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Introduction. Mycobacterium tuberculosis has held an exceptional presence in medical research, especially in recent years (1). While pulmonary infections largely remain the focus of literature and public health initiatives (2), extrapulmonary tuberculosis (EPTB) infections have been gaining prominence due to their increasing prevalence, particularly in populations characterized by a low incidence of tuberculosis (2-4). Although EPTB infections, such as tuberculous meningitis, carry a high mortality risk (5), delays in diagnosis and treatment persist. These delays are driven by their diverse, non-specific clinical presentations, coupled with the variable performance of diagnostic modalities like the Cartridge-Based Nucleic Acid Amplification Test (CBNAAT) (6-10). While general literature regarding the use and efficacy of CBNAAT in diagnosing EPTB exists (2,11-15), studies focusing on its sensitivity and specificity in paucibacillary specimens are an evolving niche (16). Since there is a greater prevalence of paucibacillary EPTB specimens in our setting, where such an analysis has not been conducted, we performed a retrospective study to understand the overarching diagnostic performance of our existing modalities in EPTB specimens. Methods. An observational retrospective study was conducted with the data collected between 2019 and 2022 at a tertiary care centre in central Kerala, India. A total of 105 specimens - categorized as pleural, lymph node, pus, tissue, cerebrospinal fluid (CSF), peritoneal and others - from presumptive EPTB cases were included in the study. The samples underwent acid-fast bacilli staining microscopy, liquid culture and CBNAAT utilizing the GeneXpert platform. The results were recorded, and the data were analysed with P<0.05 considered statistically significant. Observations and Results. In the 105 samples collected for the study, 6 (5.7%) were positive on Ziehl-Nielsen staining, 26 (24.8%) samples grew M. tuberculosis complex on liquid culture and CBNAAT detected M. tuberculosis in 16 (15.2%) specimens. The sensitivity and specificity of CBNAAT, with culture as the gold standard, were found to be 50.0 and 96.2%, respectively. The sensitivity and specificity of CBNAAT for pus, peritoneal (including tissue and fluid aspirate), pleural fluids, CSF, lymph nodes and other miscellaneous sample types ranged from 33.3 to 100% and 85.7 to 100%, respectively. Among smear-negative specimens, the sensitivity and specificity of CBNAAT were 42.9 and 96.2%, respectively, whereas in smear-positive specimens, the sensitivity increased to 80.0%. Conclusion. CBNAAT is a viable tool for the detection of M. tuberculosis; however, in paucibacillary specimens, its use along with other methods like smear and culture could lead to better identification of the mycobacteria.

Urinary tract infections are a major global health concern, with Klebsiella pneumoniae emerging as a prominent multidrug-resistant uropathogen. The increasing prevalence of extended-spectrum β-lactamase-producing strains has reduced the effectiveness of β-lactam antibiotics. In this study, 87 clinical K. pneumoniae isolates from India were analysed using whole-genome sequencing, MLST and antimicrobial resistance profiling. β-Lactam resistance was the dominant phenotype, with blaSHV and blaCTX-M identified as the most prevalent class A β-lactamase genes. Molecular docking and 100 ns molecular dynamics simulations were conducted to examine enzyme-antibiotic interactions. Meropenem exhibited the lowest binding affinity with both proteins (-2.695 kcal mol^-1 for blaSHV and -1.849 kcal mol^-1 for blaCTX-M), forming unstable complexes, particularly with blaCTX-M. These findings highlight the growing challenge of β-lactam resistance and emphasize the need for genomic surveillance and alternative therapeutic strategies against multidrug-resistant K. pneumoniae.

Stenotrophomonas maltophilia is an emerging non-fermenting Gram-negative bacillus increasingly implicated in healthcare-associated infections. Its remarkable ability to form biofilms on inert surfaces and invasive medical devices, together with its intrinsic and acquired resistance mechanisms, makes it a major therapeutic challenge in hospital settings. This study aimed to describe the epidemiological characteristics and antimicrobial susceptibility profile of S. maltophilia isolates recovered in our microbiology laboratory. A retrospective study was conducted on clinical isolates of S. maltophilia obtained from various clinical specimens. The bacterial examination was performed according to standard microbiological guidelines Over the study period, the prevalence of S. maltophilia isolates was 0.25% among all bacterial isolates (34 out of 13,959). Respiratory specimens were the most frequent source (23.53%, n=8), followed by blood cultures (17.65%, n=6). Most isolates originated from intensive care units (47.06%, n=16). Trimethoprim-sulfamethoxazole retained high efficacy with a susceptibility of 81.48%, whereas Minocycline demonstrated the highest activity, with a susceptibility rate of 100%. In contrast, the high resistance rates were observed for ceftazidime (61.90%). S. maltophilia represents an emerging nosocomial pathogen with significant therapeutic implications. Continuous monitoring of local resistance profiles, along with strengthened infection prevention measures and appropriate antibiotic use, is essential to limit the spread of resistant strains.

The rapid emergence of diseases and parasites in aquatic wildlife requires improved methodologies to identify and characterize new and future pathogens. While microinjection of pathogens directly into a model organism, such as zebrafish, enables the exploration of infection and immune response in the host, such methodology focuses primarily on identifying causative agents in events of aquatic wildlife mortality due to acute infection. Here, we present an updated protocol of infection by static immersion in larval zebrafish to investigate the possible effect of prolonged environmental exposure to an opportunistic pathogen. By controlling microbial growth and monitoring mortality over 72 h, we show that static immersion can detect minute differences in virulence profiles between and within different strains of Pseudomonas aeruginosa, an important opportunistic pathogen of animals and humans. We then conducted two sets of passive exposure virulence assays in Caenorhabditis elegans, an alternative model. We demonstrated the virulence phenotype, while showing slight differences between experimental models, and showed similar trends. We believe that passive exposure thus offers a practical host-pathogen model that simulates opportunistic infection occurring in the environment and enables the detection of minute changes in virulence between and within bacterial strains.

Introduction. Despite the established pathogenicity of Aggregatibacter aphrophilus, few clinical cases have been described in published literature. A. aphrophilus is a rare cause of prosthetic valve endocarditis and diagnosis is challenging based on nonspecific symptoms on presentation. High clinical suspicion for infective endocarditis is warranted in patients with cardiac prosthetic material and positive blood cultures. Poor dentition and dental procedures, along with other risk factors, are associated with A. aphrophilus bacteraemia. We describe a case of A. aphrophilus bacteraemia in which the diagnosis of infective endocarditis was complicated by equivocal echocardiographic findings and an initially challenging Gram stain interpretation. Case Presentation. A 59-year-old male patient with a history of bioprosthetic aortic valve replacement, atrial septal defect repair and remote dental cleaning presented to the emergency department with persistent high fevers, headaches and lethargy. Initial blood cultures were drawn and were positive for 'Gram-positive organisms', but specific morphology was challenging to determine. Subsequent testing, including MALDI-TOF, confirmed the growth of A. aphrophilus. Conclusion. Rapid identification of A. aphrophilus and recognition of the potential for infective endocarditis are paramount to ensuring adequate treatment initiation and success. This case highlights the diagnostic difficulty associated with initial Gram stain interpretation of this fastidious organism.

Mycolic acids are critical components of the Corynebacteriales cell envelope, contributing to cellular morphology, permeability and antibiotic resistance. While the core enzymes responsible for mycolic acid synthesis are relatively well described, little is known about the accessory factors that coordinate their biosynthesis and integration into the cell envelope. In this study, we investigated the role of AhfA in Corynebacterium glutamicum using genetic, biochemical and physiological approaches. Loss of AhfA resulted in broad effects on cell growth, morphology, lipid composition and cell envelope integrity, which were restored upon complementation. Our analysis suggests that AhfA acts as an early stage of mycolic acid synthesis supporting the formation of the lipid-rich outer cell envelope. Notably, a similar reduction in mycolic acid-containing glycolipids was observed in mycobacterial species, suggesting that the role of AhfA in mycolic acid metabolism is conserved across Corynebacteriales. This conservation suggests that accessory proteins involved in mycolic acid biosynthesis and homeostasis may represent potential targets for therapeutic intervention. These findings identify AhfA as a previously uncharacterized factor in cell envelope assembly and highlight the complexity of lipid integration in Corynebacteriales.

Neonatal sepsis caused by multidrug-resistant Klebsiella pneumoniae contributes substantially to neonatal morbidity and mortality, especially where treatment options and genomic surveillance are limited. Phenotypic microbiology defines susceptibility, whereas Illumina short-read whole-genome sequencing (WGS) links phenotypes to lineage, capsule, resistome, virulome and mobilome contexts. However, complete characterization of mobile genetic element (MGE) structure remains inferential. This secondary analysis re-examined 411 public K. pneumoniae genomes from the University Teaching Hospital neonatal outbreak in Zambia. The population was dominated by ST307/KL102/OL2α.2, a comparatively recent but globally expanding high-risk clone, accounting for 284 out of 411 genomes (69.1%). Minority genomes included established resistance or virulence-associated high-risk backgrounds, including carbapenemase-positive KL15/OL4 lineages and yersiniabactin-positive ST147/ST985. Extended-spectrum β-lactamase (ESBL) genotypes were widespread (396 out of 411; 96.4%) and mainly driven by blaCTX-M-15 (406 out of 411; 98.8%), whereas carbapenemase carriage was restricted to blaNDM-5-positive ST5856/ST340 (11 out of 411; 2.7%). Plasmids were the principal acquired resistance layer, as most AMR genes were plasmid-associated, and similarity analysis revealed a cohesive ST307-linked IncFIB/IncFII AA277 ESBL background, in contrast to distinct minority ESBL and IncX3 blaNDM-5 architectures. This pattern supports clonal maintenance of a stable ESBL plasmid background within ST307, while identifying IncX3 blaNDM-5 plasmids in minority lineages as a key convergence risk. Resistance genes were further structured by integron cassettes, insertion-sequence modules and plasmid cargo containing metal-, biocide- or stress-response genes. Virulence was limited and mainly ICEKp/ybt-associated, and widespread predicted prophages did not carry priority determinants. These findings show that WGS can add mechanistic resolution to routine phenotypic surveillance by identifying clone-MGE configurations that warrant targeted monitoring in neonatal K. pneumoniae outbreaks in Zambia.

Background. Africa is home to several indigenous dairy products that span the continent. These dairy products are widely consumed and cherished for their rich nutritional profile and distinctive taste, shaped by microbial fermentation. However, their microbiome remains largely unexplored, thus limiting knowledge of microbial composition, diversity and functional inference. These limitations have implications for product quality and safety and scientific documentation. Methods. The 16S rRNA dataset on five popularly consumed dairy products, Nono, Wara, Kwerionik, Ghee and Nunu, with the SRA accession number PRJNA532858, was collected for taxonomic classification and diversity analysis using the Quantitative Insights into Microbial Ecology 2 pipeline. Functional inference was conducted using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States 2 version 2. Results. In this dataset, an average of 12 phyla were observed, including Firmicutes, Proteobacteria, Actinobacteria and Bacteroidetes. At the genus level, lactic acid bacteria, particularly Lactobacillus and Streptococcus, were predominant in Nono, Wara and Nunu, with relative abundances: 57.7, 33.5 and 39.3% for Lactobacillus and 16.6, 36.7 and 39.5% for Streptococcus. Functional predictions revealed both shared and product-specific metabolic capabilities, with varying taxonomic contributions to key pathways across samples. Conclusion. Across the analysed samples, the products exhibit both conserved and distinct predicted functional signatures inferred from the 16S rRNA gene data. Particularly, those related to ATP-binding cassette transporters, amino acid metabolism, sugar utilization and biosynthetic pathways. Streptococcus dominated in the purine-rich environments of Nono and Nunu; Wara displayed lactic acid bacteria co-dominance, with a focus on peptidoglycan metabolism; Kwerionik exhibited broader microbial diversity, with a three-way taxonomic distribution; and Ghee stood out with Acetobacter-driven lipid and oxidative metabolism, showcasing the community's adaptive plasticity to distinct environmental and processing conditions. These findings suggest that traditional dairy fermentations function as complex microbial ecosystems and highlight their potential importance in food systems and microbial ecology.

'Candidatus Sarcina troglodytae' ('Ca. S. troglodytae'), a proposed bacterial species in the family Clostridiaceae, has been epidemiologically associated with a lethal disease in sanctuary chimpanzees in Sierra Leone, epizootic neurologic and gastroenteric syndrome (ENGS). As sarcinae are known to be difficult to culture, a lack of viable in vitro growth conditions has limited further characterization of this bacterium. Such studies are critical to elucidating the relationship between 'Ca. S. troglodytae' and ENGS. Here, we isolate a Gram-stain-positive, endospore-forming coccus and anaerobic bacterial strain, designated JB3^T, from brain tissue of a western chimpanzee (Pan troglodytes verus) using a reinforced clostridial medium. We describe the growth of strain JB3^T at a pH range of 6 to 9 (optimum 6) at 37 °C in an atmosphere composed of 5% H₂, 5% CO₂ and 90% N₂ and demonstrate endospore production under alkaline conditions, suggesting the possibility of an environmental reservoir. We identify ethanol as the primary non-gaseous fermentation byproduct which we hypothesize to have relevance for ENGS. Antibiotic susceptibility testing shows susceptibility to common antimicrobial agents with notable resistance to ceftriaxone. By establishing in vitro conditions for the viable culture, propagation and storage of 'Ca. S. troglodytae', this study lays the groundwork for future infection studies to test hypotheses about pathogenic mechanisms and prevention/treatment measures for ENGS.

This secondary in silico study analysed 36 Escherichia coli draft genome assemblies from Zambia to characterize the virulence gene profiles of extraintestinal pathogenic E. coli (ExPEC) and to relate them to mobilome proxies, namely plasmid replicons and prophage content. All assemblies met plausibility thresholds for E. coli (1-17 contigs; 4.85-5.77 Mb; G+C 50.47-50.96 mol%), and species identity was confirmed by average nucleotide identity to the E. coli type strain (96.6-98.9%). Using the Achtman seven-locus MLST scheme applied to the reads, 35 of 36 genomes were assigned a sequence type (ST), and 1 remained untypeable at a single locus. The most frequent type was ST69 (9 out of 36) rather than the globally dominant ST131 (6 out of 36), followed by ST405 (5 out of 36) and ST617 (5 out of 36). Phylogroup D was most frequent (12 out of 36), followed by the commensal-associated phylogroup A (8 out of 36) and the pandemic phylogroup B2 (6 out of 36). Classical intestinal-pathotype hallmarks (stx1, stx2, eae, bfpA and ipaH) were not detected. In contrast, ExPEC-associated markers were common, including iutA (27 out of 36), iss2 (26 out of 36), fyuA (24 out of 36), kpsM (23 out of 36) and ompT (18 out of 36), with a high within-genome burden (median ~193 loci; range 173-251). Gene-based rules classified 19 of 36 genomes as ExPEC and 2 as enteroaggregative E. coli, and source-informed analysis identified uropathogenic, neonatal meningitis-associated and sepsis-associated subpathotypes. IncF-family replicons predominated, sitABCD occurred in both chromosomal and IncF-plasmid contexts, and iss2 was recurrently prophage-associated. These findings indicate that extraintestinal virulence in this population is distributed and modular rather than confined to ST131. This virulence is carried across diverse lineages and phylogroups, including classically commensal backgrounds, and is mobilized by plasmids and prophages. Ultimately, these data provide a baseline for the genomic surveillance of virulent E. coli in Zambia.