Traditionally, the impact of the substrate specificity of β-lactamase produced by bacteria on β-lactam resistance (β-lactamase-mediated resistance) has been assessed by performing antimicrobial susceptibility testing (AST) on transformants carrying the cloned gene on arbitrarily chosen β-lactamase-free plasmid vectors. However, because the promoter strength and plasmid copy number of these vectors are not standardized, the resulting β-lactamase expression levels vary, further hampering cross-study comparisons. We constructed pMiniF-1 by inserting a tac promoter, a ribosome-binding site, and transcriptional terminators into the mini F plasmid, which is maintained at one to two copies per cell. This design enables controlled β-lactamase expression for accurate evaluation of β-lactamase-mediated resistance via AST. This approach enables the determination and comparison of MICs with β-lactamase expression levels controlled to be as nearly identical as possible. We performed AST on Escherichia coli DH5αΔblaEC-like transformants expressing 22 β-lactamase genes (Ambler classes A-D), including carbapenemases that were cloned into pMiniF-1. The resulting antimicrobial susceptibility profiles and MIC differences did not contradict the known substrate specificities of the enzymes. Meropenem and imipenem MICs for the IMP-6 producer were fourfold higher and eightfold lower, respectively, than IMP-1. Aztreonam MICs were similar between transformants lacking β-lactamase genes and those with class B β-lactamases. Imipenem MIC for the OXA-48 producer was ≥8-fold higher than for OXA-1, whereas cefepime MICs were 16-fold lower. We demonstrate that pMiniF-1 enables the comparison of β-lactamase substrate specificity via AST. The pMiniF-1 plasmid (Catalog No. RDB21122) is available from the RIKEN BioResource Research Center (Tsukuba, Ibaraki, Japan) (https://web.brc.riken.jp/en/).IMPORTANCEβ-lactamases play a central role in bacterial resistance to β-lactam antibiotics, and novel variants continue to emerge. However, the impact of individual β-lactamase variants on β-lactam resistance across diverse enzyme-substrate combinations remains insufficiently characterized. Traditional kinetic analyses require enzyme purification and multiple reaction assays, which hinder comprehensive experimental evaluation. Expression-based approaches have also been used to assess β-lactamase function, but conventional systems depend on unstable high-copy plasmids, raising concerns about reproducibility due to variable plasmid copy numbers. In this study, we developed pMiniF-1, a stable, low-copy plasmid that enables consistent gene expression without antibiotic selection, thereby maintaining stable copy numbers under drug-free conditions. Using 22 β-lactamase genes, we demonstrated that susceptibility testing with pMiniF-1 adequately reflects their known substrate specificity profiles. This system provides a reliable method for assessing β-lactamase substrate specificity and will facilitate comparative studies on the functional diversity and evolution of emerging β-lactamase variants.

Accurate HIV incidence estimates are essential for monitoring epidemic trends and evaluating prevention efforts. The limiting antigen (LAg) avidity enzyme immunoassay is the most widely used method to classify recent versus long-term HIV infections. The External Quality Assurance Oversight Laboratory (EQAPOL), in collaboration with Vitalant Research Institute, conducts a global External Quality Assurance (EQA) program to evaluate laboratory performance using commercial LAg kits. We assessed (i) the stability of calibrators and reagents from Sedia and Maxim kits over their shelf life, and (ii) the longitudinal consistency of EQA panel samples tested across nine proficiency rounds (2018-2023). Mixed-effects models were used to detect trends in optical density (OD) and normalized OD (ODn) values over time. No significant trends in OD or ODn were observed for either kit, and EQA panel samples showed stable reactivity across all rounds. Minor ODn shifts did not affect recency classification. Sedia kits consistently yielded slightly higher ODn values than Maxim kits, consistent with prior reports. These findings confirm the long-term reliability of both LAg assay kits and EQA panel materials, supporting the robustness of EQAPOL's quality assurance framework for accurate global HIV incidence surveillance.IMPORTANCEReliable estimation of HIV incidence depends on accurate classification of recent versus long-term infections, which in turn requires consistent assay performance and robust quality assurance. The External Quality Assurance Oversight Laboratory LAg External Quality Assurance program provides longitudinal monitoring of laboratory proficiency for two widely used commercial assays. By demonstrating the long-term consistency of calibrators, reagents, and quality control panels across multiple production lots and testing rounds, this study confirms the robustness of the global limiting antigen testing framework. These results strengthen confidence in the comparability of HIV incidence data across surveillance networks and geographic regions, supporting evidence-based decision-making for HIV prevention programs.

The use of complex cell culture model systems is becoming more common across many fields. Maintenance of these systems often includes growth factors and inhibitors not found in standard media. These added components have the potential to impact pathogen growth and development and can influence how experimental results may be interpreted. This study improves our understanding of the Toxoplasma gondii lifecycle in one of those systems. It also serves as a template for other pathogen researchers to consider influences within their own systems.

The genus Alicyclobacillus remains a persistent problem for the fruit juice industry, as its thermoacidophilic nature leads to spoilage events that cause product recalls, loss of consumer confidence, and significant economic losses. Yet remarkably little is known about the phages infecting these bacteria, with only two described to date. This study reports the first infectious phage with genomic identity to a prophage in the genus, the Alicyclobacillus phage MMB025. This finding broadens the extremely limited record of Alicyclobacillus phages and reveals integration within the sporulation sigma factor sigK, suggesting a novel link between prophage activity and sporulation regulation in Alicyclobacillus. By combining stability assays, host range testing, and genome analysis, MMB025 represents both a valuable model for phage-spore-former biology and a genetic resource for candidate lysins with potential applications in food preservation.

Mycobacterium avium complex (MAC) is a nontuberculous mycobacteria that cause chronic pulmonary infections, particularly in individuals with underlying structural lung disease. Despite prolonged multidrug therapy, treatment outcomes for MAC pulmonary disease remain suboptimal, posing ongoing clinical challenges. Although β-lactams have demonstrated efficacy against Mycobacterium abscessus, β-lactams are not currently recommended for MAC, in part due to limited supporting data. Since M. abscessus and MAC have similar cell wall structures and peptidoglycan synthesis, we hypothesized that β-lactams could be effective against MAC, especially when used in combination to achieve broader inhibition of cell wall-synthesizing enzymes, L,D-transpeptidases, and D,D-transpeptidases. In a systematic screen of clinically relevant β-lactams, we identified combinations-specifically those pairing a penem or carbapenem with a penicillin or cephalosporin-that exhibited potent bactericidal activity and synergy against MAC at clinically achievable concentrations. Of the 91 β-lactam combinations initially screened against Mycobacterium intracellulare (ATCC13950) and M. avium subsp. avium (ATCC25291), 23 pairs demonstrated synergy against M. intracellulare and 16 against M. avium subsp. avium. For M. intracellulare, most synergistic combinations reduced MICs to clinically achievable levels, and the majority of single agents and all combinations achieved ≥40% fT>MIC50. In contrast, although several combinations showed strong synergy against M. avium, their MICs often remained above therapeutic thresholds. Only a few two-drug combinations reached ≥40% fT>MIC50, indicating the need for additional antibiotics. Three β-lactam combinations achieved ≥40% fT>MIC50 against M. avium. These novel β-lactam combinations, leveraging existing antibiotics, may represent promising and immediately deployable treatment strategies for refractory MAC infections.IMPORTANCEMycobacterium avium complex (MAC) pulmonary disease is increasingly prevalent and difficult to treat, with current multidrug regimens achieving suboptimal and often unsustained responses. Unlike Mycobacterium abscessus, for which β-lactams have shown promise, β-lactams are not recommended for MAC largely due to limited data. Yet, MAC and M. abscessus share similar peptidoglycan structures and reliance on overlapping L,D- and D,D-transpeptidases for cell wall synthesis, suggesting that strategic β-lactam combinations might overcome intrinsic resistance barriers. This study provides the first systematic evaluation of clinically relevant β-lactam pairs against MAC, revealing that specific combinations-particularly those pairing a penem or carbapenem with a penicillin or cephalosporin-exhibit potent bactericidal activity and robust synergy. Several combinations achieved pharmacodynamic targets at clinically achievable exposures, especially against Mycobacterium intracellulare. These findings identify readily accessible β-lactam combinations that could be rapidly translated into new treatment strategies for refractory MAC pulmonary disease, addressing a critical unmet clinical need.

Advanced sequencing technologies and improvements in bioinformatics have provided a new way to study plant-associated microbial communities, including the use of host genomic sequencing. Our study focuses on the leaf microbiome of basin big sagebrush (Artemisia tridentata subsp. tridentata), a foundational shrub of western North America. We analyzed Illumina shotgun sequences from sagebrush leaves to investigate the metagenomes of leaf-associated microbes that were sequenced alongside their plant hosts. We aimed to profile the leaf microbiome across different sample sources (magenta box, greenhouse, and field/wild), reconstruct metagenome-assembled genomes (MAGs) where possible, and investigate functional gene annotations of the resulting MAGs, specifically with regard to the potential metabolism of sagebrush chemicals. To achieve this, Illumina shotgun sequence reads (containing both host and associated microbial reads) were mapped to the reference genomes of Artemisia tridentata, Artemisia annua, and the human reference genome to remove plant host and human-associated sequences. Host-cleaned reads were then analyzed using microbial metagenomics techniques. Taxonomic profiling revealed that Phyllobacterium and Sphingomonas were the most abundant microbial genera in greenhouse-grown plants, with very little variation among the samples. Wild, field-collected samples were much more variable and were dominated by Klebsiella and Aureobasidium species. From the co-assembly of greenhouse samples, we reconstructed two high-quality MAGs (a Phyllobacterium species and a Sphingomonas species) with >98% completion and <1% contamination. Functional annotation of these MAGs uncovered genes associated with the degradation and metabolism of camphor and other essential oils such as pinene, geraniol, and limonene, which are part of sagebrush leaf chemistry.IMPORTANCEBig sagebrush (Artemisia tridentata), the foundation species of the sagebrush steppe, has broad ecological importance because its evergreen leaves offer nutrients and shade that facilitate the establishment of diverse understory plants in arid environments. Sagebrush leaves contain various secondary metabolites, including terpenoids, flavonoids, and phenolic compounds. These chemicals contribute to the plant's defense mechanisms against herbivores and pathogens. Despite this, sagebrush hosts diverse bacterial and fungal communities. We found that the microbial metagenome-assembled genomes (MAGs) we recovered contained genes that have the potential to degrade some of the chemical compounds in sagebrush leaves that could inhibit the growth of other microbes. This is the first study to mine plant genome data using host-derived sequences to generate microbial MAGs. Our results showed that MAGs can be recovered from plant host-derived sequence data, providing a new way to explore the identity and functional capabilities of difficult-to-culture microbes.

Chronic kidney disease (CKD) is closely associated with gut microbiota dysbiosis and impaired intestinal barrier function, yet effective therapeutic strategies targeting the gut-kidney axis remain limited. This study aimed to develop a thiolated chitosan/sodium alginate-based nanoparticle system encapsulating emodin (TCS/ALG/Emodin-NPs), and to evaluate its therapeutic efficacy in CKD through modulation of gut microbiota and intestinal barrier protection. The nanoparticles were synthesized, characterized, and orally administered to CKD mice at varying doses, with irbesartan as a positive control. Our results showed that TCS/ALG/Emodin-NPs significantly improved renal function, reduced renal fibrosis, and restored gut microbiota balance, particularly increasing beneficial bacteria such as Lactobacillus. They also enhanced intestinal tight junction integrity and suppressed systemic inflammation. In a hydrogen peroxide-induced Caco-2 cell model, Emodin-NPs more effectively attenuated oxidative stress and apoptosis compared to free Emodin. The findings suggest that TCS/ALG/Emodin-NPs are a promising strategy for CKD treatment by reshaping gut microbiota and enhancing intestinal function.IMPORTANCEWhile the gut-kidney axis has emerged as a critical target in chronic kidney disease (CKD) management, therapeutic strategies leveraging this pathway remain limited. This study demonstrates that colon-targeted TCS/ALG/Emodin-NPs not only enhance drug bioavailability but also exert dual therapeutic effects by rectifying gut dysbiosis and reinforcing intestinal barrier function-key mechanisms implicated in CKD progression. The superior efficacy of TCS/ALG/Emodin-NPs over free Emodin underscores the transformative potential of nanoparticle delivery systems in overcoming the pharmacokinetic limitations of herbal medicines. This research opens new avenues for the development of microbiota-targeted, anti-inflammatory therapies that can complement existing CKD treatments and potentially slow disease progression in affected patients.

Acanthamoeba keratitis is a sight-threatening corneal infection. A delay in diagnosis and lack of amoeba-specific treatment contribute to a generally poor prognosis. Genotyping of pathogenic Acanthamoeba may offer deeper insights into the disease. Sixteen culture-positive Acanthamoeba ocular samples from 2006 to 2022 were identified and genotyped in patients from the northern and central regions of Sweden. Data from the patients' records were collected, and their respective clinical courses were reviewed. The most common Acanthamoeba genotype was T4 (11 cases), followed by T3 (4 cases) and T6 (1 case). All but one patient was a contact lens user. There were no cases of previous trauma. No statistically significant differences were found between the T4 and non-T4 groups. The influence of genotype on the clinical course warrants further investigation.IMPORTANCEThis study provides insights into the rare but serious eye infection Acanthamoeba keratitis, which can cause blindness. Our research presents data from Sweden on the genotypes of the parasite causing this infection. While the most common genotype worldwide, T4, was also predominant in our study, we found an unexpectedly high number of cases caused by the T3 genotype. This may indicate regional differences and highlights the need for ongoing research. The study also examines the link between genotype and patient outcomes. Although our findings did not establish a statistically significant correlation, knowledge of such a connection could prove valuable in efforts to improve management, and further multicenter studies may be warranted.

The NG-test CARBA 5 and Xpert Carba-R IVD kits were tested against 269 clinical Enterobacterales isolates selected for their previously determined carbapenemase content from the ATLAS Global Surveillance program, primarily from 2020 to 2022. Antimicrobial susceptibility testing by broth microdilution was performed for aztreonam-avibactam, cefiderocol, and othe agents using EUCAST 2025 breakpoints for interpretation. The CARBA 5 and Carba-R IVD kits accurately identified all NDM (93), KPC (36), and OXA-48-like (43) carbapenemases. The CARBA 5 detected all IMP (42) and 45/47 VIM, and the Carba-R detected all VIM (47) and 21/42 IMP. Aztreonam-avibactam was active against carbapenemase-producing isolates (percent susceptible [carbapenemases]): 90.7% (NDM), 92.9% (NDM+OXA-48-like), 100% (IMP, VIM, and KPC), and 88.9% (OXA-48-like). Cefiderocol susceptibility was as follows: 38.7% (NDM), 50.0% (NDM+OXA-48-like), 77.5% (IMP), 82.5% (VIM), 75.9% (KPC), and 100% (OXA-48-like). The most common carbapenemases in Enterobacterales include NDM, KPC, and OXA-48-like enzymes, all of which were accurately identified by the CARBA 5 and Carba-R kits. Aztreonam-avibactam demonstrated potent in vitro activity against isolates determined to be positive for carbapenemases by these kits, offering a potential therapeutic option when one of these isolates is encountered in the clinic.IMPORTANCEIn vitro diagnostic (IVD) kits exist that detect carbapenemases in clinical isolates of Enterobacterales. Their utility is hampered by the limited options to reliably treat infections caused by Enterobacterales that produce metallo-β-lactamases. Here, we test the utility of IVD kits for detecting carbapenemases in a recent collection of clinical Enterobacterales isolates and determine the in vitro activity of aztreonam-avibactam, cefiderocol, and other comparator agents against isolates that were positive for carbapenemases.

This work addresses a practical diagnostic gap in routine wound infection testing using panel-based PCR. In some CAP/CLIA laboratories using multiplex or panel-based PCR, detection of Staphylococcus aureus and mecA does not reliably resolve whether methicillin resistance originates from S. aureus or coagulase-negative staphylococci in polymicrobial specimens. Our approach is not a stand-alone methicillin-resistant S. aureus (MRSA) screen; it functions as a post-analytic attribution layer that probabilistically disambiguates the source of mecA within existing PCR workflows. By leveraging routinely available cycle-threshold data within a biologically gated logistic framework, this method provides transparent and auditable MRSA/methicillin-susceptible S. aureus attribution without additional instrumentation or workflow changes. Accurate attribution reduces biologically implausible MRSA calls while preserving detection of probable MRSA, supporting more rational antimicrobial stewardship in polymicrobial wound infections.