Background. Cervical screening with high-precision assays such as human papillomavirus (HPV) DNA testing is essential for the detection and treatment of precancerous lesions. HPV genotypes have different oncogenic potential and require different clinical management, illustrating the importance of extended genotyping. HPV quantification has demonstrated clinical relevance in both diagnosis and treatment. Objective. To develop a droplet digital PCR assay for the detection and quantification of 16 HPV genotypes, with comparison to a commercial test and validation on clinical samples. Methods. Primers and probes were designed to target the E6 region of 16 HPV genotypes: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, 73 and 82. Each target was evaluated to assess performance and reliability using synthetic DNA constructs, quantified international reference standards and clinical screening samples (n=303) genotyped using the Seegene Anyplex II HR HPV Detection assay. Results. Each assay demonstrated high target specificity, without cross-reactivity observed among the HPV genotypes selected. Using international molecular standards, the assay reliably detected high-risk genotypes across serial dilutions, with detection down to the level of one international unit or genome equivalent per microlitre. When applied to clinical samples with and without a histological diagnosis of cervical intraepithelial neoplasia 2 or worse (CIN2+), the assay reliably detected key HPV genotypes, with the exception of 7 of 234 (3%) of HPV-positive samples, all of which exhibited very low viral load. Conclusion. Although previous studies have described digital PCR methods targeting HPV E6, they have typically focused on a limited number of genotypes. This study expands upon existing methodology by introducing a sensitive and specific method for detection and quantification of 16 high-risk and potentially high-risk HPV genotypes.
Introduction. Gram staining is a fundamental and widely used technique in clinical microbiology because of its simplicity and diagnostic value, particularly in the diagnosis of bacterial meningitis. However, its reliability may be compromised by contamination and staining artefacts. Case Presentation. This report describes a case of pseudobacterial meningitis in a previously healthy 25-year-old woman who presented with fever, headache and nuchal rigidity. Cerebrospinal fluid (CSF) analysis revealed pleocytosis with a predominance of mononuclear cells. Gram staining revealed findings consistent with Gram-negative bacilli, prompting empiric antibacterial therapy with concurrent antiviral treatment. Comprehensive microbiological investigations, including CSF culture and broad-range 16S rRNA PCR testing, yielded negative results. Residual material or microscopic debris on supposedly ‘clean’ slides was suspected to produce artefact-like appearances that mimicked micro-organisms, leading to false-positive interpretations. Conclusion. This case highlights a potential laboratory pitfall in the interpretation of CSF Gram stains and underscores the importance of considering staining artefacts, particularly when Gram stain findings are discordant with the overall clinical and laboratory findings.
Background. Syphilis remains a diagnostic challenge due to its wide range of clinical manifestations, particularly in immunocompromised patients. Secondary syphilis may present with atypical cutaneous and neurological features, complicating timely recognition and management. High clinical suspicion is essential in such scenarios. Case Report. We describe the case of a 62-year-old man with a 6-month history of palmoplantar keratoderma, nail dystrophy, bilateral hearing loss and symmetrical proximal myopathy of the lower limbs. A comprehensive diagnostic approach, including clinical, histopathological, serological and cerebrospinal fluid (CSF) analyses, was undertaken. Skin biopsy revealed vacuolar interface dermatitis with a superficial perivascular inflammatory infiltrate, and immunohistochemistry confirmed the presence of spirochetes. Serological tests were positive for Treponema pallidum , and newly diagnosed human immunodeficiency virus (HIV) coinfection was identified. Despite a negative CSF Venereal Disease Research Laboratory (VDRL), the presence of pleocytosis, elevated protein concentration and hypoglycorrhachia supported a diagnosis of probable neurosyphilis. The patient was treated with intravenous crystalline penicillin G and initiated on antiretroviral therapy, resulting in full resolution of cutaneous and neurological symptoms. Conclusion. This case underscores the polymorphic nature of syphilis in immunocompromised individuals, particularly those with HIV infection. Clinicians should maintain a high index of suspicion for neurosyphilis even when CSF VDRL results are negative, provided that clinical and laboratory findings are supportive. Early diagnosis and appropriate treatment are critical for favourable outcomes.
Students in demanding microbiology and biomedical sciences courses often rely on suboptimal study strategies, such as massed practice, despite evidence favouring spaced retrieval and interleaving for durable learning, where durable learning is learning that is retained across longer periods of time. We developed U-Behavior, a learning management system-integrated pedagogy tool that coaches these behaviours through instruction, personalized visualizations of quiz attempt patterns [retrieval practice activity (RPA) graphs], reflections and behavioural feedback. In two experiments with identical RPAs in gateway (VMBS 100, introductory biomedical sciences) and upper division (MIP 300, general microbiology), we compared U-Behavior to low-stakes quizzing (control). U-Behavior significantly increased spacing and interleaving (large effects, ds >1.75) without raising total quiz attempts. Approximately half of U-Behavior students achieved optimal practice levels. No significant between-condition differences appeared in midterm/final exams, likely due to partial adoption. However, higher spacing scores predicted better exam performance (small-to-medium effects) beyond prior Grade Point Average (GPA) and attempt quantity. U-Behavior provides a scalable, behaviour-focused approach to enhance study quality in rigorous microbiology education.
Microbiology is often perceived by students as a challenging subject, particularly when addressing pathogenic mechanisms and disease transmission. Active learning approaches, including educational games, have been shown to enhance student engagement and conceptual understanding by integrating playful elements with scientific content. In this study, we present MicroBioCombat, a card-based educational game developed for use in secondary school microbiology education. The game comprises 81 cards representing pathogenic bacteria, transmission routes, and prophylactic or therapeutic strategies. During gameplay, students combine cards to simulate infection scenarios and corresponding defence mechanisms, with the aim of reducing their opponent’s score to zero. Beyond reinforcing microbiological concepts, MicroBioCombat promotes peer interaction, communication, and decision-making, contributing to a more dynamic and inclusive classroom environment. The game is adaptable to different educational levels and curricular contexts, and an accompanying online forum enables educators and students to share experiences and gameplay strategies, supporting collaborative and reflective learning.
Candida albicans is a yeast of major medical importance. The genome sequence for the reference isolate SC5314 has been available for many years and is displayed as a phased diploid assembly through a public community resource. As DNA sequencing technologies continue to advance, new high-quality data sets can be generated to augment existing genome resources. Here, we present Pacific Biosciences (PacBio) HiFi data sets with exceptional read length and sequencing depth. The data sets were generated from C. albicans SC5314 newly obtained from the American Type Culture Collection, thereby authenticating the isolate to a community-accessible source. The PacBio HiFi data set with a mean read length greater than 14 kb was assessed for its potential to support construction of a phased diploid genome assembly. Most chromosomes were effortlessly resolved as gapless, phased haplotypes with telomeres at both ends; however, challenging regions, including the rDNA locus on one copy of chromosome R, remained longer than could be fully resolved using these high-quality, state-of-the-art data. The PacBio HiFi reads data sets are available through GenBank and provide a valuable resource for exploring diploid loci in this important reference strain.
The Enterobacter cloacae complex (ECC) is an opportunistic Gram-negative bacterial group increasingly linked to multidrug resistance, healthcare-associated infections and diverse environmental reservoirs. However, the genomic relationships between clinical and environmental ECC populations, and the mechanisms supporting their adaptation, remain incompletely understood. This study conducted a comparative genomic analysis of 100 publicly available ECC genomes, including 62 clinical and 38 environmental isolates from different geographical regions. Analyses included average nucleotide identity, multilocus sequence typing, core-genome phylogeny, comparative genome architecture, resistome, virulome, plasmidome, integron, insertion sequence and mobile genetic element profiling. A balanced subset of 76 genomes was further used for pan-genome and source-specific functional analyses. Phylogenomic and ANI analyses showed extensive intermixing between clinical and environmental isolates, with no clear source-specific clustering. Several sequence types were shared across reservoirs, suggesting lineage overlap between clinical and environmental ECC populations. Comparative genome alignments revealed conserved chromosomal backbones together with variable accessory regions. Clinically important antimicrobial resistance genes, including bla CTX-M-15, bla NDM-1, bla NDM-5, bla KPC-2, bla KPC-4, mcr -9.1 and mcr -10, were detected, with carbapenemase genes occurring mainly in clinical isolates. Clinical genomes also carried significantly higher accessory virulence burdens than environmental genomes. Pan-genome analysis identified 18,440 gene clusters, including only 196 core genes, indicating a highly open pan-genome dominated by accessory genes. Environmental-specific genes were enriched for mobile genetic elements, while clinical-specific genes were enriched for metabolic and regulatory functions. Overall, the findings reveal strong genomic connectivity between clinical and environmental ECC populations and highlight the importance of mobile genetic elements in resistance dissemination, genome plasticity and ecological adaptation.
Accurate detection of respiratory viruses is essential for infection control, patient management and public health response. Nasopharyngeal swabs (NPS), collected by healthcare workers (HWC-NPS), remain the gold standard for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) detection but require trained personnel and can be uncomfortable for patients. Self-collected swabs, such as oral-nasal swabs (SC-ONS) and mid-turbinate swabs, offer scalable alternatives suitable for mass testing. Previous mid-turbinate swab designs included a plastic stopper and were not compatible with automated laboratory testing. This study compared the performance of (i) SC-ONS, (ii) an automation-friendly, redesigned version of mid-turbinate swab (SC-MTS) and (iii) HWC-NPS in detecting SARS-CoV-2. Between April and June 2022, paired NPS, ONS and MTS samples were collected from 100 participants at a Coronavirus disease 2019 (COVID-19) assessment centre in Hamilton, Ontario. Samples were tested for SARS-CoV-2 by reverse-transcriptase PCR. Compared to HWC-NPS, SC-ONS demonstrated 82.1% sensitivity and 100% specificity, while SC-MTS showed 75.0% sensitivity and 100% specificity. Agreement with HWC-NPS was strong for both SC-ONS (κ=0.863) and SC-MTS (κ=0.804). Agreement between SC-ONS and SC-MTS was nearly perfect (κ=0.944). Cellular material yields as well as SARS-CoV-2 viral loads were lower for self-collected swabs than HWC-NPS, while viral load comparisons revealed no significant difference between SC-ONS and SC-MTS. Our study aligns with previous work demonstrating the use of self-collected mid-turbinate swabs for the detection of respiratory viruses, while also demonstrating the compatibility of the newly designed swabs with automated laboratory instruments. Our results, while conducted in a relatively small sample, suggest that self-collected ONS and MTS are reliable alternatives to HWC-NPS, offering practical, less invasive, automation-friendly options for large-scale respiratory virus surveillance and pandemic preparedness.
The evolutionary analysis of bacterial species harbouring 16S rRNA sequences detected in the oil bean seeds of Ugba (Pentaclethra macrophylla) was carried out. The food product has a high socio-economic relevance to communities where it is consumed. Species such as Kurthia gibsonii, Stenotrophomonas geniculata and Alcaligenes nematophilus found in Ugba may have occurred in the environment and entered the sample in the field during or before harvest. The phylogenetic analysis of 35 sequences showed that some strains of the same species resolved into different monophyletic groups, suggesting species divergence or distinct evolutionary lineages. The species K. gibsonii was found to be the earliest ancestor following sequence-based ancestral analysis, suggesting that it was present in the analysed samples before other bacteria. The Ugba seeds appear to harbour a diverse group of bacteria and will benefit from metagenomic investigations as well as studies of the mechanism of survival and succession to reveal the true nature of the resident flora. This will help safeguard public health and highlight the organism's relevance to food safety surveillance and microbial evolution. Increased knowledge of the resident organisms will also lead to the improvement of fermentation techniques and enhance the quality of the final product.
We report the genome sequence of Escherichia coli GR536, a previously constructed metal-uptake-deficient strain derived from E. coli W3110. Growth of GR536 in an iron-restricted liquid medium resulted in apparent lysis during the early exponential growth phase. This effect was exacerbated in cells transformed with pBAD30, a commonly used arabinose-inducible expression vector. Whole-genome sequencing confirmed the expected gene disruptions ( entC , feoABC , mntH , zupT::cat and fecABCDE::kan ). However, comparison to E. coli W3110 identified the presence of the ϕ80 prophage (46.16 kbp) and cryptic prophage CPZ-55 (6.763 kbp), as well as the absence of cryptic prophage e14 (15.193 kbp). We also identified 9 IS-element deletions, 3 IS-element insertions, 7 other deletions or insertions and 74 candidate individual nucleotide changes. The growth defect in GR536 correlated with lysis due to the production of ϕ80 virions as determined by the inability of isolated phage to infect an E. coli strain lacking the phage receptor (∆ fhuA ) and the Bam HI digestion pattern of the purified phage DNA. We further determined that the ϕ80-dependent lysis in GR536 is exacerbated by the presence of the chloramphenicol- and kanamycin-resistance markers introduced during construction of GR536 and the pBAD30 plasmid multiple cloning site. Removal of the markers ( E. coli GR536*) and disruption of the pBAD30 multiple cloning site generated a strain that showed a 10 4 -fold reduction in ϕ80 production. Furthermore, construction of a W3110 lysogen containing the ϕ80 prophage and comparison with GR536* grown under the same conditions showed a ~10 4 -fold higher level of phage production by the parent strain, indicating that phage-dependent lysis was not increased by the deletion of the metal-uptake genes and thus independent of iron availability. These observations clarify growth conditions that limit the effects of ϕ80-dependent lysis when using GR536 to identify metal-uptake genes by complementation, specifically, removal of the antibiotic resistance markers and the avoidance of using intact pBAD30 as a negative control.
Stenotrophomonas maltophilia is a gram-negative bacterium associated with nosocomial infections and high mortality, although reported risk factors vary. We retrospectively analyzed 43 cases of S. maltophilia bacteremia at NTT Medical Center Tokyo (2002–2024). The overall mortality rate was 44.2%. Univariate analysis revealed that higher Sequential Organ Failure Assessment (SOFA) scores (p = 0.010), central venous catheterization (CVC; p = 0.028), intravenous hyperalimentation (p = 0.003), and diabetes mellitus (p = 0.033) were significantly associated with mortality. Multivariate analysis suggested SOFA score (p = 0.048) and CVC presence (p = 0.039) as independent risk factors. Appropriate antimicrobial therapy was not significantly associated with reduced overall mortality. However, it was associated with improved survival in patients with SOFA scores ≥5 (p = 0.029). Additionally, among patients with CVC, catheter removal was linked to better outcomes (p = 0.005). These findings suggest that the SOFA score and CVC use as key mortality risk factors in S. maltophilia bacteremia and indicate that antimicrobial therapy effectiveness may depend on disease severity. The study underscores a potential need to implement diverse infection control strategies to prevent S. maltophilia transmission and tailor antimicrobial therapy according to disease severity.
We describe a method using fluorescein-5-isothiocyanate (FITC)-labelled peptidoglycan sacculi to characterize peptidoglycan hydrolases in vitro . In this protocol, soluble hydrolytic products released by peptidoglycan hydrolases are separated from the insoluble FITC-sacculi, quantified and set in relation to the overall available substrate. This enables the study of hydrolase activity in end-point or time-course experiments.
Antimicrobial resistance increasingly poses a significant global health challenge. New antimicrobials are desperately needed, driving the need to explore novel sources of natural products. Snake venom has shown promise due to the complex mixture of proteins and peptides which demonstrate therapeutic utility in other aspects of healthcare. In this study, we took an iterative approach to screen 14 snake venoms against Escherichia coli and Klebsiella pneumoniae and identify venom-derived proteins with diverse activity against E. coli . Six venoms exhibited significant near-complete inhibitory activity, and four were retained after dialysis, confirming that the inhibitory effect was due to high-molecular-weight components above 3.5 kDa. Fractionation of two active venoms from Naja mossambica and Naja subfulva revealed protein fractions which have either subtle or significant antimicrobial activity. SDS-PAGE and mass spectrometry identified phospholipase A₂ isoforms in N. mossambica venom fractions and neurotoxic proteins (long and short neurotoxins) in N. subfulva venom fractions as potential antimicrobials.
Pseudomonas aeruginosa is a pathogen notorious for its antimicrobial resistance and is currently classified as a high-priority pathogen for which new drugs are needed. Tetrasodium EDTA (tEDTA) is one of the new antimicrobial compounds that have been shown to have good antibacterial and antibiofilm efficacy against P. aeruginosa. Due to the diversity and highly drug-tolerant nature of P. aeruginosa biofilms in different infection environments, it is important to carry out pre-clinical testing of new antibiofilm agents against this pathogen in media and models that accurately mimic diverse infection environments. In this study, we used different high-validity media and biofilm models that mimic chronic wounds, endotracheal tubes and cystic fibrosis lung infections to assess the efficacy of tEDTA against P. aeruginosa biofilms. We report that different infection environments influence the susceptibility of both planktonic and biofilm forms of P. aeruginosa to tEDTA. The highest tolerance to tEDTA was observed in the media and biofilm model that mimics the endotracheal tube environment. In conclusion, we show that although different infection environments influence the efficacy of tEDTA against P. aeruginosa biofilms, it has good potential for use as an alternative antimicrobial in treating P. aeruginosa-associated biofilm infections.
Fungal contamination of cereal crops threatens global food security by compromising grain quality and introducing harmful mycotoxins that endanger human and animal health. In West Africa, the long-standing ancestral use of native plant extracts to manage fungal contamination provides sustainable and locally adaptable solutions. Building on this knowledge, BioCC+, a polyherbal aqueous formulation containing Azadirachta indica, Cymbopogon citratus, Allium sativum and Capsicum frutescens, was developed to control maize contamination during storage in Côte d'Ivoire. This study evaluated the antifungal activity of BioCC+ against Fusarium graminearum and Fusarium verticillioides followed by bioactivity-guided untargeted metabolomics to explain observed effects. Crude BioCC+ extracts inhibited F. graminearum at 12.5 mg ml-1 and F. verticillioides at 25.0 mg ml-1, with the dichloromethane extract (DCMe) showing greater antifungal activity than methanol or water extracts. Based on the observed growth inhibition, DCMe was selected for fractionation using silica gel column chromatography with the following mixtures of hexane:ethyl acetate as the mobile phase: 100 : 0 for F1, 75 : 25 for F2, 50 : 50 for F3, 25 : 75 for F4 and 0 : 100 for F5, as well as 100% methanol for F6. Fractionation of DCMe resulted in three active fractions (F3, F4 and F6), with F4 showing the strongest inhibition (MIC=0.4 mg ml-1) against F. graminearum. Integration of antifungal activity assays with chemometric data through multivariate, univariate and correlation-based analysis identified six putative antifungal candidates, two known and four uncharacterized metabolites. These results provide a first step for integrating bioactivity assays with metabolomics to validate ancestral knowledge and develop community-driven practices for mitigating fungal contamination in staple crops.
The phyllosphere is host to diverse microbial communities surviving in dynamic environmental conditions and which form important relationships with their hosts. Here, we constructed metagenome-assembled genomes (MAGs) from 25 temperate forest phyllosphere samples collected in Eastern Canada. We found 423 dereplicated MAGs with completeness ≥50% and contamination ≤10%, using a combination of co-assembly strategies. The MAGs were predominantly classified into the bacterial phyla Pseudomonadota (n=197), Actinomycetota (n=88) and Acidobacteriota (n=50) and included two archaeal MAGs in the phylum Thermoproteota. These genomes can help to improve reference database entries of phyllosphere-affiliated microbes, increasing our understanding of phyllosphere microbial phylogenomic and community dynamics and the ecological roles of phyllosphere microbiomes.
Background. Cervical screening with high-precision assays such as human papillomavirus (HPV) DNA testing is essential for the detection and treatment of precancerous lesions. HPV genotypes have different oncogenic potential and require different clinical management, illustrating the importance of extended genotyping. HPV quantification has demonstrated clinical relevance in both diagnosis and treatment. Objective. To develop a droplet digital PCR assay for the detection and quantification of 16 HPV genotypes, with comparison to a commercial test and validation on clinical samples. Methods. Primers and probes were designed to target the E6 region of 16 HPV genotypes: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, 73 and 82. Each target was evaluated to assess performance and reliability using synthetic DNA constructs, quantified international reference standards and clinical screening samples ( n =303) genotyped using the Seegene Anyplex II HR HPV Detection assay. Results. Each assay demonstrated high target specificity, without cross-reactivity observed among the HPV genotypes selected. Using international molecular standards, the assay reliably detected high-risk genotypes across serial dilutions, with detection down to the level of one international unit or genome equivalent per microlitre. When applied to clinical samples with and without a histological diagnosis of cervical intraepithelial neoplasia 2 or worse (CIN2+), the assay reliably detected key HPV genotypes, with the exception of 7 of 234 (3%) of HPV-positive samples, all of which exhibited very low viral load. Conclusion. Although previous studies have described digital PCR methods targeting HPV E6, they have typically focused on a limited number of genotypes. This study expands upon existing methodology by introducing a sensitive and specific method for detection and quantification of 16 high-risk and potentially high-risk HPV genotypes.
Clostridium tetani, the causative organism of tetanus, is unusual in developed countries, including Singapore, and to our knowledge, only three known cases of C. tetani bacteraemia have been reported worldwide. This case report describes a case of C. tetani bacteraemia in a Singaporean patient without clinical manifestations of tetanus, likely arising from a sacral sore, highlighting the need for early recognition and targeted antimicrobial treatment for the condition and its complications.
Background Enterohemorrhagic Escherichia coli (EHEC) is an important etiologic agent of foodborne illness. Since the main source of EHEC is contaminated meat and fresh produce, these ingredients should be sanitized using methods that achieve efficient mitigation of bacterial load under organic-matter-rich conditions. Materials and Methods We examined the effect of chlorous acid water (CAW), which is a chlorite (HClO2)-based disinfectant, on EHEC of three major serogroups (O157, O26 and O111) and compared its bactericidal activity with that of sodium hypochlorite (NaClO). Results CAW with >25 mg/L free available chlorine (FAC) achieved a >4 log10 CFU/ml reduction in the numbers of all tested EHEC strains after 1 min of treatment, while >500 mg/L FAC in NaClO was needed to obtain the equivalent effects. CAW with low FAC concentrations (5 or 10 mg/L) showed a time-dependent increase in EHEC cell reduction even under high organic matter load, whereas NaClO required a FAC threshold to achieve an efficient bactericidal effect. Discussion The results in this study indicate that CAW is more effective than NaClO in the presence of organic matter. CAW with >20 mg/L FAC reduced the number of EHEC O157 cells adhering to bean sprouts by >3 log10 after 30 min of treatment, indicating that CAW is a suitable sanitizer for food processing.
Osteoarticular tuberculosis is a rare but serious form of extrapulmonary tuberculosis that often presents with nonspecific symptoms, leading to delayed diagnosis and increased risk of joint destruction. We report a case of knee tuberculosis in an immunocompetent patient, underscoring the diagnostic challenges and emphasizing the role of molecular techniques in the early management of this uncommon form of tuberculosis.
