Understanding the basis of fungal pathogenesis requires knowledge of pathogen biology that is built through studies of gene function and regulation. The critical first step in nearly all these studies is genetic transformation: the generation of targeted DNA sequence modifications through the introduction of exogenous DNA into the cell. For research focused on gene regulation, or where small precise mutations are desired, the maintenance of genomic context (i.e. surrounding DNA sequences) is important, as the disruption of flanking DNA elements can alter gene expression and confound results. This often makes the inclusion of selectable markers that are physically linked to the sequence of interest unsuitable and complicates the transformation process. Here, we present a co-transformation strategy in the human pathogen Aspergillus fumigatus that can be used to make precise, marker-free gene edits at a locus of interest without disturbing flanking DNA sequences. By simultaneously introducing a marker-free, modified copy of the gene of interest and a plasmid that directs the integration of a selectable marker to a different locus, this approach takes advantage of the benefits of selection, with results similar to that of a truly markerless strategy.

Introduction. Antimicrobial resistance (AMR) is a complicated public health challenge. This study aimed to obtain a baseline assessment of undergraduate health and science students’ knowledge and attitudes of antibiotic use, resistance and stewardship across European countries and to evaluate education methods.Methods. A 43-item cross-sectional multilingual survey of healthcare practitioners and undergraduates studying dentistry, medicine, nursing, pharmacy and science subjects was conducted by Public Health England (now UK Health Security Agency) in 2018 across 30 EU/EEA countries. Of the 43 questions developed for healthcare workers, a subset of 33 questions directly relevant to students was available for student completion.Results. A total of 1,222 students from 27 EU/EEA countries participated in the survey, with 50% studying medicine (379/760). The mean score across seven knowledge questions was 6.04 out of 7 (sd, 1.14). Knowledge scores differed by the degree being studied and were higher among students in the later years of their degree programme. Knowledge was significantly higher (P<0.001) in those who had received training on prudent antibiotic use and infection management. Most students had not heard about AMR awareness campaigns, including European Antibiotic Awareness Day, and felt they did not have a key role in addressing AMR.Conclusion. Although students demonstrated good overall knowledge of antibiotic use and AMR, many lacked awareness of their role in tackling AMR. Designing more effective targeted educational interventions for these students, such as curriculum development and interprofessional education and training, could be beneficial to support appropriate antibiotic use and efforts to tackle AMR.

The resurgence of syphilis has necessitated novel prophylactic strategies, such as the use of doxycycline post-exposure prophylaxis. However, the potential for increased doxycycline use to select for tetracycline resistance represents significant challenges in managing this sexually transmitted infection. This study aims to identify chromosomal mutations associated with tetracycline resistance in Spirochaetales to inform molecular surveillance tools. Whole-genome sequences (WGSs) from the Spirochaetales order, including 4,355 genomes, were analysed for the presence of mutations in 16S rRNA and non-synonymous mutations in the rpsC and rpsJ genes. The study utilized WGS from GenBank® and sequence data from the PubMLST Treponema pallidum isolate collection. Genetic resistance to tetracycline was detected using a combination of blastn searches and gene–gene analysis. A transition mutation TGA to TGG at positions 965–967 in the 16S rRNA gene was detected in 5.6% of Treponema spp. and 4.0% of Spirochaeta spp. genomes. The rpsJ gene exhibited a V57G aa substitution across a significant subset of Treponema spp. (n=14) and Spirochaeta spp. (n=1). Notably, the V57K substitution was present in Spirochaeta spp. (n=17) and Treponema spp. (n=15). The rpsC gene had the H178Q mutation and was found to be present in the Spirochaetales bacterium (n=4). The identification of putative mutations associated with tetracycline resistance in Spirochaetales provides a foundation for the development of rapid molecular tests. This study underscores the complexity of antibiotic resistance mechanisms and the critical importance of surveillance of genetic resistance determinants in the era of antibiotic prophylaxis for sexually transmitted infection management.

Background. Influenza C virus (ICV) is a lesser known member of the Orthomyxoviridae family, primarily causing respiratory tract infections in children. Co-infection with WU polyomavirus (WUPyV), a recently identified human polyomavirus, has been rarely reported. This study presents the first laboratory-confirmed case of ICV infection in Sri Lanka and its co-infection with WUPyV.Methods. Nasopharyngeal and oropharyngeal swabs were collected from children aged 3 months to 14 years with respiratory tract symptoms between November 2022 and February 2023. Samples were screened using multiplex real-time PCR (RT-PCR) and Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) RT-PCR. A nasopharyngeal swab from a 14-month-old infant showing an insignificant curve in respiratory PCR was subjected to whole-genome sequencing using the Illumina platform. Data were analysed for genomic characterization, and phylogenetic analysis was performed using the haemagglutinin-esterase gene of ICV.Results. Full-genome sequencing identified ICV and WUPyV in the sample. Phylogenetic analysis revealed that the ICV isolate belonged to the C/Sao Paulo lineage. The patient presented with mild symptoms, including fever, cough and cold, with normal inflammatory markers, and recovered with supportive care.Discussion. This case highlights the importance of considering ICV in paediatric respiratory illnesses, despite its under-diagnosis due to limited diagnostic tools. Unlike influenza A and B, ICV lacks neuraminidase, rendering neuraminidase inhibitors ineffective. The absence of ICV in current influenza vaccines further complicates preventive strategies. Co-detection of WUPyV raises questions about its role as a co-pathogen, with its clinical significance requiring further investigation.Conclusion. This report underscores the need for enhanced molecular diagnostic techniques and surveillance to better understand the epidemiology and clinical impact of ICV and its co-infections.

Tuberculosis remains a major public health concern, particularly in countries where it is still endemic. Tuberculous bursitis and tenosynovitis are rare extrapulmonary manifestations, and their association with systemic autoimmune diseases such as scleroderma is seldom reported in the literature. We report the case of a 61-year-old patient with systemic scleroderma, complicated by diffuse interstitial lung disease and treated with mycophenolate mofetil, who developed tuberculous shoulder bursitis and wrist extensor tenosynovitis. The microbiological diagnosis was confirmed by ultrasound-guided aspiration of the subacromial-subdeltoid bursa, revealing the presence of Mycobacterium tuberculosis, detected by Ziehl–Neelsen staining, GeneXpert PCR and culture. Histological analysis of synovial tissue fragments demonstrated epithelioid granulomas with caseous necrosis, confirming the tuberculous origin.

Ticks are arthropod vectors that transmit pathogens important to human and animal health. The objective of this work was to identify Uukuvirus lihanense in the metatranscriptome of hard ticks. Between October 2022 and June 2023, ticks were collected from rural areas of the Colombian Caribbean area of the departments of Córdoba and Cesar. High-throughput sequencing (next-generation sequencing) was performed using MGI’s DNBSEQ-G50RS. Bioinformatics analyses were performed in Galaxy, diamond and IQ-TREE2. A total of 766 ticks were collected; 87.33% (669/766) were Rhipicephalus microplus, 5.4% (42/766) Dermacentor nitens, 4.2% (32/766) Rhipicephalus sanguineus and 3.0% (23/766) Amblyomma dissimile. Complete and partial L and S segments of Uukuvirus lihanense (LITV) were detected in the metatranscriptome of A. dissimile, D. nitens and R. microplus. The LITV sequences found are phylogenetically related to those detected in R. sanguineus and A. variegatum from the French Antilles, in R. microplus from Trinidad and Tobago and R. microplus from Brazil. LITV was identified in D. nitens and R. microplus; the first report was in A. dissimile. Although LITV is not considered necessary in public health, the virus belongs to the Phenuiviridae family, which includes viruses of public health importance, such as Dabie banda-virus and Bandavirus heartlandense.

Whole-genome sequencing (WGS) of microbial pathogens provides a high-resolution approach to antibiotic resistance profiling, lineage classification and outbreak surveillance. Identification of SNPs across the genome by alignment against a reference genome is the highest precision method of delineating strains. SNiPgenie is a bioinformatics pipeline designed to perform the entire variant calling process across many samples simultaneously. It was developed in the context of developing WGS tools to support the tracking of infection transmission of Mycobacterium bovis in livestock and wildlife, the principal causative agent of bovine tuberculosis in these populations. SNiPgenie may, however, be applied to other bacteria where evolutionary change can be tracked accurately using SNPs. The tool comes with both a command line and a user-friendly graphical interface. It can run on standard desktop or laptop computers. SNiPgenie and its documentation are available at https://github.com/dmnfarrell/snipgenie.

Background.Pseudomonas aeruginosa is a key pathogen in cystic fibrosis (CF), driving pulmonary decline and exhibiting resistance through virulence factors and adaptive mutations. Cefiderocol (FDC) is a novel siderophore cephalosporin with activity against Gram-negative bacteria. We aimed to assess the in vitro efficacy of FDC against P. aeruginosa isolates in a CF population.Methods. The study was conducted in a tertiary hospital with a specialist adult CF service. All first isolates of significant respiratory pathogens among this cohort are cryopreserved at −80 °C. Antimicrobial susceptibility testing to FDC was performed as per European Committee on Antimicrobial Susceptibility Testing Disk-Diffusion (version 10) for all stored isolates of P. aeruginosa from 2017 to 2022 inclusive.Results. Eighty-five isolates from seventy-one patients were included. Resistance phenotypes comprised 19% (n=16) multidrug-resistant (MDR), 16% (n=14) extensively drug-resistant (XDR) and 24% (n=20) pandrug-resistant (PDR), with 24 % (n=20) exhibiting the mucoid phenotype. Overall, 85% of isolates were susceptible to FDC, with a mean inhibition zone of 25.2 mm. Antimicrobial activity was retained in 81% of MDR, 86% of XDR, 60% of PDR and 90% of mucoid isolates. Seventy-four per cent of meropenem-non-susceptible isolates remained susceptible to FDC, compared with lower susceptibility to ceftolozane–tazobactam (42%), tobramycin (36%) and ciprofloxacin (22%).Conclusion. FDC exhibited excellent in vitro activity against P. aeruginosa from adults with CF, including highly resistant and mucoid phenotypes. These findings highlight its potential as a salvage option in this high-risk population and provide the first Irish surveillance data to inform antimicrobial stewardship and future clinical use.

Chlorous acid water and sodium hypochlorite solution are effective disinfectants against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the virus that caused the pandemic. Recent studies have shown that both compounds have equivalent inactivation effects when tested on purified viruses. However, in practical applications, the presence of organic matter is common and can significantly affect disinfectant performance. We conducted several experiments comparing these two disinfectants under different conditions to better understand their practical efficacy. When an infected cell culture medium (serum-free) was used as the test virus, chlorous acid water and sodium hypochlorite solution showed reduced efficacy. This decrease was attributed to the presence of aa in the medium. Notably, sodium hypochlorite solution showed a more pronounced reduction in potency compared with chlorous acid water. In addition, we evaluated the SARS-CoV-2 inactivation effects of chlorous acid water and sodium hypochlorite solution under various organic loading conditions simulating real-world contamination scenarios such as blood, vomit and saliva. The organic materials used included BSA, SRBCs, polypeptone, FBS and artificial saliva. The results showed that chlorous acid water demonstrated superior resilience to organic matter interference compared with sodium hypochlorite solution. These results suggest that chlorous acid water may be more effective than sodium hypochlorite solution in inactivating viruses on contaminated surfaces, particularly in healthcare settings where organic contamination is common. In summary, our research suggests that chlorous acid water may be a more effective disinfectant in practical settings.

Azoles inhibit the cytochrome P450-dependent enzyme lanosterol 14α-demethylase (CYP51) that is encoded by the ERG11 gene. Azole resistance in Candida species arises through different mechanisms, like mutations in the ERG11 gene, ERG11 overexpression, CDR1,2 (Candida drug resistance) overexpression that actively efflux azole drugs, reducing their intracellular concentration and therapeutic effectiveness, and biofilm formation. We sequenced the ERG11 gene to determine mutations in the coding and non-coding regions of ERG11 in clinical isolates of Candida glabrata (Nakaseomyces glabratus) from Pakistan. Eight C. glabrata (N. glabratus) strains from our fungal strain bank (five fluconazole-resistant and three susceptible dose-dependent) were revived and used. The ERG11 gene was amplified by PCR, sequenced using the Sanger methodology and analysed using bioinformatic tools. We identified a change in nucleotide at c. -66 T/G upstream of the start codon ATG in the promoter region of the ERG11 gene in fluconazole-resistant C. glabrata (N. glabratus). Within the downstream (coding region), where numbering begins at the ATG start codon as position +1, two novel synonymous mutations at positions T300C and T834C and previously reported synonymous mutations T768C, A1023G, T1557A and A1581G were also observed. This is the first study evaluating ERG11 mutations in C. glabrata (N. glabratus) from Pakistan. The clinical significance of such uncommon ERG11 gene mutations, such as c. -66 T/G, should be explored further through correlation with treatment outcome data.