Introduction. BK polyomavirus (BKPyV) reactivation is a significant health risk among renal transplant recipients that can lead to nephropathy and allograft loss.Hypothesis/Gap statement. While the microbiota is increasingly recognized as an important determinant of viral infection and pathogenesis, as well as itself undergoing compositional changes in response to infection, the urinary microbiome has yet to be investigated in the context of BK polyomavirus reactivation.Aim. This study aimed to investigate associations between the urinary microbiome and BKPyV-DNAemia in renal transplant patients.Methodology. Shotgun metagenomics of the urinary microbiome was conducted for 22 renal transplant recipients, 11 of whom had BKPyV-DNAemia. Sequence data were analysed using two complementary approaches to identify common microbiome associations with BKPyV-DNAemia: (1) Kaiju - a DNA-to-Protein method that captures bacteria, archaea, fungi, microeukaryotes and DNA viruses and (2) MetaPhlAn4 - a DNA-to-Marker method using a reference database of specific marker genes of prokaryotes.Results. We found increased observed diversity of bacterial taxa in control patients compared to those with BKPyV-DNAemia for data analysed with MetaPhlAn4 (P=0.037) but not Kaiju (P>0.05), which followed a similar trend. Significant differences in microbial beta diversity between the control and BKPyV-DNAemia patient group were identified for the Kaiju dataset (P=0.027) but not for MetaPhlAn4 (P>0.05), with viral reads likely driving these differences in the Kaiju dataset. Both Kaiju and MetaPhlAn4 identified Proteobacteria, Firmicutes and Actinobacteria as bacterial phyla with greatest relative abundance across samples. Screening bacterial species data generated from Kaiju and MetaPhlAn4 against a database of 243 human pathogens identified 8 pathogenic species recovered from both datasets that were present in the urinary microbiome of renal transplant patients.Conclusion. The observed evidence for differences in microbiome diversity and composition associated with BKPyV-DNAemia may play an important role in its pathology and guide the development of diagnostic biomarkers. Our findings warrant further investigation across larger patient cohorts that are more evenly balanced for gender.
Prosthetic joint infections (PJIs) remain among the most devastating complications of arthroplasty, imposing substantial clinical, economic and patient burdens. Although culture-based diagnostics underpin current clinical practice, PJIs are biofilm-driven infections shaped by taxonomic diversity, spatial organization, host responses and surface interactions, meaning conventional approaches provide only a partial and often decontextualized view of the infection process. We examine how convergent methodologies can transform PJI research by integrating approaches that have traditionally been studied in isolation, including sequencing, transcriptomics, metabolomics, advanced imaging and culture-based characterization. We discuss how whole-genome sequencing, shotgun metagenomics, transcriptomic and metabolomic approaches resolve pathogen identity, functional activity and adaptive persistence and how cross-scale imaging and spatial biology techniques reveal where microbes colonize, interact and survive across implant surfaces. We highlight emerging opportunities to unify these datasets into coherent frameworks that capture both the molecular and physical dimensions of PJIs. Integrating these complementary approaches will enable a multi-layered understanding of PJIs that link composition, function and spatial organization. Ultimately, this provides a foundation for predictive diagnostics, precision antimicrobial strategies and improved implant design and supports a shift towards more effective, mechanism-informed management of implant-associated infection.
Tetracyclines are bacteriostatic antimicrobials used in the treatment of both bacterial and protozoan pathogens. They abolish protein synthesis by binding to the 16S rRNA, blocking aminoacyl-tRNA access and preventing polypeptide elongation. Tetracyclines are polyketide antimicrobials, with members of this drug family (doxycycline, minocycline and tigecycline) on the World Health Organization (WHO) list of essential medicines. Bacterial resistance to this antimicrobial family is widespread and arises via multiple mechanisms. The most common of these resistance mechanisms are tetracycline efflux, ribosomal protection proteins and the newly emerging tetracycline destructases (TDases). The rise of resistance has limited the clinical use of first- and second-generation tetracyclines; however, third-generation tetracyclines are now reserved as antimicrobials of last resort for complicated bacterial infections. However, genetically mobile TDases have now been identified in opportunistic pathogens, with this mechanism of resistance capable of inactivating third-generation tetracyclines. This presents the question, are third-generation tetracyclines doomed to fail in the same manner as first-generation tetracyclines?
Background. Histological examination and rabbit intratesticular inoculation of clinical specimens are traditionally used to diagnose syphilis and characterize the aetiological agent, Treponema pallidum. Usually, these methods provide only qualitative data of the bacterium. For quantitative analysis, dark-field microscopy and real-time PCR are commonly used but both methods have important limitations. We investigated intradermal inoculations in rabbits as a sensitive and quantitative alternative test method.Methods. We prepared 10-fold serial dilutions of a treponemal stock ranging from 4×10³ to 4 treponemes ml-1. Each dilution series was injected intradermally in two rabbits, ten sites per rabbit. Similarly, each dilution was inoculated intratesticularly into two rabbits, four dilutions in a total of eight rabbits. We scored and measured the size of lesions at the injection sites in rabbits inoculated intradermally. We monitored intratesticularly infected rabbits for orchitis. Sera from all animals were tested for seroconversion. We also compared the analytical sensitivity of animal bioassays to that of treponemal PCR.Results. The infectivity titre of T. pallidum was the same whether measured by intradermal or intratesticular inoculations, but the former used 75% fewer animals. Bacterial concentration at 50% endpoint was lower, 4 treponemes ml-1, with intratesticular route compared to 25 treponemes ml-1 using intradermal injections. Nested PCR demonstrated an analytical sensitivity of 40 treponemes ml-1, comparable to that obtained with animal bioassays. We also showed a correlation between lesion size and treponemal counts that, under standardized conditions, might be exploited to estimate the number of treponemes in a sample.Conclusions. Our studies showed that all methods tested detected T. pallidum with similar sensitivities. However, the intradermal route might be better suited to investigate clinical specimens as it offered desirable features such as treponemal quantification, minimal number of animals required and detection of live infectious bacteria, all in a single test.
Introduction. Biofilm-associated infections present a major therapeutic challenge due to their intrinsic tolerance to conventional antimicrobials. Cold atmospheric plasma (CAP) has shown promise as a non-antibiotic antimicrobial modality; however, some bacteria including Staphylococcus aureus can exhibit tolerance to plasma exposure.Gap Statement. Strategies that sensitize CAP-tolerant biofilms to plasma treatment may improve CAP efficacy, but suitable adjunctive compounds and mechanisms remain poorly defined.Aim. This study aimed to determine whether repurposed bioactive compounds could enhance CAP activity against S. aureus biofilms.Methodology. Selected compounds from the Tocriscreen™ bioactive compound library were initially screened, followed by treatment of S. aureus biofilms with KHS101 ±CAP therapy. Biofilm viability was quantified using live/dead qPCR. To probe mechanisms of sensitization, biofilms were exposed to H2O2 at concentrations equivalent to those generated by CAP, either alone, or in combination with KHS101 or conventional antibiotics. Various microscopy techniques were used to visualize the cellular impacts of KHS101, while metabolic activity and cell viability of dual therapies were determined using AlamarBlue® assay and plate count assays, respectively.Results. Short-term KHS101 treatment alone displayed modest antibiofilm activity at concentrations that inhibited planktonic growth. However, pre-treatment with KHS101 followed by CAP therapy resulted in significant reductions in viable populations in S. aureus-containing biofilms. Microscopy revealed structural perturbations consistent with cellular stress following KHS101 exposure, but also showed intact cellular ultrastructure. Mechanistic probing demonstrated that equivalent concentrations of H2O2 with KHS101 were insufficient to reproduce the enhanced efficacy observed with CAP. In contrast, H2O2 enhanced flucloxacillin activity in a strain-dependent manner, sensitizing S. aureus biofilms to otherwise sub-lethal concentrations of antibiotic.Conclusion. These findings demonstrate that tolerance of S. aureus biofilms to CAP can be overcome through dual-therapy strategies. Treatment with the repurposed compound KHS101 was associated with enhancement of CAP efficacy via an unknown mechanism. However, the inability of H2O2 to reproduce this effect highlights the importance of additional plasma-derived reactive species in mediating this dual-action killing. Together, these findings position biofilm sensitization as a central concept emerging from this study, whereby a non-lethal adjunct can lower the threshold for CAP-mediated killing without acting primarily as a direct antimicrobial.
Introduction. Oral bacteria, such as Rothia dentocariosa (Rd), can modulate external aqueous phase orthophosphate (Pi) concentrations via Pi internalization and accumulate high amounts of Pi in the form of polyphosphate (polyP).Hypothesis/Gap Statement. This study investigates whether the carbohydrate metabolism of Rd that generates acid and solubilizes Pi, derived from phosphorus-32 (32P)-labelled tooth enamel, can be internalized and incorporated into intracellular polyP. The study investigated the hypothesis that the internalization of tooth-derived Pi and polyP synthesis could be attenuated.Aim. A radionuclide 32P-labelled remineralized model examined the influence of gallein, an inhibitor of polyP kinases, on the ingestion of tooth-derived Pi and on the bacterium's cultivability.Methodology. Radionuclide 32P-labelled remineralized bovine enamel (n=6) was exposed to Rd (ATCC 17931) incubated in brain heart infusion (BHI) broth containing glucose. Attenuated total reflectance Fourier transform infrared (FTIR) spectroscopy and cross-polarization optical coherence tomography assessed the surface and subsurface characteristics of the remineralized and demineralized enamel surface. Liquid scintillation traced the 32P release from the tooth to the intracellular polyP in galleinpos/neg samples. Cell-media, supernatant, cell resuspension, and extracted polyP, from Rd exposed to the radioisotope-labelled remineralized enamel, were analysed for 32P activity at 10 and 22 h time points.Results. FTIR determined that 32P was incorporated in dental enamel lesions and was released after exposure to an acidic buffer (pH=4.5) or Rd. After 24 h of exposure, galleinpos reduced Rd's planktonic growth and increased pH by 7.45% and 7.19%, respectively, compared to galleinneg. Gallein inhibited the cultivability of planktonic Rd on BHI agar plates with a 13- to 60-fold reduction of c.f.u. ml-1 (25 µM-13.03×, 50 µM-19.58× and 100 µM-60.57×) compared to galleinneg samples. Isotope labelling demonstrated that Rd used Pi derived directly from the tooth for the benefit of the bacterium's fitness through the formation of polyP.Conclusion. Rd used the Pi derived directly from the tooth for the benefit of the bacterium's fitness. Gallein attenuated polyP accumulation, reduced bacterial cultivability and may be a potential therapy option in the prevention and treatment of primary and secondary caries.
Introduction. Bacterial vaginosis (BV) is a common gynaecological disorder characterized by an imbalance in the vaginal microbiota, leading to increased risk of infection and recurrence.Hypothesis/Gap statement. Although the role of microbiota dysbiosis in BV is established, the dynamic changes in microbial communities and inflammatory factors during BV formation and recurrence, as well as reliable biomarkers for predicting recurrence, remain inadequately explored.Aim. This study aimed to systematically analyse the changes in vaginal microbiota and inflammatory factors during different stages of BV formation and recurrence, and to identify potential biomarkers for predicting BV recurrence.Methodology. Vaginal swab samples were collected from 135 women, including 55 BV patients, 50 intermediate BV (IBV) patients and 30 healthy controls. Twenty-six recurrent BV (Re-BV) samples were obtained within 1 year after treatment. 16S rRNA gene sequencing was performed to profile the vaginal microbiota, and ELISA was used to measure IL-6, IL-10 and IL-1β levels. Statistical analyses included diversity metrics, correlation analysis and predictive modelling using LASSO regression and receiver operating characteristic curves.Results. Microbial richness was significantly higher in BV, Re-BV and IBV groups compared to controls. Lactobacillus abundance decreased progressively from IBV to BV, while Gardnerella, Prevotella, Sneathia, Fannyhessea and Dialister increased. These genera were positively correlated with vaginal pH, human papillomavirus (HPV) status and enzymatic activity. IL-6, IL-10 and IL-1β levels were elevated in BV, IBV and Re-BV groups. IL-10 and IL-1β were further increased in recurrent cases. A combined predictive model integrating microbial and cytokine markers achieved an area under the curve of 0.928 for BV recurrence.Conclusion. Distinct shifts in vaginal microbiota composition and inflammatory factor levels occur during BV formation and recurrence. The integration of microbial and cytokine profiles offers a robust approach for predicting BV recurrence, with important implications for clinical management and prevention strategies.
Infections caused by multidrug-resistant Acinetobacter baumannii are considered a threat to human and animal health. The widely studied A. baumannii strain AB5075 displays a high degree of antibiotic resistance. In this study, we experimentally validated that antibiotic resistance is largely mediated by resistance genes located on plasmid p1AB5075. We used a p1AB5075-deficient AB5075 strain to assess individual contributions of p1AB5075-encoded antibiotic resistance genes by ectopically (over-)expressing each gene in the Δp1AB5075 background. By determining individual contributions of seven p1AB5075-encoded antibiotic resistance genes, we show individual and overlapping roles of genes for aminoglycoside resistance and uncover the importance of extended-spectrum β-lactamase blaGES-11 for monobactam and cephalosporin resistance in A. baumannii AB5075. We discovered that aminoglycoside N-acetyltransferase aaC(6')-Ib3 (aacA4), which was previously shown to confer resistance to tobramycin, provides broad resistance to gentamicin, kanamycin, amikacin, streptomycin and tobramycin when overexpressed in A. baumannii AB5075. Because p1AB5075 is transferable to a wide range of environmental and clinical A. baumannii strains and non-baumannii Acinetobacter species, the relevance of our findings extends beyond A. baumannii AB5075.
Introduction. High rates of recurrent Clostridioides difficile infection (CDI) and environmental contamination are attributed to its ability to form spores. Periodontal diseases are characterized by gingival inflammation, caused by dental plaque accumulation. Hypothesis. Periodontal plaque could harbour C. difficile spores, acting as a reservoir for reinfection. Aim. Compare the prevalence and abundance of C. difficile in metagenomic sequences of saliva and dental plaque from healthy and periodontal disease patients. Methodology. Publicly available metagenomic reads from oral samples of healthy ( n =80) and periodontitis ( n =204) patients were analysed for C. difficile presence through an in-house bioinformatic pipeline. Briefly, reads underwent quality control (cutadapt/fastQC) prior to subsampling of 3 million reads (seqtk). Reads and MEGAHIT-assembled contigs were aligned to a C. difficile reference genome (ASM1888508v1) or a full non-redundant protein DIAMOND database. Outputs were filtered, annotated (Entrez Direct) and top hits identified via National Center for Biotechnology Information blast . Abundance and prevalence were compared between cohorts. Results. Low levels of C. difficile sequences were observed, with significantly higher prevalence in periodontitis (7.4%, n =15/204) vs. healthy cohorts (5.0%, n =4/80) ( P =0.0087) with reference genome alignment. Using the full non-redundant database, prevalence was also higher in periodontitis (14.2% vs. 3.8%; P =0.012), along with significantly greater average C. difficile sequence counts (0.608 vs. 0.075; P =0.018) and relative abundance (0.00029% vs. 0.0000003%; P =0.009). Conclusion. Sequences pertaining to C. difficile were detected in oral samples, with significantly more observed in periodontal disease compared to healthy cohorts. This highlights the possibility for dental plaque to act as a reservoir, potentially contributing to reinfection in CDI patients.
Introduction. Diabetes mellitus, a condition characterized by chronic hyperglycaemia, is categorized into type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM). Recent research has identified a significant association between diabetes and modifications in the gut microbiota.Gap Statement. Although numerous analyses have been conducted on the gut microbiota of patients with diabetes, regional variations due to factors such as lifestyle and dietary habits remain poorly understood.Aim. This study aimed to compare the gut microbiota structures of adult patients with T1DM, T2DM and healthy controls (HCs) in the Chaoshan region of China, providing theoretical support for gut microbiota-based targeted therapies for diabetes in the Chaoshan region.Methodology. This study enrolled 33 patients with T1DM, 35 patients with T2DM and 30 HCs in the Chaoshan region of China. Faecal samples were collected and subjected to 16S rDNA sequencing and bioinformatics analysis.Results. Our data demonstrated significant variations in gut microbiota diversity among individuals with T1DM, T2DM and HC, accompanied by changes in microbial composition across multiple taxonomic levels. Furthermore, linear discriminant analysis effect size analysis identified distinct dominant species within each group: 27 bacterial genera, including Megasphaera, were significantly enriched in T2DM patients; 27 bacterial genera, including Bacteroides, were significantly enriched in T1DM patients; and 18 bacterial genera, including Alloprevotella, were significantly enriched in the HC group. Metabolic pathway analyses using Kyoto Encyclopedia of Genes and Genomes (KEGG) and Clusters of Orthologous Genes (COG) databases demonstrated a significant enrichment of pathways and enzymes associated with starch and sucrose metabolism in both T1DM and T2DM cohorts, compared to HC. Additionally, a diagnostic model based on gut microbiota data at the class level yielded an area under the curve value of 0.861, indicating its high diagnostic efficacy in distinguishing between T1DM and T2DM. Furthermore, an analysis of the abundance of various bacterial phenotypes and probiotic species revealed notable differences among the three groups.Conclusion. This study demonstrates distinct gut microbiota composition, structure and functional profiles in Chaoshan T1DM, T2DM and HC populations, supporting microbiota modulation as a promising therapeutic strategy for diabetes.
Introduction. Endometriosis affects 10-20% of reproductive-age women. Emerging evidence links the gut microbiota to endometriosis pathogenesis, but observational studies are limited by confounding, reverse causation and uncertainty about whether reported microbial signals represent reproducible aetiological associations.Hypothesis/Gap Statement. Whether genetically predicted gut microbial genera are associated with endometriosis risk remains unclear, and available observational evidence does not establish robust causal effects after accounting for multiple testing.Aim. This study aimed to explore potential Mendelian randomization (MR)-based associations between genetically predicted gut microbiota composition and endometriosis using a two-sample MR framework.Methodology. Genome-wide association study (GWAS) summary statistics for 119 bacterial genera (MiBioGen consortium: n=18,340) and endometriosis (FinnGen: 8,288 cases, 68,969 controls) were used. Single nucleotide polymorphisms (SNPs) associated with each genus (P<5×10⁻⁵) were selected as instrumental variables after linkage disequilibrium clumping and weak instrument exclusion. The primary method was inverse-variance weighting (IVW), supplemented by four complementary methods. Benjamini-Hochberg false discovery rate (FDR) correction was applied across all 119 genera.Results. Seven genera showed nominally significant IVW associations with endometriosis (P<0.05): Lactococcus, Olsenella, Senegalimassilia, Ruminococcaceae UCG-002, Holdemania, Eubacterium ruminantium group and Anaerotruncus. Olsenella, Ruminococcaceae UCG-002 and Anaerotruncus were directionally associated with increased risk, whereas the remaining four were directionally associated with reduced risk. However, none survived FDR correction (all FDR-adjusted P=0.731). No significant heterogeneity or horizontal pleiotropy was detected.Conclusion. These findings provide exploratory and suggestive MR evidence for potential associations between specific gut microbial genera and endometriosis, rather than definitive causal evidence. As no associations survived multiple testing correction, these results should be interpreted as hypothesis-generating and require replication in larger, ancestry-matched cohorts.
Introduction. Listeria monocytogenes is a foodborne pathogen that poses a significant threat during pregnancy, frequently associated with adverse maternal and neonatal outcomes, including preterm birth, spontaneous abortion, stillbirth and neonatal sepsis.Hypothesis/Gap Statement. Despite its clinical relevance, there is a lack of whole-genome sequencing (WGS)-based data describing the genomic characteristics and circulating lineages of L. monocytogenes associated with maternal and neonatal infections in Kuwait, limiting regional epidemiological understanding.Aim. To genomically characterize L. monocytogenes isolates from maternal and neonatal clinical specimens at the Maternity Hospital, Kuwait.Methodology. Nine clinical isolates collected between 2017 and 2022, obtained from high vaginal swabs and blood cultures of unrelated mothers and neonates, were subjected to WGS. Antimicrobial susceptibility testing was performed using the VITEK-2 system. Genomic analysis included determination of sequence types (STs), clonal complexes (CCs), genomic characterization using multi-locus sequence typing (MLST), core-genome MLST (cgMLST), antimicrobial resistance (AMR) determinants and virulence gene profiles.Results. Seven isolates (S2-S8) were ST2 and belonged to CC2, whereas S1 and S9 were classified as ST3 and ST308 and belonged to CC3 and CC1, respectively. Phenotypic susceptibility profiles were correlated with genotypic findings, with a conserved resistome dominated by fosX (fosfomycin resistance) and vga(G) (lincosamide resistance). All isolates harboured conserved core virulence genes associated with stress response, quorum sensing, nutrient regulation, host cell invasion and intercellular survival. The cgMLST analysis demonstrated ≥99.5% genetic similarity among ST2/CC2 isolates, which clustered together and were genetically distinct from older local isolates (S1, S9) and international reference strains.Conclusion. This first WGS-based genomic characterization of L. monocytogenes in Kuwait demonstrates the repeated detection of ST2/CC2 across multiple maternal and neonatal cases over several years. These findings provide baseline genomic data and highlight the need for integrated, expanded genomic surveillance linking clinical, food and environmental isolates to improve source attribution, early detection and public health interventions for listeriosis.
Introduction: Cryptococcal meningitis (CM) is a leading cause of mortality among people living with human immunodeficiency virus (HIV) in Sub-Saharan Africa, requiring rapid diagnosis. Although the Food and Drug Administration (FDA)-approved Dynamiker cryptococcal antigen lateral flow assay (CrAg LFA) offers a potential point-of-care solution, its use remains limited in high-burden settings.Hypothesis/Gap statement: Despite FDA approval of Dynamiker CrAg LFA for the diagnosis of CM, the underutilization of the test in settings with a high burden of CM is of particular concern. Therefore, there is a need to validate its performance for the diagnosis of CM.Aim: To evaluate the diagnostic performance, turnaround time and cost-effectiveness of the Dynamiker CrAg LFA for CM, using cerebrospinal fluid (CSF) culture as the reference standard.Methodology: Stored CSF samples from 200 HIV-positive patients with suspected or confirmed CM (n=245) were tested using the Dynamiker CrAg LFA. Diagnostic metrics were calculated and compared to CSF fungal culture. Time to result and average test cost were also assessed.Results: The Dynamiker CrAg LFA showed a sensitivity of 100% and specificity of 84.9%, with strong agreement (κ=0.83) with CSF culture. Median time to result was 30 s compared to 3 days for culture. The average cost per test was USD 3.41 versus USD 19.80-22.40 for CSF culture.Conclusion: The Dynamiker CrAg LFA is a rapid, accurate and affordable diagnostic tool for CM and holds promise for broader implementation in resource-limited settings.
Introduction . Multidrug-resistant infections by the opportunistic pathogen Pseudomonas aeruginosa are common in healthcare settings but are increasingly difficult to treat due to high rates of antimicrobial resistance. Hypothesis/Gap Statement . Interest has developed in using R pyocins, bacteriocins produced by P. aeruginosa , as a species-specific treatment. We hypothesize that understanding the connections between disease type and R pyocin sensitivity will inform future treatment development. Aim . To evaluate the patterns of R pyocin sensitivity across a library of 247 clinical P. aeruginosa isolates and compare sensitivity profiles of strains when categorizing based on disease type, O-antigen serotype and encoded R pyocin type. Methodology . A combination of genomic analysis and R pyocin sensitivity assays using growth kinetics was conducted on 247 clinical isolates to evaluate genotype, phenotype and disease presentation in the context of R pyocins. Genomic analysis allowed in silico identification of O-antigen serotype and encoded R pyocin type, and phylogenetic comparisons revealed genetic relationships between strains. Results . Comparative analysis of clinical isolates showed that certain conditions, such as chronic infection in the lungs of patients with cystic fibrosis, increased sensitivity to R pyocins. We also observed strong correlations between encoded R pyocin subtype, sensitivity and O-antigen serotype. Finally, our data showed that a large percentage of strains had shared, broad-spectrum sensitivity to R pyocins, suggesting that most clinical isolates may be treatable with R pyocins. Conclusion . Our study reveals connections between disease type, O-antigen serotype and R pyocin sensitivity, which may inform future strain analysis and predict the effectiveness of R pyocin treatment.
Introduction. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is widely used for rapid micro-organism identification and has recently been explored for antifungal susceptibility testing (AFST).Hypothesis. Although MALDI-TOF MS has emerged as a promising tool for AFST, simplified and clinically applicable strategies for rapid fluconazole (FLZ) susceptibility detection in Candida spp. remain insufficiently validated. We hypothesized that a streamlined AFST-MS approach would demonstrate good categorical agreement (CA) with the European Committee on Antimicrobial Susceptibility Testing (EUCAST) reference method while significantly reducing turnaround time.Aim. To establish a simplified MALDI-TOF MS-based AFST approach for detecting FLZ resistance in Candida species.Methodology. Fifty-one clinical isolates and reference strains were incubated for 3 h in the presence of FLZ at two concentrations (32 and 4 µg ml-1) and in drug-free controls. Spectral profiles were compared with the EUCAST reference method.Results. Overall CA between AFST-MS and EUCAST was 85.2% (κ=0.7306). Species-specific accuracy was 100% for Candida auris, Pichia kudriavzevii (formerly Candida krusei), Candida tropicalis and Candida parapsilosis; 92.9% for Candida albicans and 40% for Nakaseomyces glabrata (formerly Candida glabrata); however, these estimates should be interpreted cautiously given the limited number of isolates per species. All discrepancies were minor errors, with no major or very major errors observed. The method reduced analysis time from 24 to 3 h and enabled presumptive FLZ susceptibility detection with good overall agreement with the reference methodConclusion. These findings support the potential of MALDI-TOF MS as a rapid adjunct tool for antifungal susceptibility assessment and may contribute to earlier therapeutic decision-making.
Introduction. Bloodstream infection is a critical cause of morbidity and mortality, and high-quality blood culture practice is essential for accurate diagnosis and antimicrobial stewardship, particularly in regional and remote healthcare settings. Gap Statement. Clinicians working in rural emergency departments (EDs) encounter unique systemic, workforce and logistical barriers to optimal blood culture practice, yet these challenges are not well characterized in the existing literature. Aim. To explore clinician-reported barriers and enablers to high-quality blood culture collection across regional EDs in Western Australia. Methodology. A qualitative study was conducted involving semi-structured interviews with doctors and nurses from three Western Australia Country Health Service EDs. Transcripts were analysed thematically to identify factors influencing blood culture ordering, collection technique, contamination and workflow integration. Results. Twenty-four clinicians participated, describing substantial delays between sample collection and laboratory processing, difficulty obtaining two sets per episode and limited feedback on contamination or blood volume adequacy. Workforce turnover, variable training and inconsistent guideline use contributed to practice variation. Key enablers included strong team culture, leadership from clinical champions, structured induction, refresher training and the use of dual-set kits and visual dashboards to provide timely, non-punitive feedback. Conclusion. High-quality blood culture practice in rural EDs is shaped by multifactorial systemic, workflow and workforce constraints and targeted, locally relevant interventions are essential to improve diagnostic yield and strengthen antimicrobial stewardship.
Introduction. Tuberculosis (TB) remains a major global health threat, and its control is hindered by prolonged treatment and increasing drug resistance. Helicase with zinc finger 2 (HELZ2) has recently been identified as an RNA-binding protein up-regulated after Mycobacterium tuberculosis (Mtb) infection, yet its functional contribution to host defence is not fully understood. Hypothesis/Gap Statement. Although HELZ2 expression increases following Mtb infection, the molecular mechanism by which HELZ2 regulates macrophage immunity, particularly autophagy-mediated bacterial clearance, remains unclear. Aim . This study aimed to elucidate the role and underlying mechanism of HELZ2 in macrophage-mediated elimination of Mtb, with a specific focus on its regulation of autophagy. Methodology. HELZ2 expression was quantified in the peripheral blood of TB patients and in Mtb-infected monocyte-derived macrophages. By knocking out and overexpressing genes, phagocytosis, intracellular bacterial survival and autophagy were analysed. Co-immunoprecipitation, chromatin immunoprecipitation and dual-luciferase assays were employed to identify HELZ2-interacting proteins and downstream transcriptional targets. Results. HELZ2 was significantly up-regulated in patient samples and infected macrophages. HELZ2 silencing impaired phagocytosis, reduced autophagic flux and increased intracellular Mtb survival. Mechanistically, HELZ2 bound to and stabilized the MYC proto-oncogene, bHLH transcription factor (MYC), which directly activated transcription of the autophagy-related gene ATG16L1. Overexpression of MYC or ATG16L1 restored the autophagy disorder caused by HELZ2 deficiency and enhanced bacterial clearance. Conclusion. HELZ2 enhances macrophage autophagy and promotes intracellular Mtb elimination by interacting with MYC and up-regulating ATG16L1. This newly identified HELZ2-MYC-ATG16L1 regulatory axis provides mechanistic insight into host defence and suggests a potential target for host-directed TB therapies.
Introduction. Respiratory syncytial virus (RSV) is a frequent cause of lower respiratory tract infections among children under 2 years old and adults over 60. Compared to other causes of viral pneumonia in adults, it is associated with a higher risk of intensive care unit admission, mechanical ventilation and 30-day mortality.Hypothesis/Gap Statement. Despite the global recognition of RSV as a cause of respiratory disease, the epidemiological information available on its distribution in Mexico and Latin America among adults is limited.Aim. To characterize the epidemiological, clinical and molecular features of RSV infections in the Mexican population during the 2023-2024 winter season.Methodology. An observational, retrospective and descriptive study was conducted including patients of all ages with RSV detected by reverse transcriptase/real-time PCR during the 2023-2024 winter season.Results. In our study, 50% of adults and 96% of paediatric patients with RSV were admitted for at least 24 h. The most frequent comorbidities in adults were systemic hypertension and diabetes. Approximately one-third of adults were over 65 years of age, and the overall in-hospital mortality in the adult group was 24%, in contrast to 6% in-hospital mortality in the paediatric population. Genomic analysis identified RSV serotype A as predominant. Complete genome sequencing revealed circulation of the A.D.1 clade, consistent with lineages from the southern USA, marking the first complete sequencing of RSV isolates from Mexican patients.Conclusion. RSV represents a significant cause of respiratory infection in Mexico across age groups, with substantial mortality in adults. The identification of the A.D.1 clade underscores the importance of continuous epidemiological and molecular surveillance for understanding RSV circulation patterns in the region.
