated macrophages, revealing a novel mechanism of avian cathelicidins and supporting HDPs as alternatives to antibiotics for controlling avian colibacillosis.
The Journal of antimicrobial chemotherapyRomain Palich, Minh P Lê, Antonella Castagna, Clotilde Allavena, Andrea Antinori, Diana Canetti, Juan Tiraboschi, Claudine Duvivier, Esteban Martinez, Pere Dom…
BACKGROUND: We evaluated the efficacy and safety of a once-daily oral two-drug regimen (2DR) combining doravirine and raltegravir in virologically suppressed people with HIV (PWH). METHODS: DORAL is a multicentre, open-label, randomized phase II trial conducted in France, Italy, and Spain. Virologically suppressed adults receiving stable antiretroviral therapy (ART), naïve to doravirine and without raltegravir resistance, were randomized (2:1) either to switch immediately to doravirine (100 mg) plus raltegravir (1200 mg) or to continue baseline ART until week 48 before switching to doravirine plus raltegravir. The primary endpoint was virological failure (VF; confirmed plasma HIV-1-RNA ≥50 copies/mL) at week 48. Secondary endpoints included maintenance of virological suppression through W96, immunological outcomes, safety, and pharmacokinetics in a genital sub-study. RESULTS: Among 114 participants (median age, 57 years; median CD4 count, 700 cells/mm3; median duration of virological suppression, 10 years), one VF occurred by week 48 in the immediate-switch group (1.3%; 95% CI, 0.0-6.9); none occurred in the delayed-switch group. No additional VF was observed through week 96. Immunological parameters remained stable during follow-up. Three adverse events considered related to doravirine and raltegravir led to treatment discontinuation. One grade 3 adverse event (anxiety), considered related to study treatment, was reported. In the genital sub-study, drug concentrations exceeded inhibitory thresholds in most samples, with infrequent detection of low-level seminal HIV-1-RNA. CONCLUSIONS: This proof-of-concept phase II switch trial supports the feasibility of a once-daily doravirine plus raltegravir 2DR in selected PWH, particularly when minimizing drug-drug interactions or avoiding NRTI exposure is a priority.
The Journal of antimicrobial chemotherapySamantha Poloni, Adeline Gagnon, Benoit Valot, Alexandre Meunier, Didier Hocquet, Xavier Bertrand, Adrien Biguenet, Kevin Bouiller
BACKGROUND: This study aimed to describe penicillin G susceptibility, blaZ diversity, and genetic determinants of the cefazolin inoculum effect (CfzIE) among MSSA bloodstream isolates. METHODS: The first three non-duplicate MSSA isolates recovered each month from blood cultures at our French university hospital between January 2010 and December 2023 were selected. Whole-genome sequencing was performed using the Illumina NextSeq platform to characterize blaZ and its genetic context. Susceptibility to penicillin G and the CfzIE were assessed using phenotypic methods. RESULTS: Among 488 MSSA isolates, 373 (76.5%) carried blaZ, of which 119 (31.9%) exhibited CfzIE. Thirty BlaZ allotypes were identified overall; 26 were included in the genetic determinant analysis after exclusion of isolates with multiple or truncated blaZ. In multivariable analysis, BlaZA3 was strongly associated with CfzIE (aOR, 15.72; 95% CI, 3.33-74.24; P < 0.001), as was CC7 (aOR, 5.48; 95% CI, 1.45-20.69; P = 0.011). Among BlaZA3 isolates, CfzIE was mainly observed with complete blaR1. Among 122 isolates with a penicillin G inhibition zone ≥26 mm, seven carried blaZ, including six with sharp zone edges and truncated blaZ sequences. CONCLUSION: CfzIE was mainly associated with a BlaZA3-related genetic background, particularly in the presence of complete blaR1, rather than with blaZ type alone. Approximately one-quarter of MSSA bloodstream isolates were susceptible to penicillin G, and rare phenotype-genotype discrepancies were associated with truncated blaZ sequences.
BMC complementary medicine and therapiesMohamed T Selim, Nashaat N Mahmoud
BACKGROUND: Nerium oleander L. (N. oleander) is a species of shrub or small tree in the Apocynaceae family that has high levels of digitalin linear activity and cardiac glycosides, especially nerine and oleander. The study was researched to assess the in vitro proximate compositions, phytochemical analysis, and antimicrobial activity of the dried stems, leaves, and flowers of N. oleander extracted using various solvents. METHODS: Petroleum ether, ethyl acetate, acetone, methanol, and water extracts of the various solvents were tested for total phenol, flavonoid, and tannin contents using various phytochemical assays, while the antimicrobial activity was evaluated using the agar well diffusion method against six bacterial and fungal strains. RESULTS: The flowers had a higher moisture content (9.40%), whereas the stems had a higher ash content (18.58%) than the leaves and flowers. The flowers also had higher lipid, carbohydrate, and crude protein contents (4.33, 66.16, and 15.29%, respectively). The leaves of the N. oleander produced a maximum yield followed by flowers and stems, respectively. The highest percentage extractive yield was demonstrated by water extracts, which were followed by methanol, acetone, ethyl acetate petroleum ether extracts. The findings of the study showed that alkaloids, flavonoids, tannins, glycosides, steroids, coumarins, quinones, phenols, cardiac glycosides, and terpenoids were found by phytochemical analysis of N. oleander in different parts, while saponins and anthraquinones were completely absent in all parts. On the other hand, anthocyanins are present only in flowers and completely absent in stems and leaves. The greatest levels of phenol content, flavonoids and tannins were found in acetone extract of the flowers (157.12 ± 17.62 mg GAE/g, 187.43 ± 15.91 mg QE/g, and 89.93 ± 18.77 mg TAE/g, respectively), while the petroleum ether extract of stems had the lowest amount (5.45 ± 2.65 mg GAE/g, 6.43 ± 0.29 mg QE/g and 3.45 ± 2.05 mg TAE/g, respectively). Antimicrobial tests revealed the extract's ability to inhibit several Gram-positive bacteria (Staphylococcus aureus ATCC 6538 and Bacillus subtilis ATCC 6633), Gram-negative bacteria (Pseudomonas aeruginosa ATCC 9027, Salmonella Typhimurium ATCC 14028 and Escherichia coli ATCC 11229), and eukaryotic strains such as unicellular fungi (Candida albicans ATCC 10231). CONCLUSION: These results highlight the potential of N. oleander extracts as natural antimicrobials.
Archives of microbiologyAdriano Souza Santos Monteiro, João Ricardo Filho, Vívian Santos Galvão, Vanessa Tibolla Moretto, Ana Beatriz Purificação, Lee Senhorinha Almeida Andrade, Cami…
Klebsiella pneumoniae poses a significant public health threat, but the relationship between community-acquired (CA) infections and environmental isolates remains poorly understood. Therefore, this study aimed to assess whether K. pneumoniae from CA infections share microbiological and molecular features with isolates recovered from an urban river in Salvador, Brazil. A total of 115 isolates, including 65 from CA infections and 50 from river water, were analyzed using antimicrobial susceptibility testing, β-lactamase and virulence gene detection, and multilocus sequence typing (MLST). Resistance rates were similar between groups, including multidrug resistance (24.6% vs. 22.0%; p > 0.05) and carbapenem resistance (4.6% vs. 4.0%; p > 0.05). β-lactamase gene detection was consistent with observed resistance patterns, with blaCTX-M detected in 18.5% of CA isolates and 12.0% of river isolates, while blaKPC-2 (3.1% vs. 2.0%) and blaNDM-1 (1.5% vs. 2.0%) occurred at comparable frequencies. However, hypervirulent K. pneumoniae isolates were identified only among CA infections (4.6%). MLST revealed high clonal diversity, with 88 sequence types (STs), including 14 novel STs, with most STs being singletons. Four STs were shared between groups, including the high-risk lineages ST11 and ST307. These findings indicate that clinically relevant K. pneumoniae lineages, including high-risk, occur beyond healthcare settings, with urban aquatic environments serving as potential environmental reservoirs.
Current microbiologyAline Madeira Marques Saraiva, Amália Raiana Fonseca Lobato, Emanoele Saraiva Pereira, Maria Paula Cruz Schneider, Diego Assis das Graças, Adonney Allan de Oli…
Acinetobacter baumannii is a high-priority pathogen due to its global dissemination and antimicrobial resistance. This work describes the first report of A. baumannii ST-15 isolates belonging to the International Clone 4 group, that exhibited an MDR profile with the co-production of OXA-23 and OXA-58 enzymes. Strains were isolated from an intensive care unit in a hospital of the Brazilian Amazon. Eight strains were recovered from an intensive care unit, showing high clonal stability across diverse clinical sources, including tracheal aspirates, urine, and blood, which underscores the high versatility and systemic dissemination potential of this lineage.The blaOXA-23 and blaOXA-58 genes were detected in all strains. A. baumannii isolates AB_OXA58_IEC07 and AB_OXA58_IEC02 were selected for hybrid genome sequencing and plasmid reconstruction, respectively, as representative samples of the outbreak. The reconstructed mobilome revealed a genome size of 4,110,050 bp, a GC content of 39.2%, and 3846 coding sequences for the reference strain, while four distinct plasmids were fully resolved.The blaOXA-58 gene was detected flanked by ISAba3 and IS6-like elements.pdif modules were identified in another plasmid, together with mobilization and replication genes. The tet39 gene was also identified in a novel plasmid that is likewise mobilizable. VFDB found several genes related to biofilm formation and virulence. The identification of these high-risk genetic determinants within a monoclonal outbreak, involving diverse clinical sites, emphasizes the successful persistence of the ST-15 lineage in the hospital environment. This study describes a rare co-production of oxacillinases, highlighting the importance of constant genomic surveillance in monitoring the emergence of antibiotic-resistant pathogens that could pose a threat on a regional or global scale.
PLoS pathogensIchiro Misumi, Itoe Shiota, Tomoyuki Shiota, Jingyu Zhao, John M Cullen, Stanley M Lemon, Jason K Whitmire
Epidemiological studies show that more men are hospitalized for severe hepatitis A than women. However, the extent to which social and behavioral differences versus intrinsic biological mechanisms account for this disparity in disease incidence remains unclear. To better define how sex influences infection outcome, we challenged male and female Ifnar1-/- mice with HAV and quantified virus replication and liver injury. Male mice replicated and shed more virus than female mice, and demonstrated both accelerated and more severe liver injury, as evidenced by increased serum alanine aminotransferase (ALT) activity, greater immune cell infiltration in the liver, and higher intrahepatic expression of chemokines and interferon-stimulated genes. Sex differences were apparent in young and older adult mice, indicating that age did not significantly influence these effects. Ovariectomized female mice had higher intrahepatic viral loads, greater serum ALT elevation, and more severe pathology, compared to surgical controls. Conversely, ovariectomized mice given estradiol implants showed reduced viral loads and ALT activity, indicating that estrogen exerts a protective effect. Female Ifnar1-/- mice lacking hepatocellular expression of estrogen receptor were phenotypically similar to those expressing Esr1, indicating that direct ESR1 signaling in hepatocytes is not essential for protection against pathogenesis. Collectively, these findings indicate that sex influences the pathogenesis and outcome of HAV infection and that female sex hormones protect against severe liver injury.
The Journal of antimicrobial chemotherapyXia Sun, Wenyan Lu, Haoyu Ge, Yuling Qin, Dailin Yuan, Xu'ai Lin, Yuefeng Rao, Stijn van der Veen
OBJECTIVES: The global emergence of ceftriaxone-resistant Neisseria gonorrhoeae threatens current first-line treatment and necessitates alternative therapeutic options. Tigecycline, a last-line glycylcycline antibiotic approved for complicated infections, was evaluated for repurposing against N. gonorrhoeae through in vitro and in vivo activity studies. METHODS: Antimicrobial susceptibility testing was performed for 616 clinical isolates (2011-2024) from Hangzhou, China, and bactericidal activity was determined in time-kill assays. A mouse vaginal tract infection model was used to evaluate in vivo tigecycline activity. Chequerboard assays were performed to assess interactions between tigecycline and ceftriaxone. RESULTS: Tigecycline demonstrated potent activity (MIC50: 0.125 mg/L; MIC90: 0.25 mg/L), including against tetracycline-resistant (tetracycline MIC50: 2 mg/L; MIC90: 64 mg/L) and high-level ceftriaxone-resistant (ceftriaxone MIC50: 0.03 mg/L; MIC90: 0.125 mg/L) strains. Time-kill assays (2 strains) revealed concentration-dependent bactericidal activity, with 4×MIC achieving ≥105-fold killing. In the mouse vaginal tract infection model, high-dose tigecycline (25 mg/kg) significantly enhanced bacterial clearance, eliminating infection within 3-6 days compared to 8-12 days in vehicle controls, and substantially reduced bacterial burden. Chequerboard assays against 21 isolates (10 ceftriaxone-susceptible, 11 ceftriaxone-resistant) demonstrated indifferent interactions between tigecycline and ceftriaxone (fractional inhibitory concentration indices 1.03-2.56), indicating no antagonism. CONCLUSIONS: These findings establish that tigecycline exhibits potent, rapid bactericidal activity against N. gonorrhoeae, including against high-level ceftriaxone-resistant strains, with robust in vivo efficacy. They support further investigation of tigecycline as a potential repurposing candidate for multidrug-resistant gonorrhoea, although its clinical role remains to be defined.
PloS oneAna Tsutsunava, Nestani Tukvadze, Zaza Avaliani, Galo A Goig, Levan Jugheli, Sebastien Gagneux, Russell R Kempker, Nino Maghradze
Emerging resistance to delamanid (DLM) and pretomanid (Pa) threatens drug-resistant tuberculosis (DR-TB) treatment outcomes. The lack of rapid molecular diagnostics, due to limited knowledge of resistance-conferring mutations, along with standardization challenges for critical concentrations (CC) in phenotypic drug susceptibility testing (pDST) hinder resistance detection. Here, we explore the correlation between minimum inhibitory concentrations (MICs) and genomic variants of DLM and Pa resistance in Mycobacterium tuberculosis (Mtb). Seventeen Mtb isolates phenotypically resistant to DLM detected through routine pDST using the Mycobacterial Growth Indicator Tube (MGIT) system from 2021-2024 were selected from the National Reference Laboratory biobank in Tbilisi, Georgia. DLM/Pa MICs were determined using the Resazurin Microtiter Assay (REMA) with a DLM concentration range of 0.008-2 µg/ml, and a broader range of 0.125-32 µg/ml was utilized for Pa. To identify mutations associated with DLM/Pa resistance, isolates were subjected to whole genome sequencing (WGS). Of the seventeen Mtb isolates phenotypically resistant to DLM, eight (47%) manifested a MIC value below the DLM CC (0.06 µg/ml), with only one carrying a mutation in a gene associated with DLM/Pa resistance (fbiC). Among the remaining nine isolates with MICs above the CC, the premature stop codon mutation W88* in ddn gene predominated, present in five cases and associated with elevated DLM and Pa MICs (0.125-2 µg/ml and 2-8 µg/ml, respectively). Three isolates (19%) harbored novel, previously undescribed genetic variants, all within genes previously linked to resistance DLM/Pa (G35fs in ddn, Q317R and Q279H in fgd1, and C565W in fbiC). Our results support DLM and Pa resistance association with W88* and W678G mutations in ddn and fbiC genes respectively, currently graded as "uncertain significance" in the WHO catalogue of Mtb drug resistance-conferring mutations. Additionally, we observed previously undescribed mutations exclusively within genes linked to DLM/Pa susceptibility, prompting further investigation of their role in resistance.
PLoS neglected tropical diseasesStuart D Blacksell, Khanh Kim Le, Paola De Benedictis, Morgane Gourlaouen
Lyssaviruses cause rabies in mammals, a viral encephalomyelitis that is almost always fatal once clinical symptoms develop and can be transmitted to humans. Given this high fatality rate, strict biosafety measures are essential during laboratory handling of potentially infectious materials. This review critically assesses current biosafety practices in rabies diagnostic and research laboratories by examining principal risk factors, transmission pathways, and the effectiveness of existing preventive measures. We conducted a comprehensive review of the literature on biosafety risks associated with handling rabies virus and related lyssaviruses, including the use of personal protective equipment, sample handling procedures, and chemical and physical methods for virus inactivation. Particular attention was given to documented cases of laboratory-acquired rabies and their implications for laboratory safety. Although laboratory-acquired infections remain rare, their occurrence highlights important gaps in current biosafety practices. The effectiveness of disinfection and inactivation methods varies widely, especially in resource-limited settings, and key diagnostic procedures, such as the direct fluorescent antibody test, require validation under biosafety constraints. Significant knowledge gaps remain regarding infectious dose, the effectiveness of decontamination methods, and diagnostic standardisation. Strengthening evidence-based biosafety protocols and validating inactivation methods are essential to ensure safer rabies diagnostics and to support effective rabies control, particularly in low-resource settings.
Brazilian journal of microbiology : [publication of the Brazilian Society for Microbiology]Mayara S Tavares, Patrícia G Paulino, Daniele S Juliano, Roberto Laureano-Melo, Huarrisson A Santos, Rosa H Luchese
This research aimed to access the incidence of Listeria sp. and L. monocytogenes in frozen ready-to-eat (RTE) fish products immediately after purchasing, and after refrigerated storage. Out of 72 kani-kama samples, 17 (23,6%) and 16 out of 48 samples (33,3%) of smoked salmon samples, tested positive for Listeria sp. by the traditional culture method, whereas 45,8% and 56.2% were positive by PCR, for kani-kama and smoked salmon samples, respectively. Applying qPCR, 21 samples of kani-kama out of 72 (29.2%) and 23 out of 48 samples of smoked and sliced salmon (47.9%) were positive for L. monocytogenes. Quantification was performed by culture to account only for viable cells, and confirmation was carried out via PCR and qPCR for Listeria sp. and L. monocytogenes, respectively. The average counts of L. monocytogenes did not exceed 2 Log CFU/g, both in kani-kama or smoked salmon. However, 15.3% of individual kani-kama samples and 14,6% of smoked salmon samples showed counts of up to 3.2 Log CFU/g, some even immediately after purchasing. Higher number of positive samples was observed during refrigerated storage compared to the number of samples analysed immediately after purchasing, indicating that refrigeration for a long time can pose an additional health threat. Unlike kani-kama, which is a heat-processed product, a higher background microbiota count was detected in smoked salmon. However, this background microbiota apparently did not influence the growth of Listeria spp.
HIV research & clinical practiceJesal Gohil, Marcelino Molina, Ali Ahmed, Simon Collins, Ming Jie Lee, Tamara Elliott, Gary Whitlock, Marta Boffito, Piyumika Godakandaarachi, Julie Fox, Amand…
INTRODUCTION: Evaluation of the efficacy of immunotherapeutic strategies aimed at achieving durable antiretroviral treatment (ART)-free control within a clinical trial relies on analytical treatment interruptions (ATIs). ATIs involve stopping ART to assess post-intervention efficacy with frequent viral load monitoring. These trials rely on participant commitment but also involve uncertainty about the timing and experience of viral rebound, transmission risk, and impacts on personal and sexual relationships. Understanding participants' lived experiences is critical to participant involvement and retention and to designing ATI studies that are ethical, feasible, and acceptable. METHODS: We conducted a qualitative study involving semi-structured interviews with 20 cisgender men living with HIV enroled in the phase 2 double-blinded randomised placebo-controlled RIO trial of HIV-specific broadly neutralising antibodies between January and May 2025 in the United Kingdom. We transcribed, coded and analysed the interviews using thematic analysis. RESULTS: The participants described being motivated by scientific altruism but had worries about stopping ART. During the ATI, participants found breaking their daily pill-taking routine challenging, yet a majority described feelings of liberation thereafter. The participants preferred once weekly viral load testing, as it reassured safety, which was coupled with support from researchers, who buffered anxiety during periods of viral rebound. ATIs had a notable impact on personal relationships, including avoidance of dating, reduced intimacy and sexual pleasure while off ART. Easy access for partners without HIV to free PrEP, PEP, and condoms was welcomed by all participants. Restarting ART was difficult following the ATI, but was resolved with pre-trial preparation supporting adherence. The participants with lengthy ATIs remained enthusiastic but were challenged by uncertainty that required adjustment of their life plans. Finally, participants expressed mixed views regarding the use of placebo arms in trials but accepted a second ATI period after receiving broadly neutralising antibodies open-label. CONCLUSIONS: Participants described being part of an ATI trial as both liberating and demanding. The findings indicate that acceptability is influenced by predictable, frequent weekly HIV viral load monitoring, timely results communication, and access to free prevention, with clear concerns about relationship impacts during viral rebound, and clearly communicated pre-defined ART restart criteria.
Veterinary research communicationsDeepak B Rawool, Radhakrishna Sahu, Niveditha Pollumahanti, Laxman R Chatlod, Yogesh P Gadekar, Vishnuraj M R, Jess Vergis, Nagendra Nath Barman, Sukhadeo B Ba…
In this study, a total of 2818 food samples of animal origin collected from 18 states of India using a stratified random sampling method were processed for isolation of Salmonella spp. PCR serotyping was performed to identify non-typhoidal serovars. All confirmed Salmonella isolates were screened for antibacterial susceptibility and for their virulence potential using the Galleria mellonella larvae. Microbiological screening identified 58 Salmonella spp. isolates (2.06%). Variation in isolation rate was not evident in various types of samples that were screened, and the place of isolation. On PCR serotyping, 39.65% were identified as S. Typhimurium, 32.76% as S. Enteritidis, and the remaining 27.59% were other Salmonella serovars. Notably, 44.83% of the Salmonella isolates exhibited MDR pattern with a MAR index of > 0.2. All the isolates were virulent to G. mellonella larvae. Findings of the study warrants further investigations and appropriate preventive cum control measures to ensure food safety.
Current microbiologyPeibo Zheng, Zhe Shao, Xiaoyi Wei, Shucheng Liu, Xiaohong H Sun, Weiqing Q Lan
This study investigated the synergistic antibacterial mechanism of ginger essential oil (GEO) and rosemary essential oil (REO) against Shewanella putrefaciens. Antibacterial efficacy and interactions were assessed by determining the minimum inhibitory concentration (MIC), fractional inhibitory concentration (FIC) index, and growth curves. Membrane integrity was evaluated using confocal laser scanning microscopy (CLSM), leakage of intracellular components, and alkaline phosphatase (AKPase) activity. Structural damage was observed via scanning electron microscopy (SEM). The effects on antioxidant defense and energy metabolism were analyzed by measuring superoxide dismutase (SOD), succinate dehydrogenase (SDH), and malate dehydrogenase (MDH) activities. Biofilm inhibition capacity was also examined. Results demonstrated a synergistic effect (FIC index = 0.375). The combination of GEO and REO caused severe membrane damage, intracellular leakage, inhibition of key enzyme activities, and effective biofilm inhibition. The synergistic mechanism involves membrane disruption, suppression of metabolic pathways, and biofilm inhibition.
Magnetic nanoparticles (NPs) are maintained in the body for a significantly longer duration than small molecule antibiotics, potentially facilitating prolonged therapeutic effects. In this study, a novel nanoantibiotic formulation (l-glu-Fe3O4-Amp NPs) was prepared, and its antibacterial efficacy was evaluated. For this purpose, l-glutamic acid (l-glu) was used for surface modification of iron oxide nanoparticles (Fe3O4 NPs), and ampicillin (Amp) was then conjugated l-glu-Fe3O4 NPs structure. Fourier transform infrared analyses revealed that the final nanoantibiotic formulation l-glu-Fe3O4-Amp NPs was successfully prepared. The size distribution of the formulation was determined to be 164 nm by ZETAsizer. It exhibited a controlled drug release profile over a certain period of time, with a slightly increased release and then exhibited a stable profile. Agar well diffusion assay and the LIVE/DEAD BacLight fluorescent assay showed that the formulation had an antibacterial effectiveness against both Bacillus cereus and Staphylococcus aureus. The formulation has a lower minimal inhibitory concentration value against S. aureus. It significantly increased the accumulation of reactive oxygen species in both bacteria when compared with the Amp group. Its cellular uptake and bacterial interaction were confirmed by confocal and transmission electron microscopy images. The in vitro assay revealed that this formulation is nontoxic to fibroblast cells. This study highlights the potential of l-glu-Fe3O4-Amp NPs conjugate as a potentially effective antibacterial agent. This is the first report on the use of l-glu as a surface modifier of Fe3O4 NPs and Amp. Use of l-glu increases the drug-carrying capacity of NPs and reduces the possible side effects of Amp.
Infection with the hepatitis delta virus (HDV) causes the most aggressive type of viral hepatitis and frequently leads to liver cirrhosis and hepatocellular carcinoma. Immunological aspects associated with HDV infection are poorly studied, and quantitative identification of anti-HDV antibodies may be important for understanding the disease. A quantitative, chemiluminescence-based ELISA was developed and validated using 399 serum samples (199 HDV-Ab positive, 200 healthy controls). Serum levels of HDV-RNA, HBV-DNA and hepatic enzymes (ALT, AST) were measured. Statistical analyses included ROC analysis, distribution analysis, Student's t-test and correlation analysis. ELISA demonstrated linear quantification of anti-HDV antibodies with high specificity (Sp = 100%) and sensitivity (Se = 100%), detecting down to 0.5 ng/mL in pure standards. The mean anti-HDV antibody levels were 67,622 ng/mL overall, 70,883 ng/mL in males and 64,043 ng/mL in females. Antibody levels followed a normal distribution, despite population heterogeneity. Student's t-test revealed significant gender differences in antibody levels and ALT (p = 0.0128 and p = 0.0006), whereas HDV-RNA, HBV-DNA and AST did not differ significantly. Comparisons of the high versus low HDV-RNA subgroups showed no significant differences in antibody levels. Correlation analysis indicated no significant associations between anti-HDV antibody levels and HDV-RNA, hepatic enzyme levels. This study presents a robust quantitative ELISA for detecting anti-HDV antibodies. The humoral immune response, as reflected by antibody levels, is independent of the viral load and liver injury markers. Gender-related differences in antibody and ALT levels were observed. These findings provide a foundation for precise evaluation of HDV-specific immunity in chronic infections.
MicrobiologyOpenTalita Vilalva Freire, Ana Carolina de Freitas Marques, Valéria Dos Santos Gonçalves, Iluska Senna Bonfá Moslaves, Mônica Cristina Toffoli-Kadri, Arthur Felici…
Bacterial resistance to antibiotics represents a critical global health challenge, demanding urgent alternative strategies to manage infections caused by multidrug-resistant (MDR) pathogens such as Acinetobacter baumannii, Staphylococcus aureus, Escherichia coli, and Klebsiella pneumoniae. A promising approach to address this problem involves the use of combination therapies, in which plant-derived natural products represent an important reservoir of bioactive compounds with the potential to act as effective adjuvants, restoring or enhancing antibiotic efficacy. In this study, the ethanolic extract of Mouriri elliptica (EME) leaves was investigated for its antimicrobial and synergistic potential. EME showed moderate inhibitory activity against both standard and MDR strains, with the most significant effect against A. baumannii (MIC 78.1-312.5 µg/mL). Synergistic interactions were observed with conventional antibiotics, particularly ciprofloxacin against K. pneumoniae, reducing MIC values up to fifteen-fold, and with ampicillin against E. coli and S. aureus. The extract also inhibited biofilm formation, with 68% inhibition for E. coli at 500 µg/mL and 72.04% for S. aureus at 250 µg/mL, with atomic force microscopy images corroborating these findings. LC-MS profiling identified 41 compounds, including chlorogenic acid derivatives, flavonoids, ellagic acid derivatives, and triterpenes. Acute toxicity evaluation in mice at 2000 mg/kg revealed no adverse effects. Collectively, these findings support the potential of M. elliptica as a natural antibacterial adjuvant for enhancing the effectiveness of selected antibiotics against MDR pathogens, warranting further chemical, mechanistic, and in vivo investigation.