Pathogens doi: 10.3390/pathogens15070673

Authors: Reuven Yosef

Domestic cats (Felis catus) are ubiquitous companion animals that provide substantial psychological and social benefits to children and adults alike, but they also serve as reservoirs and vectors for a wide range of zoonotic pathogens. Close physical contact between cats and children, frequent use of shared environments such as homes, playgrounds, and sandboxes, and still-developing hygiene behaviours increase opportunities for exposure to protozoa, helminths, bacteria, fungi, and ectoparasite-borne agents. This review synthesizes current evidence on key feline-associated zoonoses of pediatric concern—including Toxoplasma gondii, Toxocara cati, Ancylostoma spp., Dipylidium caninum, Bartonella henselae, Salmonella enterica, Campylobacter jejuni, Pasteurella multocida, Microsporum canis, flea-borne Rickettsia species, and rabies—with emphasis on transmission routes, clinical manifestations, and risk modifiers in children, pregnant women, and immunocompromised individuals. Within a One Health framework, we also summarize global publication trends on feline zoonoses, discuss how urban cat ecology and management (including free-ranging cats in child-frequented environments) may shape pediatric risk, and outline practical prevention strategies centred on hygiene, veterinary care, and targeted education for caregivers and children.

Pathogens doi: 10.3390/pathogens15070672

Authors: D. P. Grosa D. Zavadska Z. Freimane L. Karele G. Karelis

Background: Tick-borne encephalitis (TBE) is an endemic neuroinfectious disease prevalent in parts of Europe and is often associated with persistent neurological and cognitive sequelae. The aim of this study was to evaluate the long-term outcomes and predictors of post-encephalitic sequelae in adult patients with TBE in Latvia. Methods: A retrospective cohort with prospective follow-up was used that included 105 adult patients hospitalized with laboratory-confirmed TBE between 2018 and 2024. The patients’ clinical and demographic data were extracted from medical records, and reassessments were performed ≥6 months after discharge using structured clinical and neurological evaluations for neurocognitive, subjective, and neurological sequelae. Disease severity was classified using the Mickienė and Bogovič criteria, and sequelae severity was defined according to the Bohr criteria for post-encephalitic syndrome (PES). Results: Sequelae were observed in 52/105 (49.5%) patients and were more frequent in meningoencephalitis than in meningitis cases (18/25 [72.0%] vs. 33/77 [42.9%]). The most common persistent symptoms were impaired concentration (33/52 [63.5%]), fatigue (29/52 [55.8%]), and sleep disturbances (21/52 [40.4%]). Neurological sequelae included tremor (23/52 [44.2%]), vertigo (11/52 [21.2%]), and hearing impairment (5/52 [9.8%]). According to the Bohr criteria, most of the patients had mild sequelae (42/52 [80.8%]), while 10/52 [19.2%] had moderate sequelae; no severe cases were observed. In the multivariable analysis, increasing age was independently associated with greater sequelae severity (OR = 1.045 per year; 95% CI, 1.015–1.073; p = 0.003). Sex, comorbidities, biphasic disease, and length of hospital stay were not significant predictors. Acute neurological manifestations, particularly paresis (p = 0.002) and tremor (p = 0.019), were associated with worse outcomes. Although the disease severity scores correlated with sequelae in unadjusted analyses, neither the Mickienė nor the Bogovič classification independently predicted outcomes after adjustment. Conclusions: Nearly half of the hospitalized patients with TBE included in this study developed long-term sequelae, which were predominantly neurocognitive and mild in severity. Age was the primary independent predictor of worse outcomes, while acute neurological deficits such as paresis and tremor also indicated increased risk. These findings highlight the substantial burden of post-encephalitic syndrome and the need for structured long-term follow-up in TBE survivors.

Pathogens doi: 10.3390/pathogens15070671

Authors: Antonios Synapalos Anastasia Diakou Stefanos Sgardelis

Brown bear populations in Greece face multiple threats, and parasitic infections may pose an additional risk to these vulnerable animals. This study represents the first comprehensive assessment of endoparasite occurrence, prevalence, and seasonality in brown bears in Greece, in relation to geographical location and the animal’s different physiological phases. A total of 918 faecal samples were collected over a three-year period from regions with brown bear presence in Greece. For each sample, the date of collection and the coordinates of the site were recorded. Samples were examined using sedimentation, flotation, and McMaster techniques, while the Baermann method was additionally applied to a subset of 195 samples. Spatial and temporal patterns in parasite occurrence and diversity were analysed using generalised additive models (GAMs). Ten parasitic taxa were identified, with Baylisascaris transfuga being the most prevalent (39.8%), followed by Crenosoma spp. (26%), Uncinaria spp. (18.09%), and Dicrocoelium dendriticum (14.38%). Less prevalent taxa included Eucoleus aerophilus, Sarcocystis spp., Toxascaris leonina, Eimeria spp., Linguatula serrata, and Taeniidae. Μixed infections, involving two or more parasites, were detected in 22% of the samples. The prevalence of B. transfuga was higher in late autumn, with high-risk infection areas identified in both late summer and autumn. In contrast, Uncinaria spp. and D. dendriticum showed no seasonal variation, while D. dendriticum exhibited spatial clustering patterns similar to B. transfuga but without clear seasonal trends. These findings highlight the widespread occurrence and complexity of parasitic infections in Greek brown bears. Continued long-term monitoring is essential to improve understanding of transmission dynamics and the ecological processes shaping parasite distribution in this animal species.

Pathogens doi: 10.3390/pathogens15070670

Authors: Peter Collignon John J. Beggs Jan M. Bell Denise Daley Elizabeth Roughead on behalf of the Australian Group on Antimicrobial Resistance

Background: Antimicrobial resistance (AMR) is widely considered to be driven by antimicrobial consumption through within-host selection. However, whether this mechanism adequately explains population-level patterns of resistance in invasive infections remains uncertain. If antimicrobial use is the dominant determinant, resistance should be highest in demographic groups with the greatest exposure. Methods: We conducted a retrospective analysis of 44,792 community-onset Escherichia coli bloodstream infection episodes identified through national Australian surveillance data (2013–2024). Resistance prevalence across individual antimicrobials and composite multidrug resistance panels was analysed by age and sex. These data were compared with community antimicrobial dispensing derived from the Pharmaceutical Benefits Scheme. Mean resistance was modelled as a function of age and sex. Results: Antimicrobial use was substantially higher in females than males (~23% overall) and increased markedly with age, with individuals aged ≥80 years receiving approximately three times more antimicrobials than those aged 25–30 years. In contrast, resistance was consistently lower in females across most antimicrobials and composite measures. Resistance demonstrated an inverted U-shaped age distribution, peaking at 30–40 years before declining in older age groups. From early adulthood to older age, antimicrobial dispensing increased threefold, whereas mean resistance declined by approximately 20%. These patterns were consistent across antimicrobial classes, years, and jurisdictions. Conclusions: These findings show that demographic patterns of antimicrobial resistance in community-onset E. coli bloodstream infections are not well explained by a simple population-level consumption model. These findings should be interpreted as important hypothesis-generating insights. Although antimicrobial exposure remains important for individual-level selection, the observed discordance between prescribing and resistance suggests that other factors, including differences in transmission pathways, healthcare contact, disease prevalence, community sanitation and socioeconomic circumstances may also significantly shape resistance patterns.

Pathogens doi: 10.3390/pathogens15070669

Authors: Raikhan Mussagalieva Ziyat Abdel Zauresh Zhumadilova Aigul Abdirassilova Svetlana Issaeva Bolatbek Baitursyn Nurbol Shaki Beck Abdeliyev Dinmukhammed Otebay Tatyana Meka-Mechenko

Camels are increasingly recognized as an important epizootological and epidemiological link in natural plague foci, contributing to the transmission of Yersinia pestis from wildlife to humans. In the Republic of Kazakhstan, where natural plague foci occupy up to 40% of the territory, the rapid growth of camel populations may significantly enhance epidemiological risks. The aim of this study was to perform a comprehensive assessment of the role of camels in the epizootology and epidemiology of plague based on retrospective data (1907–2003) and contemporary monitoring (2000–2025), including spatial analysis and risk zoning. A total of 64 cases of camel plague and 43 epizootic foci were identified during the historical period. More than 400 human cases, including fatal outcomes, associated with infected camels were documented, with direct contact during slaughter and meat processing accounting for 94.7% of infections. Spatial analysis and epidemiological zoning revealed a heterogeneous risk distribution, with western and southern regions representing the highest-risk areas. Serological investigation (n = 2726) showed 75.6% seropositivity, likely reflecting substantial population immunity largely associated with vaccination. Despite increasing camel population size (from 227.7 to 304.0 thousand heads in 2020–2025), vaccination coverage varied between 32.0% and 51.0%, reflecting risk-based preventive strategies. The absence of recent camel plague cases supports the effectiveness of integrated control measures, including vaccination, surveillance, and the establishment of protective zones. These findings suggest that camels remain an important component of plague transmission systems and should be systematically integrated into surveillance programs within a One Health framework.

Pathogens doi: 10.3390/pathogens15070668

Authors: Alexander O. Frolov Anna I. Solovyeva Marina N. Malysheva Maria E. Belokon Grigory N. Machakhtyrov Varvara A. Machakhtyrova Anatoly A. Bondarev Maria S. Maximova Anna I. Ganyukova

The subgenus Megatrypanum Hoare, 1964, with the type species Trypanosoma theileri Laveran, 1902, comprises stercorarian trypanosomes of mammals. A substantial portion of this subgenus consists of T. theileri-like trypanosomes parasitizing cervids and bovids worldwide. Similar to most other members of the genus Trypanosoma that lack obvious economic importance, the biology of T. theileri-like trypanosomes remains poorly understood. In particular, fundamental aspects such as their host specificity, host–parasite interactions, and the morphology of developmental stages have been studied only to a limited extent. In this work, we provide a detailed description of the development and cellular organization of T. theileri-like trypanosomes in the horsefly Hybomitra montana using transmission electron microscopy (TEM). We show for the first time that T. theileri-like trypanosomes possess a well-developed cytostome–cytopharyngeal complex, morphologically similar to those in other stercorarian trypanosomes. This complex is present in the studied trypanosomes at the epimastigote stage and degrades during metacyclogenesis. In the host ileum, epimastigotes and trypomastigotes at different stages of metacyclogenesis are embedded in a fibrillar matrix that isolates them from the gut lumen. This promotes their accumulation in the vector, thereby increasing the efficiency of future infection of the vertebrate host, which occurs via contamination of the oral mucosa.

Pathogens doi: 10.3390/pathogens15070667

Authors: Marianna Imparato Annalisa Buonanno Angela Maione Monica Matuozzo Chiara D’Ambrosio Andrea Scaloni Marco Guida Emilia Galdiero Elisabetta de Alteriis

This study describes extracellular vesicles (EVs) isolated from the culture supernatant of a Kluyveromyces marxianus strain deriving from an artisanal sourdough. Previous work had clearly shown the probiotic properties of the yeast isolate and its antagonistic activities against clinical fluconazole-resistant Candida albicans strains. Characterization of the isolated EVs by nanotracking particle analysis showed they had a mean diameter of 157.7 nm. Proteomic characterization of the purified EVs identified a complex array of 100 proteins. Both C. albicans planktonic growth and biofilm formation were inhibited by K. marxianus EVs, as well as adhesion and invasion of Candida cells in the vaginal epithelial A-431 cells. In the same cell model, K. marxianus EVs exerted an immunomodulatory effect affecting the secretion of pro-inflammatory and anti-inflammatory cytokines. Further, the expression of C. albicans SAP2 and SAP6 genes, coding for two aspartyl proteases involved in the invasion and damage of the epithelial mucosa, was affected by the presence of the yeast EVs. Overall, the results of this study show that K. marxianus EVs retain, at least in part, the beneficial features of the live microorganism, representing a postbiotic cell-free alternative preparation potentially useful for the management of C. albicans vaginal infections.

Pathogens doi: 10.3390/pathogens15070666

Authors: Silvia Garattini Lorenza Romani Luana Coltella Tommaso Alterio Stefania Mercadante Costanza Tripiciano Maia De Luca Sara Chiurchiù Laura Cursi Francesca Ippolita Calò Carducci Cristina Russo Carlo Federico Perno Alberto Villani Andrea Pietrobattista Stefania Bernardi Laura Lancella

Pediatric acute liver failure (PALF) is a rare but life-threatening condition characterized by rapid clinical deterioration and high mortality. Viral infections represent a major etiology of PALF, although the causative agent remains unidentified in a substantial proportion of cases. Human Enteroviruses (EVs) are typically associated with self-limiting illnesses; however, they may rarely cause severe systemic disease, including fulminant hepatitis, particularly in neonates and young children. We describe the case of a 4-year-old previously healthy male who presented with acute fulminant hepatitis secondary to systemic Echovirus 25 (E25) infection, with concomitant Epstein–Barr virus (EBV) co-infection of recent onset. The diagnosis was established through multiplex PCR on cerebrospinal fluid, blood, stool, and nasopharyngeal aspirate, with serotype confirmation by the Italian National Institute of Health. The patient required intensive supportive care including therapeutic plasma exchange (TPE), continuous kidney replacement therapy (CKRT), and intravenous immunoglobulins (IGIV). Despite initial clinical deterioration and placement on the liver transplant list, the patient achieved complete hepatic recovery and was discharged after fourteen days of hospitalization without requiring transplantation. This case highlights the importance of prompt virological workup including enterovirus PCR in children presenting with acute liver failure of undetermined etiology and supports the use of extracorporeal therapies as a bridge to recovery.

Pathogens doi: 10.3390/pathogens15070665

Authors: Jose Arturo Molina-Mora Ravi Kant

Background: Bacterial adaptation to environmental and chemical stress involves coordinated, system-level responses collectively described as perturbome. Understanding conserved elements within core perturbomes may reveal strategic vulnerabilities for antimicrobial development. Methods: In this study, we implemented an integrative framework combining functional and comparative genomics, drug–target interactions and molecular docking to prioritize conserved stress-response targets in Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. Results: A total of 147 genes from previously defined core perturbomes were analyzed through interactome reconstruction and functional enrichment. Interactome and functional analyses revealed significant connectivity and functional clustering, primarily associated with molecule biosynthesis, translation, transcriptional regulation, and energy metabolism. Orthology-based comparative genomics identified six conserved orthogroups shared across at least two species, representing key stress-adaptive nodes including fatty acid synthesis initiation, metabolic stress buffering, transcription termination (Rho), ATP synthesis, peptidoglycan remodeling, and UDP-glucose-mediated envelope biosynthesis. Drug–target interaction analyses suggested that these conserved proteins are modulated by enzymatic inhibitors, metabolite analogs, or active-site competitors. Structural and docking analyses focused on a selected protein, FabF (β-ketoacyl-ACP synthase II) and confirmed catalytically coherent binding of cerulenin within the active site, with high concordance between experimentally resolved and AlphaFold-predicted structures, supporting the reliability of structure-based prioritization. Conclusions: Overall, the results demonstrate that bacterial stress responses converge on evolutionarily conserved metabolic and regulatory elements essential for homeostasis and tolerance to perturbations, being the first work integrating core perturbome data from different microorganisms. The proposed perturbome-informed framework provides a rational strategy to identify robust, broad-spectrum antimicrobial targets and highlights opportunities for drug repurposing and future experimental validation.

Pathogens doi: 10.3390/pathogens15070664

Authors: Laura Vitiello Stefania Proietti Dolores Limongi Carla Prezioso Caterina Mammi Massimiliano Caprio Fabrizio Maggi Guido Antonelli Stefano Bonassi Patrizia Russo

Chronic obstructive pulmonary disease (COPD) is characterized by systemic inflammation, immune dysregulation, and increased susceptibility to infections. Obesity may influence these processes and has been proposed as a potential contributor to the so-called “obesity paradox”, although its effects on immune competence, viral burden, and survival are not yet fully understood. Seventy patients with severe to very severe COPD (GOLD stage 3–4) were stratified according to BMI (<30 vs. ≥30 kg/m2). Clinical and functional parameters were assessed together with biomarkers of oxidative stress, DNA damage, systemic inflammation, and T-cell subsets. A comprehensive viral panel, including Torque Teno virus (TTV), was also analyzed. Five-year survival was evaluated using Kaplan–Meier curves and Cox regression models. Patients with BMI ≥ 30 showed higher lymphocyte counts and increased CD4+ and CD8+ T-cell levels, accompanied by lower systemic inflammatory indices. No significant differences were observed in oxidative stress or DNA damage markers. In addition, TTV viremia (≥4 log10 copies/mL) was more frequently observed among patients with lower BMI. Despite these differences, five-year survival did not significantly differ between the two groups. These findings suggest that BMI alone may have limited value as a predictor of outcomes in patients with advanced COPD. Conversely, immune-inflammatory indices and viral burden, particularly TTV viremia, could provide complementary information for risk assessment and may deserve further investigation as potential tools for personalized patient stratification.

Pathogens doi: 10.3390/pathogens15070663

Authors: Marat Kumar Symbat Suleimenova Sardor Nuralibekov Yermukhammet Kasymbekov Temirlan Sabyrzhan Kuanysh Isbekov Saule Assylbekova Victor Fefelov Berik Pangereyev Kobey Karamendin Aidyn Kydyrmanov

Freshwater amphipods of the genus Gammarus are important trophic components of aquatic ecosystems and are increasingly considered a potential bioresource for aquaculture. However, their role in the maintenance and transmission of infectious agents remains poorly understood. This study evaluated the presence of major crustacean and fish pathogens in Gammarus lacustris populations from Kazakhstan and characterized associated viral communities using metagenomic sequencing. Six pooled samples collected from freshwater ecosystems across Kazakhstan were screened using PCR and RT-PCR assays targeting World Organisation for Animal Health (WOAH)-listed pathogens, including White Spot Syndrome Virus, Taura Syndrome Virus, Infectious Myonecrosis Virus, Aphanomyces astaci, and Aphanomyces invadans. In parallel, high-throughput sequencing (Illumina NovaSeq) was performed to assess virome composition and structure. No WOAH-listed pathogens were detected, suggesting a low detectable occurrence of major notifiable agents under the conditions of the present study. Metagenomic analysis revealed a virome dominated by RNA viruses, particularly picorna-like viruses (Picornaviridae), Dicistroviridae, and Marnaviridae. Phylogenetic and genome organization analyses identified potentially novel or highly divergent viral lineages within Picornavirales. Collectively, these findings suggest a favorable epizootiological profile of G. lacustris populations while highlighting freshwater amphipods as hosts of diverse and partially uncharacterized viral communities relevant to aquatic disease surveillance and aquaculture biosecurity.

Pathogens doi: 10.3390/pathogens15070662

Authors: Rolf Claesson Jessica Radu Zeinab Razooqi Anders Johansson Jan Oscarsson

The oral pathogen Filifactor alocis encodes a repeats-in-toxin (RTX) protein, FtxA, that is encoded by the ftxA gene; it is present in approximately 50% of known isolated strains from various infected oral sites, including periodontitis, peri-implantitis, and root canal infections. It has been determined from PCR assessment of periodontally diseased cohorts in Ghana and Australia. Based on current knowledge, ftxA appears to be associated with both the progress and severity of periodontitis. This finding could potentially be linked to enhanced levels of ftxA-positive F. alocis, relative to ftxA-negative strain, and/or, in addition, a synergy between ftxA-positive strains and other periodontal pathogens. The exact mechanism remains unclear but may depend on an FtxA-mediated shifting of the host cell response toward immunosuppression. The main objective of the present work was to evaluate the prevalence and loads of F. alocis and the presence of ftxA in subgingival plaque in patients recruited for periodontal treatment in Sweden. This observational study included all samples that were received from external clinics over one full year (n = 71 patients). Our findings revealed that F. alocis was carried by 49 (69%) of the individuals, with the prevalence of ftxA amounting to 42.9% (n = 21). In 32 of the 71 samples, F. alocis could be quantitatively assessed. In this sub-population of F. alocis-positive patients, high loads of the bacterium were not related to age, and high loads were more frequently observed upon carriage of ftxA. The presence of, and co-colonization with, F. alocis with four additional periodontal pathogens was also evaluated. F. alocis was notable in that it co-colonized with all of the other species. Moreover, it was detected alongside two and even three of the other species within the same sample.

Pathogens doi: 10.3390/pathogens15070661

Authors: M. Fernanda San Martin Nicol Quiroga Arnau Casanovas-Massana Carezza Botto-Mahan Antonella Bacigalupo Pedro E. Cattan Patricio Arroyo Juan Contardo Rodrigo Salgado Esteban Yefi-Quinteros Juana P. Correa

Although pathogenic Leptospira DNA has been detected in water and soil from different climatic regions, information from Mediterranean-climate agricultural systems remains limited. This study characterized the environmental detection of pathogenic Leptospira DNA in water and soil samples from irrigated agroecosystems of central Chile, evaluating spatial and seasonal variation and associations with selected physicochemical variables. A total of 605 samples were collected from eight agricultural sites during spring 2019, summer 2020, and winter 2021. Samples were analyzed by real-time PCR targeting lipL32. Overall, 29.1% of samples were PCR-positive, and pathogenic Leptospira DNA was detected in all sites and seasons. Soil samples showed higher positivity than water samples (34.5% vs. 21.4%), and positivity was higher in summer (41.7%) than in spring (22.7%) or winter (19.3%). Water temperature and turbidity were the only physicochemical variables that differed between positive and negative samples, whereas the binomial generalized linear mixed model (GLMM) showed that season and sample type were associated with PCR positivity after accounting for site-level clustering. These results show that pathogenic Leptospira DNA can be widely detected in irrigated agricultural systems from a Mediterranean-climate region, suggesting that soil, seasonality, irrigation practices, and other site-level characteristics should be considered in future studies on the environmental ecology of pathogenic Leptospira.

Pathogens doi: 10.3390/pathogens15070660

Authors: Ricardo Lopes Andreia Garcês Vanessa Silva Hugo Lima de Carvalho Filipe Sampaio Gonçalo Barros Cátia Fernandes Ana Patrícia Lopes Cátia Marques Luís Cardoso Elsa Leclerc Duarte Ana Cláudia Coelho

In 2022, the World Health Organization (WHO) published the Fungal Priority Pathogens List (FPPL), yet its application has remained largely focused on human medicine, with limited consideration of animal hosts and veterinary diagnostics. This retrospective study aimed to characterise the occurrence of WHO critical- and high-priority yeasts in veterinary clinical submissions in Portugal within an explicit One Health framework. All yeast-positive submissions received by a Portuguese veterinary diagnostic laboratory between 2019 and 2026 were reviewed. Isolates were identified phenotypically, by an automated identification system and by MALDI-TOF mass spectrometry. Data on host species, sample type and year of submission were analysed using standard descriptive and inferential statistics. Among 2033 mycological submissions, 219 were yeast-positive. Out of these, 82 isolates (37.4%) corresponded to WHO critical- or high-priority taxa, most frequently Candida albicans, followed by Nakaseomyces glabratus, Cryptococcus neoformans var. neoformans, the Candida parapsilosis complex and Candida tropicalis. The remaining 137 isolates (62.6%) corresponded to non-WHO taxa, among which the most frequent were Papiliotrema laurentii and Debaryomyces hansenii (n = 21 each; 15.3%), followed by the Stephanoascus ciferrii complex (n = 15; 11.0%), Meyerozyma guilliermondii and Candida sake (n = 12 each; 8.8%), and Wickerhamomyces anomalus (n = 9; 6.6%). WHO-prioritised taxa were recovered predominantly from ornamental birds, as well as from dogs, cats and marine mammals. These findings demonstrate that FPPL-listed yeasts are regularly detected among yeast-positive veterinary diagnostic submissions and highlight ornamental birds as prominent hosts within this dataset.

Pathogens doi: 10.3390/pathogens15070659

Authors: Mairi Ziaka

The gut–microbiota–brain axis (GMBA), an intricate network connecting the gastrointestinal (GI) tract and the brain, plays a pivotal role in maintaining overall health and influencing disease processes. The human gut microbiota, comprising over 3000 bacterial species, regulates immune responses, hormonal signals, and metabolite production, maintaining homeostasis under normal conditions. Dysbiosis, or microbial imbalance, has been linked to various central nervous system (CNS) disorders, including Alzheimer’s disease (AD), Parkinson’s disease (PD), multiple sclerosis (MS), and autism spectrum disorder (ASD). Given the growing interest in this topic and the limited effectiveness of current therapeutic strategies for managing patients with AD, the purpose of the current narrative review is to analyze the pathophysiological role of the GMBA in the pathogenesis of AD and assess potential therapeutic strategies targeting the GMBA, particularly the microbiome and its metabolites. A comprehensive literature search was conducted using PubMed, Scopus, and Web of Science to identify clinical studies, experimental research, and review articles examining the GMBA in health and AD, as well as related therapeutic strategies. The search terms included “Alzheimer’s disease”, “neuroinflammation”, “amyloid-beta”, “tau”, “gut–brain axis”, “microbiome”, “short-chain fatty acids”, “probiotics”, “prebiotics”, and “fecal microbiota transplantation”. In AD, altered gut microbiota composition is associated with neuroinflammation, neurodegeneration, and exacerbation of disease progression. Probiotics have shown potential in enhancing cognitive function and reducing neuroinflammation by modulating microbiota composition and influencing brain-derived neurotrophic factor (BDNF) levels. Prebiotics, through their impact on gut microbiota and metabolite production, also offer therapeutic promise by improving cognitive function and mitigating neuroinflammation. With its historical and modern applications, fecal microbiota transplantation (FMT) may represent a potential strategy for addressing dysbiosis and its neurological implications. This manuscript focuses on GMBA and its effects on neuroinflammation, neurodegeneration, and CNS health while emphasizing the need for further research into microbiome-based therapies and the gut–brain relationship in patients with AD.

Pathogens doi: 10.3390/pathogens15070658

Authors: Mehmet Karakuş Ayda Yılmaz Mert Okbay Metin Pekağırbaş Ozge Erisoz Kasap

Sand fly midgut microbiota plays a critical role in shaping Leishmania development and vector competence, yet functional evidence from natural vector populations remains limited. In this study, sand flies were collected between 2020 and 2022 in Cukurova region, Türkiye to characterize the gut bacterial composition of Phlebotomus tobbi and evaluate the anti-leishmanial potential of cultivable isolates. A total of 1739 sand flies were captured (878 females, 861 males), of which Ph. tobbi was the predominant species (n = 1312). 16S rRNA amplicon sequencing (V4–V6) showed that the gut microbiota was dominated by Proteobacteria, with Erwinia aphidicola/persicina representing the most abundant species across all analyzed groups. Fourteen cultivable bacterial species were identified by MALDI-TOF MS, including Serratia liquefaciens, Pantoea agglomerans, and Micrococcus luteus. Functional XTT assays against Leishmania infantum promastigotes demonstrated variable inhibitory activity among isolates. The strongest leishmanicidal effects were observed with S. liquefaciens (32.3%) and M. luteus (28.8%). Morphological examination confirmed promastigote rounding and cell death in isolates showing >25% activity. These findings define the gut bacterial landscape of Ph. tobbi in an endemic region and identify bacterial taxa with in vitro anti-leishmanial activity, highlighting their potential for future microbiota-based or paratransgenic control strategies.

Pathogens doi: 10.3390/pathogens15060657

Authors: Sveinung Wergeland Sørbye Mona Antonsen Elin Richardsen

Cervical intraepithelial neoplasia grade 1 (CIN1) is usually managed conservatively, but uncertainty remains about the subsequent risk of clinically significant high-grade disease, particularly after repeated CIN1. We conducted a retrospective population-based cohort study using anonymized cervical cytology, HPV, and histopathology records from Northern Norway from 2011 to 2025. We described temporal trends in screening-related outcomes and estimated the 5-year risk of CIN3+ after histologically confirmed CIN1 diagnosed in a single calendar year or in two consecutive calendar years. Across 2011–2025, the annual datasets comprised 334,471 screening records; 35,796 had ASC-US+ cytology (10.7%), 29,723 had a positive HPV test (8.9%), 35,416 underwent biopsy (10.6%), and 7870 were diagnosed with CIN2+ (2.4%). HPV positivity increased from 0.9% in 2011 to 15.7% in 2025, whereas CIN2+ detection peaked at 3.1% in 2018 and declined to 1.8% in 2025. In person-based analyses, the 5-year risks after CIN1 diagnosed in a single calendar year versus two consecutive calendar years were 4.3% versus 3.4% for CIN3+, 0.2% versus 0.1% for cervical cancer, and 15.4% versus 14.3% for CIN2+. Repeated CIN1 was not associated with higher subsequent CIN3+ risk, supporting conservative, risk-based follow-up after CIN1 biopsy.

Pathogens doi: 10.3390/pathogens15060656

Authors: Ashwaq Abdullah Alanezi Waleed E. Elawamy Huda Khalaf Alshammri Eman Ali Elkordy Ahmed E. Taha

Ventilator-associated pneumonia (VAP) is linked to high mortality rates, especially in developing countries. This cross-sectional survey study was conducted across three central hospitals in the Hail region of Saudi Arabia, King Salman Specialist Hospital, Hail General Hospital, and King Khalid Hospital, to assess the knowledge and adherence of intensive care unit (ICU) healthcare practitioners to the ventilator bundle (VB) for VAP prevention. It also looked at the practitioners’ perceived barriers to effective VB deployment. The study (n = 86) revealed significant disparities in VAP prevention knowledge across educational levels regarding the recommended degree of head-of-bed (HOB) elevation (p < 0.001), the use of endotracheal tubes with extra lumens for subglottic drainage (p < 0.001), and the protective effects of 0.12% chlorhexidine gluconate antiseptic oral rinse (p = 0.019). Professional experience significantly influenced knowledge of non-standard VB components (p < 0.001), the recommended frequency of awakening and spontaneous breathing trials (SBTs) (p < 0.001), and knowledge of extra-lumen tubes (p = 0.038) and kinetic beds vs. standard beds (p = 0.005). Significant differences were found between professional categories regarding knowledge of hand hygiene performance (p = 0.032), the correct degree of HOB elevation (p = 0.007), and patient positioning (semi-recumbent vs. supine) (p = 0.023). Years of experience significantly impacted reported compliance with institutional VB (p = 0.013), adherence to oral care protocols (p = 0.035), and the assessment of sedation depth (p = 0.002). While basic measures like HOB elevation practice and DVT prophylaxis showed universal reported compliance (100%), significant performance gaps were identified in more complex tasks, such as interrupting continuous sedative infusions and performing SBTs as recommended (p < 0.001), particularly among novice practitioners. The primary implementation barrier preventing full compliance with the VB was identified as educational deficit, which was prioritized as the most important area for quality improvement, highlighting the need for targeted training for newly hired ICU staff.

Pathogens doi: 10.3390/pathogens15060655

Authors: Eşe Aslan Başbulut Demet Yavuz Tülin Demir

Background: Lyme borreliosis is a multisystemic tick-borne infection caused by Borrelia burgdorferi sensu lato complex. Renal involvement is well documented in dogs with Lyme nephritis, whereas human renal manifestations have been reported only rarely, mainly as isolated case reports of glomerular disease. Because a proportion of chronic kidney disease (CKD) cases remain unexplained, we aimed to determine the possible contribution of previous exposure to idiopathic CKD. Methods: The study included 45 patients and 45 age and sex matched controls. Serum samples were tested for anti-Borrelia IgG using enzyme-linked immunosorbent assay (ELISA). Positive or borderline ELISA results were further evaluated by Western blot (WB) analysis. Results: Overall, anti-Borrelia IgG seropositivity was 5.55% with WB. In the CKD group, ELISA positivity was 11.1% and WB-confirmed positivity was 8.9%. In the control group, ELISA positivity was 4.4% and WB-confirmed positivity was 2.2%. (p > 0.05). All confirmed patients were living in rural areas, engaged in farming and/or animal husbandry, and reported repeated tick exposure. Conclusions: IgG seropositivity was numerically higher among patients with CKD of unknown etiology than among healthy controls; however, this difference was not statistically significant. Larger, risk-factor-adjusted studies are needed to clarify whether Borrelia exposure may be associated with unexplained CKD.

Pathogens doi: 10.3390/pathogens15060654

Authors: Nathalia de Assis Pereira Juliane Saldanha Thiago Borges Fernandes Semedo Guilherme Siniciato Terra Garbino Rogério Vieira Rossi Sofia de Souza Pereira Gomes Sayanne Luns Hatum Thállitha Samih Wischral Jayme Vieira Rafael Felipe da Costa Vieira Jansen de Araújo Edison Luiz Durigon Daniel Moura de Aguiar

In this study, we conducted a molecular investigation of hemotropic Mycoplasma spp. in bat species captured in the northern region of the Pantanal biome, Mato Grosso State, Brazil. Tissue samples were screened by qPCR targeting the 16S rRNA gene. Positive samples were subsequently subjected to conventional PCR assays targeting partial fragments of the 16S rRNA (~900 bp) and 23S rRNA (~800 bp) genes. Hemoplasma DNA was detected in four bat species: Glossophaga soricina, Molossops temminckii, Molossus rufus, and Desmodus rotundus. Phylogenetic analyses based on partial 16S and 23S rRNA gene sequences demonstrated that the detected hemoplasmas clustered predominantly with previously described bat-associated hemoplasmas from Brazil and other countries in the Americas. Notably, the detection in M. temminckii represents, to our knowledge, the first molecular evidence of hemotropic Mycoplasma infection in this bat species. These findings expand current knowledge regarding the occurrence, host range, and genetic diversity of hemotropic Mycoplasma spp. in bats from the Pantanal biome and contribute to wildlife surveillance efforts in this ecologically important region.

Pathogens doi: 10.3390/pathogens15060653

Authors: Wenting Sheng Linfeng Chen Junyu Ma Muhammad Saqlain Muhammad Hammad Latif Ke Zhang Jingyuan Yang Muhammad Nosherwan Wei Zhu Lili Xu Dandan Wu Yonghong Zhou Chaojie Xie Houyang Kang Tzion Fahima Yinghui Li

The einkorn wheat group, comprising ancient diploid species (2n = 14, AA), including Triticum monococcum, Triticum boeoticum, and Triticum urartu, represents a valuable source of genetic variation for improving disease resistance in wheat. To develop a practical platform for introgressing powdery mildew resistance into bread wheat, we screened 21 einkorn accessions with Blumeria graminis f. sp. tritici (Bgt) race E09 and identified seven resistant donors. Because direct hybridization between diploid einkorn (AA) and hexaploid wheat (AABBDD) is constrained by genomic divergence and poor cross-compatibility, we crossed resistant einkorn accessions with susceptible durum wheat and induced chromosome doubling in the F1 hybrids to generate synthetic durum–einkorn amphiploids. Nine amphiploids were obtained. Chromosome counts and genomic in situ hybridization confirmed the expected genomic constitution (AABBAA) in most lines, with limited variation in chromosome number in two amphiploids. Phenotyping against Bgt race E09 showed that three amphiploids retained high resistance, four showed moderate resistance, and two were moderately susceptible. Marker analysis identified five einkorn accessions contain known Pm genes such as Pm60, Pm60b, and PmNCA6/Pm37, as well as their derived amphipliods. Two einkorn accessions and their derived amphiploids may harbor novel Pm genes. Field evaluation of the agronomic traits of these amphiploids indicated some improvement in tillering, spike length, and seed weight. Moreover, these amphiploids had better seed-setting rates in crosses and backcrosses with common wheat. These synthetic durum–einkorn amphiploids thus offer a new bridging platform for transferring alien genes from diploid einkorn to hexaploid common wheat, providing valuable genetic resources for wheat-breeding programs.

Pathogens doi: 10.3390/pathogens15060652

Authors: Vitor Duque

Orthohantaviruses are zoonotic pathogens belonging to the family Hantaviridae and are responsible for significant human disease. These infections are characterized by acute systemic illness, vascular dysfunction, and, in severe cases, hemorrhage and multiorgan failure. Depending on the viral species involved, infection may result in hemorrhagic fever with renal syndrome (HFRS) or hantavirus cardiopulmonary syndrome (HCPS), both of which are associated with substantial morbidity and mortality. Rodents act as natural reservoirs, maintaining viral persistence in endemic ecosystems and enabling sporadic spillover to humans through exposure to infected excreta or contaminated environments. This review synthesizes current knowledge on rodent reservoir competence, hantavirus replication strategies, pathogenesis, clinical manifestations, ecological drivers of transmission, public health implications and future therapeutic developments and challenges. Understanding these mechanisms is essential for enhancing surveillance, risk assessment, and preventive strategies against orthohantavirus infections.

Pathogens doi: 10.3390/pathogens15060651

Authors: Madeline R. Bolding Sarah C. Mozden Olivia R. Pimentel Makaela M. Montoya Jessica Z. Kubicek-Sutherland Nileena Velappan

Proteins are the most common targets for antibody discovery and vaccine development, but their sequence variability can limit the breadth of resulting antigens. Lipids represent an alternative class of antigens due to their structural conservation and roles in host–pathogen interactions. Here, we describe the development and optimization of an in vitro antibody selection workflow using lipid-containing nanodiscs as antigen presentation platforms to enable phage and yeast display selections under conditions adapted for these non-protein targets. Lipopolysaccharide (LPS) nanodiscs were first used as a model system to evaluate selection strategies, including competitive and subtractive approaches to reduce non-specific binders, yielding peptide and single-chain variable fragment (scFv) binders that were affinity matured to improve binding signals. The same approach was subsequently used to select scFv antibodies that recognize lipid nanodiscs prepared from Yersinia pestis membrane lipid extracts. These antibodies show binding to lipid nanodiscs derived from Y. pestis, with evidence of selectivity relative to control nanodiscs. Overall, this work establishes a workflow for antibody selection against lipid-containing nanodisc antigens and highlights practical considerations associated with these targets. The approach may be useful for generating affinity reagents to membrane-associated lipids, although further characterization is required to define antigen specificity and functional activity.

Pathogens doi: 10.3390/pathogens15060650

Authors: Amanda Cristina Machado Carloto Ana Carolina Jacob Rodrigues Mariana Barbosa Detoni Ellen Mayara Souza Cruz Virgínia Márcia Concato-Lopes Rodolfo Bento Balbinot Fabrício Seidy Ribeiro Inoue Yuri Barreiros Arthur Poester Cordeiro Pedro Henrique Hermes de Araújo Claudia Sayer Paulo Emílio Feuser Celso Vataru Nakamura Ivete Conchon-Costa Danielle Lazarin-Bidóia Wander Rogério Pavanelli

Leishmaniasis is a neglected tropical disease caused by parasites of the genus Leishmania. Curcumin (CUR) is a polyphenol with several biological properties, including antimicrobial effects. However, its low bioavailability remains a challenge, and nanoencapsulation may represent a useful strategy to overcome this limitation. This study aimed to evaluate, in vitro, the antipromastigote activity of free CUR and the antiamastigote effect of CUR nanoparticles and their association with antimoniate, as well as to elucidate possible mechanisms of action. Free CUR directly inhibited promastigote proliferation, with an IC50 of 25 µM at 24 h. CUR induced mitochondrial hyperpolarization, increased the production of reactive oxygen species (ROS) and nitric oxide (NO), and enhanced lipid peroxidation and the accumulation of lipid droplets in promastigotes. These alterations were associated with autophagic and apoptotic processes, morphological and ultrastructural changes, DNA fragmentation, and cell cycle arrest. Free CUR also reduced the viability of BALB/c peritoneal macrophages, and this effect was attenuated after nanoencapsulation. Free CUR, CUR nanoparticles, and their association with antimoniate (AM) reduced both the percentage of infected macrophages and the number of intracellular amastigotes at all tested concentrations, with increased NO production observed at the highest concentrations of free CUR. Altogether, our findings suggest that CUR exerts leishmanicidal activity against promastigotes by disrupting oxidative metabolism and triggering autophagic and apoptotic pathways, while amastigote elimination appears to occur through mechanisms independent of oxidative stress.

Pathogens doi: 10.3390/pathogens15060649

Authors: Ardian Ramadani Giacomo de Marco Oscar Vazquez Elio Paris Andreas Tsoupras Christina Steiger Romain Dayer Dimitri Ceroni

Osteoarticular infections (OAIs) present a significant diagnostic and therapeutic challenge to paediatric clinical practice. When evaluating suspected paediatric OAIs, the principal pathogens commonly considered are Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae and Kingella kingae. However, advances in molecular diagnostic techniques, particularly polymerase chain reaction assays and next-generation sequencing, have considerably improved the detection of less commonly identified pathogens responsible for OAIs. Consequently, familiarity with fastidious, emerging, exposure-related and uncommon bacterial pathogens is essential to ensure accurate diagnoses and appropriate therapeutic interventions. This review summarises bacterial pathogens responsible for OAIs in immunocompetent children that are fastidious, emerging, exposure-related or otherwise less commonly encountered. We describe their microbiological characteristics, clinical phenotypes, diagnostic pitfalls and organism-specific diagnostic strategies.

Pathogens doi: 10.3390/pathogens15060648

Authors: Brayan Villazana-Calderon Hilda Jiménez-Wences J. Noé García-Chávez Imelda Martínez-Ramírez Claudia González-Espinosa Gloria Fernández-Tilapa Marcela Lizano J. Omar Muñoz-Bello

The continuous expression of HPV oncogenes E6 and E7 contributes to the maintenance of the cervical cancer (CC) phenotype by altering gene expression programs involved in tumor progression and aggressiveness. MicroRNAs (miRNAs) have emerged as critical regulators of gene expression in CC, including miR-218-5p, which has been described as a tumor suppressor. In this study, we investigated the impact of HPV-16 oncoproteins E6 and E7 on the regulation of miR-218-5p expression and its target gene PIK3C2A, as well as their functional and clinical relevance in CC. We found that miR-218-5p expression is significantly reduced in HPV-16-positive CC cell lines, while PIK3C2A expression is increased. Silencing the expression of the E6/E7 oncogenes in Ca Ski cells restored miR-218-5p levels and reduced PIK3C2A expression. Conversely, overexpression of the E6 and E7 oncogenes in C-33 A cells significantly decreased miR-218-5p expression and increased PIK3C2A expression. Functional assays performed on C-33 A cells expressing E6 and E7 revealed that ectopic expression of miR-218-5p suppresses cell proliferation and migration, effects that are partially mediated by PIK3C2A. Bioinformatics analysis showed that low miR-218-5p expression and high PIK3C2A expression are associated with reduced overall survival in patients with cervical cancer. Our findings identify the miR-218-5p/PIK3C2A axis as a novel regulatory pathway modulated by HPV-16 oncoproteins E6 and E7 that contributes to CC cell proliferation and migration. Furthermore, miR-218-5p and PIK3C2A emerge as potential prognostic biomarkers in CC.

Pathogens doi: 10.3390/pathogens15060647

Authors: Ze-Yun Xu Guo-Qing Chen Jing Xue Yu-Xin Chi Rui Jian Wen-Ping Guo

Ticks are widely distributed in China and can carry and transmit a variety of pathogens that potential to cause serious impacts on public health and the economy. Little is known about the broader spectrum of Coxiella-like endosymbiont (CLE) in ticks under natural conditions in China. The aim of this study was to detect, analyze, and characterize phylogenetically CLE found in ticks in Hebei Province, China. A total of 947 ticks collected from Hebei Province were identified as Haemaphysalis longicornis based on morphological characteristics and cytochrome c oxidase gene PCR analysis of extracted DNA. Subsequently, DNA was analyzed via PCR for the IS1111 gene (frequently associated with Coxiella burnetii), and the amplified DNA was then sequenced and analyzed phylogenetically using a set of primers targeting the 16S rRNA, groEL, and rpoB genes. A total of 8.24% (78/947) of ticks from the Chengde, Baoding, and Cangzhou regions were positive in the IS1111 PCR. Phylogenetic analysis using the 16S rRNA, groEL, and rpoB genes revealed the presence of CLE in Ha. longicornis ticks from these regions and the formation of two distinct clades, suggesting horizontal gene transfer events. Our results strengthen the growing evidence that CLE, not Coxiella burnetii, is ubiquitously associated with ticks across diverse geographic locations—a distinction critical for accurately interpreting tick microbiome surveys and avoiding false assumptions of zoonotic risk.

Pathogens doi: 10.3390/pathogens15060646

Authors: Kabir Brar Bahar E. Mustafa Ian Beveridge Charles Gauci Abdul Jabbar Abdul Ghafar

Ticks are haematophagous ectoparasites and vectors of a diverse range of pathogens, exerting substantial impacts on wildlife, domestic animals and public health. In Australia, despite the country’s rich and unique biodiversity, a comprehensive understanding of ticks and tick-borne pathogens associated with wildlife remains limited. Environmental change, urban expansion and climate variability are increasingly disrupting wildlife habitats, potentially intensifying interactions between wildlife hosts, ticks and humans. A broad evidence synthesis of studies published between January 1940 and March 2024 was conducted, retrieving 133 eligible records from Web of Science, CABI Abstracts and PubMed databases. Fifty tick species parasitising 160 wildlife species were identified, predominantly from the genera Ixodes, Amblyomma and Haemaphysalis. The most commonly reported hosts included marsupials, particularly bandicoots, wallabies and possums, with notable tick species being Ixodes tasmani, Ixodes holocyclus and Amblyomma triguttatum. Microorganism records were relatively limited and mostly represented molecular detections or reported associations, including Babesia, Borrelia, Coxiella, Rickettsia and Theileria species, rather than confirmed vector competence, reservoir status or pathogenicity. Key limitations included geographic sampling biases towards eastern Australia, limited molecular identification of ticks and infrequent pathogen screening, particularly regarding the ecology, epidemiology and molecular diversity of host–vector–microorganism interactions. Improved surveillance, expanded molecular characterisation, and integrated One Health investigations are required to better understand the ecological and public health significance of these host–vector–microorganism interactions.

Pathogens doi: 10.3390/pathogens15060645

Authors: Damani N. Bryant Nicole A. Lurndahl Maddy Ellis-Cramer Sarah C. Gresch Marc D. Schwabenlander Peter A. Larsen Tiffany M. Wolf Stuart S. Lichtenberg

Infectious prion adsorption on metal, minerals, wood, and plastic is well documented, raising the specter of food safety hazards for meat packing workers, sport hunters, and consumers. We previously demonstrated that sodium hypochlorite, and to a lesser extent, potassium peroxymonosulfate, and hypochlorous acid can decontaminate prion-contaminated nonporous surfaces. However, the extent to which chemical aging of surfaces affects subsequent recoverable prion seeding activity is unknown. In this study, we investigated the potential for four chemical decontaminants known for their anti-prion activity (sodium hypochlorite [bleach], hypochlorous acid [Briotech], potassium peroxymonosulfate [Virkon-S], and Wex-Cide-128) to alter the surfaces of steel knives and the subsequent prion decontamination efficacy of each. We found that hypochlorous acid, sodium hypochlorite, and potassium peroxymonosulfate corrode the surfaces of steel knives, resulting in significant physical alterations. Knives exposed to hypochlorous acid exhibited the most substantial corrosion (rust), which is consistent with its oxidizing effects. Oxidation of the knife surface was corroborated by complementary energy-dispersive X-ray spectroscopy data trends. Scanning electron microscopy data indicate corrosion is apparent after minimal exposure to oxidizing agents. Finally, we used the real-time quaking-induced conversion assay on swabs collected from chemically aged knife surfaces to evaluate recoverable surface-associated CWD-prion seeding activity detected by RT-QuIC after prion exposure and decontamination. Our results indicate decreased recoverable prion seeding activity from knife surfaces aged with 40% bleach. We also observed some recoverable seeding activity post-decontamination on knives chemically aged with 10% bleach and Wex-Cide-128, but largely similar efficacy to prior studies. This implies that existing chemical prion decontaminants are likely effective after repeated use on steel surfaces.

Pathogens doi: 10.3390/pathogens15060644

Authors: Ozgur Celebi Hugh Dyson Thomas R. Laws Fatih Buyuk Mehmet Doganay Mitat Sahin Les Baillie

During construction of the Baku–Tbilisi–Kars railroad between Kars City in Türkiye and Tbilisi in Georgia, blasting operations in an anthrax-endemic region disrupted a burial pit containing carcasses of cattle that had died of anthrax. Railroad workers expressed concerns that release of this material could result in them developing anthrax infection. We therefore undertook a seroprevalence study six months later to seek evidence of exposure to Bacillus anthracis spores. We used an optimised Enzyme-Linked Immunosorbent Assay (ELISA) to screen serum for antigen-specific IgG antibodies to the anthrax toxin subunits Protective Antigen (PA) and Lethal Factor (LF). Stepwise linear regressions and t-tests were performed to compare results from railroad workers (n = 64) with a group of long-term Kars City residents (urban dwellers, n = 16), who had no history of possible contact with anthrax antigens. Anti-PA IgG concentrations were higher (p = 0.038) in railroad workers than in urban dwellers, but anti-LF IgG concentrations did not differ (p = 0.932) between the two groups. The anti-PA response is known to be dominant, and the difference was small. The lack of LF response did not preclude an antibody response to B. anthracis. Following the blasting operations, no cases of anthrax infection occurred in either railroad workers or villagers living nearby, suggesting that the spore exposure (evidenced by higher antibody titres) was at levels insufficient to initiate clinical infection. The elevated PA-specific antibody responses in railroad workers compared with urban dwellers might be consistent with the former having had previous subclinical exposure to B. anthracis. In anthrax-endemic regions, therefore, construction activities that involve blasting or large-scale excavation may pose risks of occupational exposure to Bacillus anthracis spores.

Pathogens doi: 10.3390/pathogens15060643

Authors: Eva Spada Federica Bruno Germano Castelli Roberta Perego Noemi Cerutti Fabrizio Vitale Vito Biondi Luciana Baggiani Daniela Proverbio

Feline leishmaniosis (FeL) caused by Leishmania infantum is increasingly recognized in endemic areas, but factors influencing susceptibility in cats remain incompletely understood. Because blood group antigens may modulate host–pathogen interactions, this study evaluated whether feline AB blood system phenotypes are associated with L. infantum seropositivity and/or molecular positivity in cats from Italy. Exploratory analyses further assessed whether blood phenotype was associated with the magnitude of indirect fluorescent antibody test (IFAT) antibody titres or with real-time PCR (qPCR) parasite load. In this retrospective cross-sectional study, cats were classified as L. infantum-positive when they had an IFAT titre ≥1:80 and/or a positive qPCR on blood or lymph node aspirates. Feline AB blood typing was performed by tube agglutination, with type B and AB samples confirmed by immunochromatographic testing and back typing. A total of 706 cats were included. Overall, 67/706 cats (9.5%) were classified as L. infantum-positive. Blood phenotype distribution was 83.1% type A, 10.1% type B, and 6.8% type AB. L. infantum positivity was detected in all three phenotypes, and no evidence of association was found between blood phenotype and L. infantum positivity, IFAT seropositivity, qPCR positivity, IFAT titre, or qPCR parasite load. After adjustment for region, blood phenotype remained not significantly associated with L. infantum positivity. These findings suggest that feline AB blood system phenotypes were not associated with L. infantum infection in this feline cohort. Future studies should investigate whether blood phenotype may influence other aspects of FeL, such as clinical expression or disease outcome.

Pathogens doi: 10.3390/pathogens15060642

Authors: Bo Guan Le Zhang Chunling Zhang Jing Huang

Acinetobacter baumannii is a significant pathogen of hospital-acquired infections, and its multidrug resistance (MDR) and extended drug resistance (XDR) have become increasingly severe, posing a global public health challenge. This article provides a narrative review of the major resistance mechanisms of Acinetobacter baumannii, including β-lactamase production, efflux pump overexpression, target site modification, reduced membrane permeability, and biofilm formation. Additionally, it summarizes the current main drugs and their target sites for treating MDR Acinetobacter baumannii infections, with a focus on the mechanism of action, antibacterial activity, and clinical research progress of the novel fully synthetic fluorocycline antibiotic—eravacycline. Eravacycline inhibits protein synthesis by high-affinity binding to the bacterial ribosomal 30S subunit and demonstrates activity against multidrug-resistant Acinetobacter baumannii (excluding Pseudomonas aeruginosa), providing a potential novel therapeutic option for MDR/XDR Acinetobacter baumannii infections. Finally, the article outlines future research directions and treatment strategies. Due to the narrative nature of this review, no systematic methodology (e.g., PRISMA) was applied, and the available clinical evidence, particularly for CRAB infections, remains limited.

Pathogens doi: 10.3390/pathogens15060641

Authors: Sony Yean Didot Budi Prasetyo Sovanncheypo Chao Linavin Vuth Matthieu Prot Artem Baidaliuk Sarah Bonnet Etienne Simon-Loriere Sébastien Boyer

In Cambodia, livestock production plays an important role in the national economy and food security, yet tick-borne diseases remain an underrecognized constraint on animal health and productivity. Domestic animals may also serve as reservoirs of zoonotic pathogens in this predominantly rural setting. To address the lack of baseline molecular data on tick-borne pathogens in Cambodia, we conducted a cross-sectional study of ticks collected from November 2022 to April 2023 across 24 provinces. Ticks were collected from various hosts and environments, including cats, cattle, dogs, goats, pangolins, pythons, wild pigs, and bat cave floors, representing urban, rural, farm, wildlife rescue center, and forest fringe habitats. A total of 1526 ticks belonging to nine species were pooled into 352 samples and screened using conventional PCR (cPCR) targeting Anaplasma, Ehrlichia, Babesia, and Coxiella. Additionally, a subset of Rhipicephalus microplus ticks was analyzed using metatranscriptomic next-generation sequencing (NGS). Rhipicephalus microplus ticks collected from cattle tested positive for Anaplasma marginale (1.1% of pools) and Ehrlichia minasensis (0.9% of pools), whereas Rhipicephalus linnaei ticks collected from dogs were positive for Anaplasma platys (0.3% of pools) and Babesia canis (2.0% of pools). A high prevalence of Coxiella-like endosymbionts (15.6% of pools) was found in R. microplus from both cattle and goats. Metatranscriptomic analysis also identified six tick-associated viruses in R. microplus from cattle; with Guangdong tick manly virus being the most dominant (32.5% of samples); followed by Zhangzhou Totiv tick virus 1 (15.0%), Jingmen tick virus (5.0%), and Mogiana tick virus; Rhipicephalus-associated rhabdo-like virus; and Rhipicephalus-associated flavi-like virus; each at 2.5%. These findings provide the first molecular evidence of numerous bacterial, protozoal, and viral pathogens circulating in R. microplus and R. linnaei in Cambodia. The study highlights the need for integrated One Health surveillance to better understand, prevent, and control tick-borne diseases in the region.

Pathogens doi: 10.3390/pathogens15060640

Authors: Leah Liu Wang Kendra Alfson J. J. Patten Marc E. Mattix Yenny Goez-Gazi Sean N. Avedissian Robert A. Davey Ricardo Carrion Shi-Hua Xiang

Orthoebolavirus causes severe Ebola virus disease (EVD) and deadly outbreaks in humans. This infection occurs through macropinocytosis and trafficking to late endosomes or lysosomes that utilize the receptor Niemann–Pick C1 (NPC1) to enter the cell cytoplasm. We designed peptide inhibitors based on the NPC1 receptor to target the NPC1 binding site to block viral entry. The results indicated that the ligand-based peptide inhibitors showed potent inhibition activities in vitro studies against pseudotyped or replication-competent Orthoebolavirus. Therefore, we further evaluated one of them in a mouse model challenged with mice-adapted Ebola viruses, which showed some protection efficacy compared with the control group. This study suggests that ligand-based peptides are encouraging inhibitors in the development of inhibitors against Ebola virus infection.

Pathogens doi: 10.3390/pathogens15060639

Authors: Cassandra D. Pauling Deborah M. Anderson

Yersinia pestis is the causative agent of bubonic plague, a zoonotic disease that is primarily transmitted by infectious fleas. Plague is endemic in regions around the world, including the United States, where optimal climate conditions support a stable, enzootic sylvatic transmission cycle. Epizootic outbreaks periodically occur with rapid spread of disease that increases the risk of human exposure. As fleas are ectotherms that are responsive to environmental conditions, it is likely that transmission efficiency varies under different ecological conditions, with optimal conditions capable of supporting rapid spread of disease while sub-optimal conditions may promote lower levels of transmission. To test this, we experimentally infected Xenopsylla cheopis with Y. pestis using a membrane feeder in order to define the impact of varying temperature, humidity and mammalian blood sources on infection and transmission. We show that environmental factors and host blood source are key factors influencing colonization, bacterial aggregation, and transmission rates, with variation in the responses seen depending on the experimental conditions. The combined data illustrate the impact of ecological factors on Y. pestis flea infection and suggests that optimal conditions involving the vector–host–pathogen interface are needed for enhanced transmission rates and the rapid spread of infection that occurs during epizootic outbreaks.

Pathogens doi: 10.3390/pathogens15060638

Authors: Joseph Capobianco Chin-Yi Chen Yiping He

Accurate detection of Campylobacter in chicken liver is hindered by strong matrix inhibition. This study evaluated sample-processing strategies to improve droplet digital PCR (ddPCR) quantification of Campylobacter coli and Campylobacter jejuni in chicken liver. Mechanical homogenization (Stomacher) and enzymatic/mechanical dissociation (gentleMACS), with and without 8 μm filtration, were compared. Particle-size analysis showed that filtration, especially following gentleMACS treatment, produced smaller, more uniform particles and reduced variability. Percent-degradation assays confirmed that gentleMACS achieved substantially greater tissue disruption than Stomacher homogenization. The multiplex ddPCR assay, which simultaneously targets C. coli and C. jejuni, produced droplet counts comparable to single-target reactions, indicating minimal interference between targets under the conditions tested. In inoculated liver samples, gentleMACS processing yielded droplet counts similar to those obtained from pure cultures, whereas unprocessed liver caused severe matrix interference and inconsistent quantification. Furthermore, gentleMACS-treated samples exhibited strong log-to-log linearity for quantifying C. coli and C. jejuni, enabling detection near 1 genome copy equivalent per reaction. Overall, the results indicate that enzymatic/mechanical dissociation combined with fine-pore filtration improves ddPCR detection of Campylobacter species in chicken liver.

Pathogens doi: 10.3390/pathogens15060637

Authors: Ivan Matevski Zagorka Popova Hristovska Igor Djadjovski Kiril Krstevski

Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne zoonotic disease of significant public health concern, particularly in endemic regions. However, data on the distribution and circulation of Crimean-Congo hemorrhagic fever virus (CCHFV) in animal populations remain limited, despite their importance for assessing virus circulation and infection patterns. A cross-sectional seroepidemiological study was conducted in North Macedonia (N. Macedonia) to determine the seroprevalence rates of CCHFV in small ruminants and to identify areas at increased risk of virus circulation. A total of 1992 sera samples from sheep and goats were tested for Crimean-Congo hemorrhagic fever virus (CCHFV) antibody using a commercial enzyme-linked immunosorbent assay (ELISA). Data on species, age, animal origin, and risk factors (questionnaire) were collected and analyzed. Overall, true seroprevalence, calculated by adjusting the apparent seroprevalence according to the diagnostic performance of the ELISA assay in order to estimate the actual prevalence in the studied population, was 25.02% (493/1992; 95% CI 23.11–26.94). Sheep showed higher seroprevalence rates, 27.34% (397/1452; 95% CI 25.11–29.69), compared to goats, 17.78% (96/540; 95% CI 14.78–21.23). Seroprevalence rates varied markedly across regions, ranging from 2.41% to 49.80%, with the highest values observed in the Eastern and Vardar regions. Seroprevalence rates increased with age, reaching the highest values in animals aged ≥ 5 years, 27.27% (81/297; 95% CI 22.52–32.60). Small ruminants, particularly sheep, may serve as useful indicators for defining the high-risk areas for CCHFV transmission, providing valuable support for a One Health approach.

Pathogens doi: 10.3390/pathogens15060636

Authors: Giorgio Galletti Sara Salvato Stefania Calò Maria Ottaiano Maria Beatrice Boniotti Marco Tamba

Animal tuberculosis in cattle (TB) has been controlled in many European countries through long-standing eradication programs, yet sporadic breakdowns continue to occur in officially tuberculosis-free (OTF) areas, challenging the sustainability of disease freedom. This study aimed to identify and quantify herd-level and area-level risk factors associated with TB occurrence in Italian OTF regions in order to support risk-based surveillance strategies. A national longitudinal open-cohort study was conducted using data from the Italian Veterinary Information System, including approximately 300,000 herd–year observations from 2022 to 2025. The outcome was the occurrence of at least one TB breakdown per herd–year, analyzed using a discrete-time hazard modeling approach based on a binomial generalized linear mixed model with province-level random effects. The incidence of TB remained very low but increased over time, and significant spatial clustering was observed. Higher TB risk was associated with larger herd size, a previous history of TB, non-OTF herd status, proximity to recent breakdowns, number of animals purchased, transhumance practices, and a shorter time since acquisition of OTF status at provincial level. These findings highlight that, even in disease-free contexts, TB risk is heterogeneous and driven by identifiable factors, supporting the refinement of targeted, risk-based surveillance to maintain OTF status over time.

Pathogens doi: 10.3390/pathogens15060635

Authors: Hyo-Ju Yoon Gyoo-Cheon Kim Seoul-Hee Nam

Background: Oral candidiasis is an opportunistic infection caused by Candida albicans (C. albicans), highlighting the need to evaluate candidate substances that ensure both antifungal efficacy and mucosal safety. This study aimed to assess the potential of Sambucus williamsii var. coreana (S. williamsii var. coreana) extract as a naturally derived antifungal agent for topical oral application by investigating its antifungal activity against C. albicans and its cytotoxicity in human keratinocyte (HaCaT) cells. Methods: S. williamsii var. coreana was extracted with 70% ethanol, concentrated, and freeze-dried. The extract was prepared at concentrations of 1–40 mg/mL and applied under 6 h and 24 h exposure conditions. Antifungal activity against cultured C. albicans was evaluated using colony-forming unit (CFU) analysis, while cytotoxicity in HaCaT cells was assessed via the Water-soluble Tetrazolium Salt-1 assay after incubation at 37 °C in 5% CO2 for 2 h. Statistical significance was analyzed using Student’s t-test, ANOVA, and Tukey’s HSD test (p < 0.05). Results: The S. williamsii var. coreana extract exhibited concentration- and time-dependent antifungal activity. A 99.99% inhibition of C. albicans was observed at 5 mg/mL. No detectable CFUs were observed at 30 mg/mL after 6 h and at 10 mg/mL after 24 h. HaCaT cell viability decreased in a concentration-dependent manner, with the half-maximal inhibitory concentration determined to be 10 mg/mL. Conclusions: The extract of S. williamsii var. coreana exhibited concentration- and time-dependent in vitro antifungal activity against C. albicans. However, the concentration associated with no detectable CFUs overlapped with the cytotoxic concentration range in HaCaT cells, indicating that further studies are required to define an appropriate concentration range for potential oral application.

Pathogens doi: 10.3390/pathogens15060634

Authors: Basant Mostafa Gabr Mona Abd El-Aziz Gadallah Wafaa Abd Elaziz Sama Metwally Raghda Gabr Mashaal Rasha A. Abd Ellatif Ahmed G. Elkhouly Hanan Salem Amira E. Oraiby Bsant S. Kasem Sherif Abdelbaky Reham M. Elkolaly Marwa S. Taha

To improve clinical decision-making about Carbapenem-resistant Gram-negative bacteria (CR-GNB) infections and halt the spread of resistant microbes, quicker and less expensive diagnostic techniques are required. Thus, the purpose of this study was to thoroughly evaluate the diagnostic efficiency (sensitivity, specificity, and concordance) of direct-from-specimen multiplex lateral flow immunoassay (LFIA) across diverse raw clinical specimens and pathogen types from critically sick patients. A total of 300 non-duplicate samples were tested to detect CR-GNB. Five major Carbapenemase genes were detected directly from the specimen using carbapenem-resistant K.N.I.V.O. detection K-Set and from culture using culture-enhanced multiplex PCR. Turnaround time (TAT) of each method was calculated. The direct LFIA revealed 100% specificity for NDM, KPC, and IMP enzymes in all tested clinical matrices (blood, urine, and respiratory samples). The study demonstrated 100% sensitivity and specificity with perfect categorical agreement (κ = 1.000) for the blaKPC in the Klebsiella pneumoniae and for blaOXA-48 and blaIMP in the Acinetobacter baumannii; however, sensitivity of blaVIM was significantly diminished across all isolates and samples. TAT decreased significantly (p < 0.001) from 30 to 70 h to about 50 min. The tested direct LFIA facilitates the prompt enhancement of lifesaving tailored antibiotic treatment for severe illnesses.

Pathogens doi: 10.3390/pathogens15060633

Authors: Kentaro Kato Mizuki Kudo Hideaki Unno Tomomitsu Hatakeyama Hiroshi Tachibana

The galactose/N-acetyl-D-galactosamine (Gal/GalNAc)-inhibitable lectin of Entamoeba histolytica plays essential roles in host cell adhesion and cytotoxicity. The intermediate subunit lectin-1 (Igl1) contributes to these functions, but its molecular state under different environmental conditions remains unclear. In this study, we found that Igl1 is present as multiple fragments in the culture supernatant of trophozoites, whereas a single major species corresponding to intact Igl1 was detected in cell lysates. Notably, the molecular sizes of both intact Igl1 and its extracellular fragments differed depending on culture conditions, with larger apparent sizes observed under co-culture with Caco-2 cells. These differences were not explained by changes in transcript levels, protein folding, or N-terminal truncation. Fragmentation of Igl1 was suppressed by a cysteine protease inhibitor, indicating extracellular generation. These findings demonstrate that host-cell-associated conditions alter the molecular size of Igl1 and that extracellular protease-dependent processing generates multiple Igl1 fragments, providing new insights into the regulation of this key virulence factor. The presence of extracellular fragments further suggests a potential contribution to host tissue damage during amoebiasis.

Pathogens doi: 10.3390/pathogens15060632

Authors: Francesco De Maria Francesco Branda Giancarlo Ceccarelli Fabio Scarpa Massimo Ciccozzi Alessandro Russo

Orthohantavirus infections are classically associated with hemorrhagic fever with renal syndrome (HFRS) in Eurasia and hantavirus cardiopulmonary syndrome (HCPS) in the Americas. However, accumulating evidence indicates that the clinical spectrum is considerably broader, with frequent involvement of organ systems beyond the kidney and lung. Hepatic manifestations, in particular, may mimic acute viral hepatitis, leading to diagnostic challenges and underrecognition. This paper synthesizes published evidence on hepatic involvement in orthohantavirus infection, with a focus on clinical presentation, pathogenic mechanisms, differential diagnosis, biomarkers, and public health implications. Relevant literature was identified through searches of peer-reviewed articles, with emphasis on studies reporting hypertransaminasemia, hepatitis-like illness, and liver injury in confirmed hantavirus infections. Mild to moderate elevations in aminotransferases are common during acute orthohantavirus infection, and in some patients the clinical picture may be dominated by fever, thrombocytopenia, and hepatitis-like abnormalities, closely resembling dengue, leptospirosis, or classical viral hepatitis. Hepatic injury appears to result primarily from systemic endothelial dysfunction, immune-mediated inflammation, and microvascular leakage rather than direct hepatocytopathic effects. Emerging biomarkers of severity, including thrombocytopenia, neutrophil-to-lymphocyte ratio, soluble thrombomodulin, and IL-6 trans-signaling, reflect widespread vascular and inflammatory activation. Diagnostic delays are frequent, particularly in non-endemic regions, due to low clinical awareness and overlapping features with more common febrile hepatotropic syndromes. Orthohantavirus infection should be considered in the differential diagnosis of acute febrile illness with unexplained hypertransaminasemia and thrombocytopenia, especially when epidemiological clues suggest rodent exposure or compatible environmental contexts. Recognizing hepatic involvement as part of a systemic endothelial syndrome may improve diagnostic accuracy, reduce underreporting, and facilitate earlier supportive management. Increased awareness among hepatologists, infectious disease specialists, and emergency physicians is warranted.

Pathogens doi: 10.3390/pathogens15060631

Authors: Lucio Fortelny Manfred Marschall

Herpesvirus infections belong to major pathogens in the human population. This study aimed at evaluating diagnostic data for eight human herpesviruses, based on datasets derived from a large European tertiary care center. Specifically, we analyzed 118,692 herpesvirus submittals to the Diagnostic Division of the Virological Institute, University Hospital Erlangen (UKER), Germany, between July 2014 and June 2024. Our points of focus were the following: (i) the frequencies of herpesvirus diagnostic results with positivity rates, (ii) departments representing main sample submitters, (iii) the specific importance of intensive care units (ICUs), (iv) the COVID-19 pandemic period, and (v) distinct properties of sample types. Overall, we are stating the highest frequencies of diagnostic assessment for herpes simplex virus (HSV), human cytomegalovirus (HCMV), and Epstein–Barr virus (EBV) infections, pointing to their dominant relevance for clinical practice. Notably, HCMV submittals (46.6% of total), together with EBV (26.2%) and HSV (15.7), accounted for almost 90% of all herpesviral diagnostic samples during this period. Within these key groups, HCMV, EBV and HSV showed positivity rates of 14.5%, 35.0%, and 18.5%, respectively. Concerning a main input of sample submittals, two departments were predominant in our center, i.e., the Departments of Haematology–Oncology and Anaesthesiology. These included patients under multifold types of treatment associated with an increased risk of herpesvirus reactivation or primary infection. Furthermore, another high portion of submittals was noted for ICUs and external sources. In addition, a numerical, transient increase in herpesvirus diagnostic submittals, from various sources, was shown for the COVID-19 pandemic years (mostly 2021) as compared to other periods. Combined, these data underlined the importance of clinical monitoring of herpesvirus infections, particularly for high-risk patients, and the steady need of improvements in preventive measures, therapeutic options, and safe diagnostic tools.

Pathogens doi: 10.3390/pathogens15060630

Authors: Ana-Maria Marin Dan-Cornel Popovici Maria Monica Florina Moraru Gianluca Marucci Adriano Casulli Francesco Celani Sorin Morariu Anamaria Plesko Narcisa Mederle

Trichinellosis is an important parasitic zoonosis caused by nematodes of the genus Trichinella, affecting numerous carnivorous and omnivorous mammal species. In Europe, wildlife represents the main reservoir of Trichinella spp., contributing to the maintenance of the sylvatic cycle and acting as a potential source of infection for domestic animals and humans. In Romania, Trichinella spiralis, Trichinella britovi, and Trichinella pseudospiralis have been reported in domestic animals and wildlife, with T. britovi being frequently associated with the sylvatic cycle and wild carnivores. The aim of this study was to investigate the occurrence and molecular characterization of Trichinella larvae isolated from muscle tissues of European badgers (Meles meles) originating from different areas of Romania. Overall, muscle samples collected from 24 European badgers from six Romanian counties were examined using the artificial digestion method. Recovered larvae were subjected to PCR-based species discrimination by multiplex polymerase chain reaction (PCR). Trichinella larvae were detected in one of the 24 examined European badgers, originating from Arad County, Western Romania. Molecular analysis confirmed the presence of T. britovi, the species most commonly identified in wild carnivores from temperate regions. Although T. britovi has previously been reported in the European badger in Romania in a specimen from Central Romania, the present finding represents, to the best of our knowledge, the first detection of this parasite–host association in Western Romania. The low infection prevalence and the detection of T. britovi in the European badger indicate circulation within the sylvatic cycle and highlight the need for continued wildlife monitoring, given the zoonotic potential of this parasite.

Pathogens doi: 10.3390/pathogens15060629

Authors: Jinhua Yu Bingchen Jiang Juan Xiong Xiaojing Xu Liping Xu Yuanying Jiang Hui Lu

Azole resistance in Candida albicans is an increasing clinical challenge. Upc2 is a key transcription factor regulating ergosterol biosynthesis, but its additional roles in azole tolerance remain unclear. This study investigated whether Upc2 contributes to azole resistance through pathways beyond ergosterol synthesis. Chemical sensitivity screening, RNA sequencing, flow cytometry, and molecular assays were performed to compare wild-type C. albicans and the upc2Δ/upc2Δ mutant under fluconazole (FLC) treatment. The UPC2 gene deletion affected physiological processes that are dependent on the calcineurin signaling pathway and led to an overall negative enrichment trend in the unfolded protein response (UPR) pathway gene set. Mechanistically, the UPC2 gene deletion impaired unconventional splicing of HAC1 mRNA, leading to accumulation of unfolded proteins and phenotypically its deletion enhanced sensitivity of C. albicans to FLC in planktonic growth, hyphal development, and biofilm formation. Our findings reveal that Upc2 regulates proteostasis in C. albicans, and its absence enhances FLC efficacy by disrupting the UPR pathway. Targeting Upc2-mediated UPR signaling may represent a promising strategy to combat azole resistance.

Pathogens doi: 10.3390/pathogens15060628

Authors: Jiahui Chen Huaying Wang Ying Li Yuyi Xiao Yi Yan Yifei Zhang Xiaoxia Lu

Pediatric respiratory infections remain among the leading causes of emergency department visits, hospitalization and pediatric intensive care unit (PICU) admission. Although most acute respiratory infections in children are viral, clinical manifestations overlap substantially among viral, bacterial and atypical pathogens, creating diagnostic uncertainty and promoting empirical antimicrobial use. Rapid antigen tests, nucleic acid amplification tests, multiplex respiratory panels and metagenomic sequencing have expanded the ability to detect pathogens within clinically actionable timeframes. However, evidence from pediatric emergency trials indicates that rapid pathogen detection alone does not necessarily reduce antibiotic prescribing or healthcare costs. These findings suggest that the value of rapid diagnostics depends less on analytical breadth than on whether testing is applied to the right child, in the right clinical scenario and within a predefined decision pathway. This narrative review reorganizes the evidence around a scenario-driven top-pathogen framework. Top pathogens are defined as organisms that, in a specific age group, syndrome, season or care setting, have high prevalence, severe disease potential, transmissibility, treatment implications, antimicrobial resistance relevance or infection-control value. We discuss how top-pathogen testing should differ across emergency triage, inpatient ward management, severe pneumonia, PICU care, hospital-acquired pneumonia, ventilator-associated pneumonia and outbreak settings. We further examine the economic mechanisms through which rapid testing may generate value, including reduced unnecessary antibiotics, timely antiviral therapy, optimized isolation, shorter length of stay, reduced repeated testing and prevention of healthcare-associated transmission. Finally, we propose implementation principles centered on diagnostic stewardship, antimicrobial stewardship, local epidemiology and real-world cost-effectiveness evaluation. A scenario-driven top-pathogen strategy may provide a practical bridge between broad syndromic testing and precision infectious disease management in children.

Pathogens doi: 10.3390/pathogens15060627

Authors: Luis Gil-Gallardo Marina Simon Páez Javier Nieto Martínez Maria Delmans Flores-Chavez María Asunción Iborra-Bendicho Manuel Segovia-Hernández

Strongyloidiasis is a neglected tropical disease that frequently coexists with Chagas disease (CD) among individuals from Latin America. Serology is increasingly used for the diagnosis and post-treatment follow-up of strongyloidiasis; however, comparative data on commercially available assays remain limited. We conducted a cross-sectional evaluation in a cohort of fifty-five patients with previously treated CD attending a Tropical Medicine Unit in Spain. All patients underwent serological screening for Strongyloides stercoralis using two commercial ELISA assays: the Strongyloides IgG ELISA kit (DRG Instruments GmbH) (DRG) and Anti-Strongyloides ELISA IgG (Euroimmun Medizinische Labordiagnostika AG) (Euroimmun). A composite reference standard defined infection as seropositivity in combination with eosinophil count (>0.5 × 103 eosinophils per µL) or seropositivity in both assays. Infected individuals received ivermectin and were reassessed one-year post-treatment. Thirty-seven patients (67.3%) met the criteria for strongyloidiasis. Concordance between the assays was substantial (κ = 0.80). According to the composite study definition, both assays identified most patients as infected. At one-year follow-up, significant reductions were observed in DRG and Euroimmun indexes, as well as in eosinophil count (all p < 0.001). Treatment response rates were 83.3% with DRG and 73.3% with Euroimmun. Both ELISA assays showed comparable performance in identifying patients at risk of strongyloidiasis and in monitoring serological response after ivermectin treatment, supporting their usefulness in the management of patients with CD and suspected strongyloidiasis. Routine serological screening should be considered in this high-risk population.

Pathogens doi: 10.3390/pathogens15060626

Authors: Cristina Gay Lucía Ramos-Merino Elena Sandoval David Couto-Mallón Andrés Perissinotti María García-Vieites Eduardo Barge-Caballero Marta Farrero Marta Hernández-Meneses María G. Crespo-Lerio

Background: Infections remain a major complication in patients with left ventricular assist devices (LVADs). Enterococcal infections are increasingly recognized and are therapeutically challenging because of antimicrobial tolerance, biofilm formation on prosthetic material, and the frequent impossibility of device removal. Objective and method: We report two clinical cases of Enterococcus faecalis LVAD infection and a PRISMA-based systematic review of cases published between 1996 and 2025 that were identified through PubMed/MEDLINE. Primary outcomes were clinical cure, relapse or recurrence, mortality, and device removal. Results were synthesized descriptively. Results: Seventeen cases were analyzed, including 15 previously reported cases and two new cases. E. faecalis accounted for 47% of infections, Enterococcus faecium for 29%, and Enterococcus spp. without species identification for 24%. Bacteremia was the most common presentation, occurring in 76%, whereas LVAD-associated endocarditis occurred in 18%. Driveline or pocket involvement was reported in 41%. Suppressive antimicrobial therapy (SAT) was used in 29%, often as a bridge to heart transplantation. Cultures from explanted LVAD components were positive in 47% of cases despite prolonged antimicrobial therapy. Recurrence or relapse and overall mortality each occurred in 18% of patients. Conclusions: Enterococcal LVAD infections may be associated with microbiological persistence despite prolonged therapy. SAT may help control infection in selected patients and bridge to heart transplantation when device removal is not feasible.

Pathogens doi: 10.3390/pathogens15060625

Authors: Bac V. G. Nguyen Tu Thien Nhat Nguyen Bang Chau Ngoc Tu Hung Van Cao Bich Ngoc Thi Nguyen Thanh Tri Vu Gia-Phong Vu Hoai Thu Le Phuoc Vinh Nguyen

Vulvovaginal candidiasis (VVC) is a common fungal infection among reproductive-age women and is increasingly challenged by the emergence of non-albicans Candida species and reduced azole susceptibility. This prospective cross-sectional study investigated 235 symptomatic reproductive-age women attending two healthcare facilities in Ho Chi Minh City, Vietnam, to determine VVC prevalence, Candida species distribution, pregnancy-associated patterns, antifungal susceptibility, and diagnostic performance. Vaginal swabs were cultured on Sabouraud Dextrose Agar and CHROMagar™ Candida, while species identification was confirmed by PCR-RFLP targeting the ITS region. Susceptibility to fluconazole and clotrimazole was assessed using the disk diffusion method. Candida spp. was detected in 55.7% of participants. C. albicans accounted for 50.3% of isolates, whereas non-albicans Candida species represented 49.7%, indicating a substantial species shift. VVC was more frequent among pregnant women, particularly in the third trimester. Most C. albicans, C. tropicalis, and C. parapsilosis isolates remained susceptible to azoles; however, C. glabrata showed markedly reduced susceptibility to fluconazole and clotrimazole. CHROMagar™ Candida reliably identified C. albicans but misclassified several non-albicans Candida isolates compared with PCR-RFLP. These findings highlight the need for routine species-level diagnosis, antifungal susceptibility testing, and strengthened VVC surveillance in reproductive and antenatal healthcare settings in Vietnam.

Pathogens doi: 10.3390/pathogens15060624

Authors: Salah-Eddine Laasli Abdelfattah A. Dababat Fouad Mokrini Elhassan Mayad Amal Hari Honglian Li Hajar El Hamss Mustafa İmren Muhammad Amjad Ali Faheem Shehzad Baloch Rachid Lahlali

Root-knot nematodes (Meloidogyne javanica and M. incognita) constrain olive (Olea europaea L.) production and require safer alternatives to chemical nematicides. Despite the growing interest in microbial biocontrol agents, limited information is available on how application timing and microbial consortia influence nematode suppression and host biochemical responses in olive. This greenhouse study evaluated endophytic Trichoderma (T. harzianum) and Bacillus (B. subtilis), applied alone or in combination, either before or after nematode inoculation, to determine how biocontrol treatment timing influences nematode suppression, plant performance, and host biochemical responses. Across both nematode species, preventive application consistently outperformed curative application, and the combined treatment delivered the strongest overall protection. Preventive co-application reduced nematode infection, improved root and shoot growth, attenuated oxidative damage, and preserved chlorophyll more effectively than single-agent or post-inoculation treatments. Spectroscopic (Fourier Transform Infrared (FTIR)) and multivariate analyses further showed that effective treatments were associated with lower nematode-associated protein and lipid signals and stronger signatures of structural defense in root tissues. Although M. javanica caused greater physiological disruption than M. incognita, both species responded to the same general treatment pattern. These findings show that early establishment of microbial biocontrol agents is critical for durable suppression of root-knot nematodes in olive and supports preventive microbial consortia as a promising strategy for integrated nematode management.

Pathogens doi: 10.3390/pathogens15060623

Authors: Zhiqiang Hu Jiali Li Ase Hailai Ran Guan Xinhong Li Xi Chen Yiqun Chen Mingyu Fan Zengwen Huang Guangwen Yan Chaoyun Yang

Avian influenza virus (AIV), a zoonotic pathogen capable of cross-species transmission, poses a significant global health threat due to its rapid evolutionary adaptation. This review consolidates evidence from the past decade on AIV-autophagy interactions, emphasizing mechanistic insights and therapeutic potential. Research indicates that various AIV strains can trigger autophagosome formation via viral components, although the completeness of autophagic flux is not fully understood. These virus–host interactions are notably influenced by viral genotypes (e.g., H5N1 vs. H9N2) and host species (avian vs. mammalian). Current studies suggest that modulating autophagy may reduce AIV-induced acute lung injury, with pharmacological agents showing potential in mitigating inflammatory responses. We systematically explore three research areas: (1) strain-specific mechanisms of autophagy induction, (2) host-specific autophagic responses in poultry and human models, and (3) the therapeutic potential of stage-specific autophagy manipulation. This synthesis clarifies critical knowledge gaps, particularly the need for standardized autophagic flux assessment in avian cells, while providing a conceptual framework for developing autophagy-targeted strategies against AIV pathogenesis.

Pathogens doi: 10.3390/pathogens15060622

Authors: Konrad Wojnarowski Paulina Cholewińska Dongqing Zhao Yoshikazu Hasegawa Daniela Denk Dušan Palić

Rapid and accurate diagnosis of infectious diseases in aquaculture is essential for preventing major economic and ecological losses. Traditional culture-based methods focus on isolation of individual pathogens, and often are burdened with extended processing times, particularly during investigations of polymicrobial infections. Application of Oxford Nanopore Technologies (ONT) sequencing offers a rapid, culture-independent workflow for the identification of bacterial and fungal pathogens directly from fish tissues. Swab and organ samples from four cases (1: Salmo spp.; 2: Cyprinus carpio; 3: Salvelinus fontinalis; 4: Heniochus acuminatus) were analyzed using ONT long-read sequencing for metagenomic screening and bioinformatic classification. The results revealed case-, species-, and tissue-specific microbial profiles, with external tissues showing higher microbial diversity and internal organs enriched in pathogenic taxa. Dominant pathogens included Streptococcus iniae, Aeromonas hydrophila, Pseudomonas spp., and Saprolegnia parasitica, alongside opportunistic zoonotic bacteria such as Escherichia coli and Acinetobacter baumannii. We demonstrate the potential for diagnostic application of ONT sequencing in investigations and detection of multi-pathogen infections, including assessments of microbial community structure changes during disease outbreaks in aquatic species. The presented workflow enables rapid, cost-effective, and comprehensive pathogen profiling, supporting early disease surveillance and improved management in aquatic veterinary practice.

Pathogens doi: 10.3390/pathogens15060621

Authors: Jakub Niestępski Jakub Marek Baran Zuzanna Waszak Joanna Jarzębska Damian Grusiecki Maja Śmigielska Magdalena Marczyńska Maria Pokorska-Śpiewak

Background: International travel and global mobility have led to an increasing number of pediatric patients presenting with infections acquired outside Europe. However, epidemiological data on imported infectious diseases in children remain fragmented, with substantial heterogeneity across studies. This systematic review aimed to synthesize current evidence on the spectrum, distribution, and epidemiological characteristics of imported infections in children and adolescents returning to Europe. Methods: A systematic review was conducted in accordance with PRISMA 2020 guidelines and registered in PROSPERO (CRD420251245531). PubMed, Scopus, and the Cochrane Library were searched for studies published between 2010 and December 2025. Studies reporting laboratory-confirmed or clinically diagnosed imported infections in pediatric populations (0–18 years) returning to Europe were included. Data were extracted and synthesized descriptively, with separate analyses for malaria-specific, non-malarial, and syndromic cohorts. Results: A total of 31 studies was included. Malaria was the most consistently reported infection, predominantly caused by Plasmodium falciparum and most frequently reported in association with travel to sub-Saharan Africa, which accounted for the largest proportion of reported exposures in clinical cohorts. Visiting friends and relatives (VFR) was the dominant travel category, representing between 45.8% and 87.4% of cases in disaggregated malaria cohorts and a substantial proportion across non-malarial studies. Non-malarial infections—including gastrointestinal, bacterial, parasitic, and viral diseases—represented a substantial proportion of cases in mixed and post-travel cohorts, with gastrointestinal illness frequently constituting the leading diagnosis. Considerable heterogeneity was observed across studies in terms of design, diagnostic approaches, and reporting practices, precluding formal meta-analysis. Conclusions: Imported infections in pediatric travelers encompass a broad and heterogeneous spectrum extending beyond malaria alone. Diagnostic approaches should integrate travel history, geographic exposure, and clinical presentation. Standardized pediatric-specific surveillance and harmonized reporting are needed to improve comparability and support more accurate epidemiological assessment.

Pathogens doi: 10.3390/pathogens15060620

Authors: Yesica Gisel Candia Vanesa Nahirñak Alejandra Badaracco Humberto Debat María Elena Schapovaloff Nicolás Bejerman

The yerba mate psyllid, Gyropsylla spegazziniana, represents a major pest affecting yerba mate production, leading to considerable economic losses. Although several aspects of its ecology and management have been investigated, little is known about the viruses associated with this insect pest. In this study, we conducted the first RNA high-throughput sequencing (HTS) analysis of G. spegazziniana to examine its virome, uncovering a diverse array of previously undescribed RNA viruses. Our analysis led to the identification of five novel viruses spanning different viral lineages. These include representatives with evolutionary affinities to beny-like, picorna-like, and sobemo-like viruses, provisionally designated as Gyropsylla spegazziniana beny-like virus 1 (GSBlV1), Gyropsylla spegazziniana picorna-like virus 1 (GSPlV1), and Gyropsylla spegazziniana sobemo-like virus 1-3 (GSSlV1-3), respectively. Phylogenetic analysis of the bi-segmented, highly divergent sobemo-like viruses revealed that these viruses are grouped with other insect-associated sobemo-like viruses. The beny-like virus clustered together with other insect-associated beny-like viruses, whereas the picorna-like virus clustered together with psyllid-associated picorna-like viruses. Overall, these findings demonstrate that G. spegazziniana harbors a complex and previously uncharacterized virome. This work provides a foundation for further research into the ecological significance, evolutionary patterns, and possible use of these viruses in biological control strategies of this major pest within yerba mate agroecosystems.

Pathogens doi: 10.3390/pathogens15060619

Authors: Manao Ozawa Motoshi Kawano Shoko Iwamoto

We conducted whole-genome sequencing to investigate serotypes, virulence-associated genes, antimicrobial resistance determinants, and genetic relationships among Glaesserella parasuis isolates from diseased pigs in Japan, focusing on underrecognized aspects of disease epidemiology and control. Although Glässer’s disease is well recognized in swine production, its epidemiology remains incompletely understood, particularly regarding the relationship between serotype, genotype, and pathogenicity. Serotypes 5 or 12 (5/12) (28.9%) were predominant, followed by serotype 7 (10.8%). Phylogenetic analysis based on core-genome single nucleotide polymorphisms and cluster analysis classified the isolates into three genetic groups, with no clear association between serotype and genetic grouping. One genetic group tended to exhibit a lower proportion of severe clinical cases compared with the others, with a statistically significant difference observed in one comparison but not in the other. These findings provide evidence suggesting genotype-associated differences in disease severity, indicating that pathogenic potential may be more closely linked to genetic background than to serotype. These findings suggest a potential limitation of serotype-based vaccine strategies. Although 86.7% of isolates lacked antimicrobial resistance genes, resistance determinants were identified on contigs predicted to be of plasmid origin. These results indicate that antimicrobial resistance, while not widespread, may be underestimated and could disseminate. Overall, our findings highlight underexplored aspects of Glässer’s disease relevant to improving control and prevention.

Pathogens doi: 10.3390/pathogens15060618

Authors: Harry Tagbor Joseph Osarfo Doris Okyere Ekoue Kouevidjin Matilda Aberese-Ako Gifty Dufie Ampofo

Despite declining malaria transmission, maternal anaemia and low birthweight (LBW) remain high in Ghana, suggesting that non-malarial determinants now dominate these outcomes. A cohort of 5197 pregnant women was enrolled at eight health facilities across Ghana’s Ashanti and Volta regions. Independent predictors of anaemia at term and LBW were identified by multivariable logistic regression using multiple imputation by chained equations. Anaemia at term prevalence was 60.57% and LBW was 11.54%. No intestinal helminths were detected. Anaemia at antenatal care (ANC) booking was the strongest predictor of anaemia at term (aOR 3.98; absolute risk increase of 31.4 percentage points). Three or more iron and folic acid doses and eight or more ANC contacts each reduced risk by only four to six percentage points. For LBW, household poverty (absolute risk increase 8.8%), short maternal stature, and male foetal sex dominated. Intermittent preventive treatment of malaria in pregnancy with sulfadoxine–pyrimethamine (aOR 0.65) remained protective despite booking parasitaemia being non-significant. The Volta region had higher anaemia (71.1%vs. 47.7%) but lower LBW (9.6%vs. 14.4%) than Ashanti, with markedly higher parasite densities among infected women, consistent with waning naturally acquired immunity. Persistent anaemia and LBW reflect nutritional depletion, structural poverty, and increased malaria vulnerability under declining transmission. Pre-conception nutrition, social protection, and sub-microscopic malaria surveillance must complement clinic care.

Pathogens doi: 10.3390/pathogens15060617

Authors: Li Wei Chengjia Xie Qingnan Ren Mengfan Zhi Song Shen Xiufeng Gu Qiang Feng Tianyong Sun

Indole, a volatile metabolite produced by bacterial tryptophanase (encoded by tnaA) during tryptophan metabolism, contributes to oral malodor and may influence the progression of periodontitis. However, the genetic features underlying strain-specific indole production and its association with bacterial virulence remain unclear. Analysis of a previously published periodontitis cohort revealed that periodontitis severity was associated with salivary indole-related metabolic signatures, which were positively correlated with the abundance of Porphyromonas gingivalis (P. gingivalis). Further analysis showed that W83, a reference strain previously reported to exhibit relatively high virulence-associated characteristics, produced significantly higher levels of indole than ATCC 33277 under the experimental conditions. Comparative genomic analysis of 36 complete P. gingivalis genomes showed that the amino acid sequences of TnaA were highly conserved. However, the transposase region adjacent to tnaA differed among strains: previously reported high-virulence strains, including W83, W50, and A7436, harbored the IS5-family transposase ISPg1, whereas several low-virulence reference strains carried the IS982-family transposase IS195. In saliva samples from periodontitis patients, ISPg1 expression was positively correlated with tnaA expression, and both were associated with periodontal clinical parameters. Together, these findings indicate that transposase-associated genomic variation near tnaA is associated with strain-specific tryptophan-indole metabolism, virulence-associated gene expression, and periodontal clinical parameters, while direct causality remains to be established in future functional studies.

Pathogens doi: 10.3390/pathogens15060616

Authors: Yongmei La Fan Li Cunjuan Duan Jinjiao Kong Haipeng Zhang Hongli Tan Baoxiang Li Youhong Zhong Shilong Yang Peng Wang Liyuan Shi

To determine whether Yersinia pestis (Y. pestis) signals were present in the soil, 78 soil samples were collected from 10 counties identified as Rattus tanezumi plague foci and another six counties in non-foci areas in Yunnan, China, from August 2024 to May 2025. Nucleic acids were extracted using the DNeasy PowerSoil Pro Kit (Qiagen) and detected with a Bio-Rad QX200 droplet digital PCR (ddPCR) system targeting two Y. pestis genes (caf1 and ypo0392). Our sampling design also considered two additional dimensions: nine soil types and three habitat types. The raw data obtained from ddPCR were copy numbers. A gene was considered positive when its copy number exceeded the limit of detection (LoD). A sample was considered positive if the copy number of ypo0392 exceeded its LoD (regardless of caf1), or if both genes exceeded their respective LoDs. No correlation was detected between the positivity rate of Y. pestis and the division of sampling sites into plague focus and non-focus regions (p = 0.758). Similarly, the Mann–Whitney U test revealed non-significant differences in pathogen copy number across the two site categories, with p = 0.603 for the caf1 gene and p = 0.372 for the ypo0392 gene. No statistical difference in positivity rate was found for either soil types or habitat types. However, for both the grouping variables, a statistical difference in copy number was observed. Our results indicated that quantifying nucleic acid abundance by copy number provided richer information than a simple positive/negative determination. The detection of Y. pestis signals was associated with soil type, rather than with whether an area was classified as a focus or non-focus region. Accordingly, future research on the preservation mechanism of Y. pestis should not be restricted to the concept of natural foci but should adopt a broader perspective.

Pathogens doi: 10.3390/pathogens15060615

Authors: Bożena Kowalczyk Agnieszka Szuster-Ciesielska Rafal Luchowski Wiesław I. Gruszecki Piotr Koper Markus Petzold Jacek Tarasiuk Sofia Barigelli Ermanno Federici Marta Palusińska-Szysz

Legionella pneumophila is widely recognized as the principal organism responsible for Legionnaires’ disease. Modifications of surface-exposed lipopolysaccharide in L. pneumophila are key determinants of bacterial adaptation to host cells. Using a mutant strain deficient in N-methylation of legionaminic acid and fluorescence-based biophysical analyses, it was demonstrated that N-methyl groups linked to the acetimidoylamino group of legionaminic acid modulate the physicochemical properties of the bacterial cell surface and influence the interaction of L. pneumophila with macrophages. Loss of N-methyl groups reduced the efficiency of bacterial interaction with THP-1-derived macrophages and impaired intracellular proliferation. In addition, the presence of N-methyl groups in legionaminic acid contributes to increased TNF-α production in THP-1 macrophages stimulated with L. pneumophila sg1 LPS, without affecting IL-6 induction, suggesting that N-methylation of legionaminic acid may skew the early pro-inflammatory response toward TNF-α-dominated signaling during L. pneumophila infection.

Pathogens doi: 10.3390/pathogens15060614

Authors: Mauro Serricchio Muriel Fragnière Jochen Gottschalk Caroline Tinguely Christoph Niederhauser

Transmission of malaria by blood transfusion is rare in non-endemic countries but can lead to serious complications in blood recipients. Increasing travel to and immigration from regions at risk for tropical diseases poses a challenge to blood donation services, which are striving to reduce the number of blood donor deferrals while ensuring a high level of blood safety. National guidelines of the Blood Transfusion Service of the Swiss Red Cross demand that donors at risk are serologically tested for malaria antibodies. Here, we summarize the numbers of malaria tests performed and the results obtained since the introduction of mandatory testing in Switzerland in 2007. From malaria-positive donors, information on travels to endemic areas and place of origin, or if malaria symptoms were experienced and if prophylaxis was taken, was requested in a post-donation questionnaire.

Pathogens doi: 10.3390/pathogens15060613

Authors: Ewa Zastempowska Magdalena Twarużek Jan Grajewski Henryka Lassa

Enterococci are among the most frequently isolated environmental bacteria that cause mastitis in cows. This study aimed to determine the prevalence of virulence genes, as well as phenotypic and genotypic antibiotic resistance, among eighty enterococcal isolates from cases of bovine mastitis in Polish herds. The presence of virulence and antibiotic resistance genes was determined by PCR. E. faecalis isolates were found to carry more virulence genes than E. faecium isolates, including the efaAfs (100%), ace (98.1%), gelE (86.5%), asa1 (63.5%), esp (57.7%) and cylA (17.3%) genes. The efaAfm gene was the only virulence gene detected in E. faecium isolates. This study revealed that E. faecalis showed a higher virulence gene burden. The ermB gene was present in 90.9% of the Enterococcus spp. that were phenotypically resistant to erythromycin. Almost all tetracycline-resistant Enterococcus isolates carried the tet(M) gene (94.3%), either alone or in combination with the tet(L) and tet(O) genes. Three isolates harboured vanC genes and were susceptible to vancomycin (MIC = 4 μg/mL). The results confirm the high level of antimicrobial resistance of enterococci isolated from cows with mastitis and indicate the genes that may be responsible for this resistance.

Pathogens doi: 10.3390/pathogens15060612

Authors: Noman Ali Nan Wu Engin U. Akkaya Mahinur S. Akkaya

Powdery mildew fungi are major obligate biotrophic plant pathogens, that cause widespread disease in agricultural and natural ecosystems worldwide, but a comparative structural view of their secretomes across multiple genera has remained limited. Here, we performed computational structure prediction and comparative analysis of 7545 secretome candidates from 26 isolates representing five genera (Blumeria, Erysiphe, Golovinomyces, Parauncinula, and Podosphaera) using AlphaFold2-based structure prediction, structural annotation against CATH, SCOPe, and PDB, Foldseek-based clustering and network analysis, structure-based grouping of RALPH (RNase-like proteins associated with haustoria) candidates, and comparison with defined fungal effector structural families. The predicted secretomes showed comparable model confidence across isolates and revealed a conserved structural core composed of recurrent microbial ribonuclease, immunoglobulin/fibronectin-like, glycosidase-related, and other enzyme-associated folds, with MoHrip2-like representing the most prominent shared fold among defined fungal effector structural families. Structural clustering and network analysis identified a prominent RALPH-centered component with additional conserved and lineage-enriched communities. RALPH candidates formed a structurally diverse repertoire that could be partitioned into 15 topology-defined groups, several linked to previously characterized powdery mildew effectors. Blumeria was structurally distinct, showing expansion of RALPH-associated components and the absence of multiple fold/domain categories retained in dicot-associated genera. Together, these results establish a comparative structural landscape of powdery mildew secretomes and provide a framework for future functional, evolutionary, and genomics-driven studies of conserved and lineage-associated secretome candidate.

Pathogens doi: 10.3390/pathogens15060611

Authors: Amany E. Gomaa Eric Parperides Xin-Qiu Yao Gabriela Espinoza Vergara Ziomara Jurado Hernan Garcia-Ruiz

Potyvirus rapae (turnip mosaic virus, TuMV) is widely used as a model system in plant–virus interaction studies. The TuMV RNA genome encodes 11 proteins, some of which remain poorly characterized, while the functions of others are well defined. Studying individual proteins in isolation may not recapitulate native expression levels, subcellular localization, and interaction with host factors during virus replication and movement. An alternative approach is to tag individual viral proteins in the context of an infectious clone. Epitope tags may alter protein functions and affect viral replication, movement, or a combination of essential steps, thus leading to changes in pathogenicity. Because they have central roles in viral infection, here we measured the effect of individually tagging the helper component proteinase (HC-Pro) and nuclear inclusion protein b (NIb) with a 6His-3xFLAG tag. Epitope tags were placed at the N-terminus of HC-Pro and the N- and C-termini of NIb within a TuMV infectious clone carrying coding sequences for the green fluorescent protein (TuMV-GFP). Constructs carrying a tagged HC-Pro displayed pathogenicity similar to that observed for TuMV-GFP in Nicotiana benthamiana and Arabidopsis thaliana plants. In contrast, infectivity of NIb-tagged clones became temperature sensitive and, even at the permissive temperature, showed reduced pathogenicity compared to TuMV-GFP. Providing a silencing suppressor in trans did not restore infection efficiency, suggesting reduced viral fitness due to structural or functional disruption caused by the epitope tags. Structural models generated using AlphaFold2 showed no effect of the tag on HC-Pro. In contrast, structural models illustrated tag interference with the NIb catalytic site. AlphaFold2 was further used to predict the structural impact of several tags on NIb and to predict the effect of a 6HIS-3xFlag tag on all other TuMV proteins. This study provides a broadly applicable framework for selecting suitable epitope tags to mark viral proteins and maintain function in the context of virus infection.

Pathogens doi: 10.3390/pathogens15060610

Authors: Yalewayker Asrat Biruk Bayleyegn Mark Willcox Nicole Carnt Binod Rayamajhee

Background: Acanthamoeba is a free-living protozoan widely distributed in the environment and causes Acanthamoeba keratitis, skin, and brain disease. Acanthamoeba can exchange genes, potentially increasing antimicrobial resistance and virulence. Therefore, this systematic review aimed to summarize published studies on horizontal gene transfer (HGT) between Acanthamoeba and its intracellular microorganisms and to evaluate the impact of HGTs on the pathogenicity of Acanthamoeba. Methods: This systematic review was conducted following the recommended reporting guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) statement guideline. The electronic databases PubMed, Embase, and Web of Science were used to search for relevant published research articles. Results: Nineteen studies that fulfilled the inclusion criteria were included in this systematic review. A total of 14 (73.6%) studies reported evidence of HGT involving Acanthamoeba, and five studies of the nineteen (26.3%) analysed the presence of intracellular microorganisms on the pathological effects of the host Acanthamoeba. Horizontally transferred genes were predominantly reported from Pseudomonas species, Legionella pneumophila, and Chlamydia species. Conclusions: HGT can occur among intracellular microorganisms and their host Acanthamoeba. Acanthamoeba harbouring intracellular microbes showed enhanced pathogenic effects on human corneal epithelial cells and in a mouse model. However, heterogeneity among the included studies precluded meta-analysis. Studies using clinical and environmental samples are needed to characterize the horizontal transfer of virulence and antimicrobial resistance genes.

Pathogens doi: 10.3390/pathogens15060609

Authors: Alaine M. L. Catão Dulcimê Gonçalves Dorta Walquíria Arruda Cristian Montalva Christian Luz

Leptolegnia chapmanii is an oomycete pathogen of mosquito larvae. We investigated whether nutritional factors promoting cyst germination in vitro are associated with early infection events and instar-specific susceptibility in Aedes aegypti. Cysts of the Brazilian isolate ARSEF 12829 germinated rapidly in soybean seed extract, sunflower seed extract and minimal medium supplemented with yeast extract, whereas basal minimal medium did not promote germination. In sunflower seed extract, germination increased significantly with incubation time; in minimal medium, germination at 24 h was much higher with ≥0.2% yeast extract than with 0.1%. In third-instar larvae, a few cysts attached to the cuticle during the first 30–60 min, with no external germ tubes observed. At 3 h, melanized hyphal structures were detected in the midgut, and histological sections showed germinated and ungerminated cysts in the endoperitrophic space, with hyphae crossing the peritrophic matrix and midgut epithelium toward the hemocoel. Mortality increased with cyst concentration and exposure time and decreased with larval instar. At 3.3 × 103 cysts/mL, final mortality reached 100% in L1–L3 and 91.2% in L4 larvae. These results link rapid cyst germination with early midgut invasion and high larvicidal activity.

Pathogens doi: 10.3390/pathogens15060608

Authors: Kim Ruiz-Arellanos Maria Camila Cardenas-Fernández Silvana Bonilla

Helicobacter pylori (H. pylori) antimicrobial resistance is a key driver of eradication failure in children and adults. Recognizing factors associated with H. pylori antimicrobial resistance may help identify individuals at risk for treatment failure and tailor management. We conducted a retrospective cohort study of patients with H. pylori-positive gastric biopsy culture and available antimicrobial susceptibility data at a large pediatric tertiary care center between 2020 and 2025. Descriptive statistics and logistic regression models were used to assess associations among demographic characteristics, prior treatment history, and antimicrobial resistance. Of 174 patients (56.1% male, median 14 years), 50.3% were resistant to at least one antimicrobial. Resistance rates included metronidazole (28.3%), clarithromycin (18.5%), rifampin (12.7%), fluoroquinolones (11.6%), amoxicillin (4%), and tetracycline (0.6%). The multidrug resistance rate was 17.3%. Prior H. pylori treatment was the most consistent predictor of antimicrobial resistance on both bivariate and multivariable analyses. Race was independently associated with metronidazole resistance, with Black patients exhibiting the highest rates and significantly greater odds compared to White patients. Hispanic ethnicity was also independently associated with increased odds of metronidazole resistance. Careful consideration of prior treatment history, race, and ethnicity is warranted when managing pediatric H. pylori infection, given their association with increased antimicrobial resistance.

Pathogens doi: 10.3390/pathogens15060607

Authors: Putu Prathiwi Primadharsini Shigeo Nagashima Masaharu Takahashi Kazumoto Murata Hiroaki Okamoto

Hepatitis E virus (HEV) infection is generally self-limiting in immunocompetent individuals but may progress to chronic infection in immunocompromised patients, underscoring the need for effective antiviral therapies. Although ribavirin is currently used off-label for HEV treatment, its associated adverse effects highlight the need for safer alternatives. In this study, we screened an anti-viral compound library comprising 800 compounds using three HEV reporter systems designed to target distinct stages of the viral life cycle. Candidate compounds were further evaluated in PLC/PRF/5 cells using both acute and chronic infection models with wild-type genotype 3 HEV (HEV-3). Antiviral activity was assessed by measuring HEV RNA levels in culture supernatants. Elbasvir, a known inhibitor of hepatitis C virus (HCV) non-structural protein 5A (NS5A), was identified as the most potent candidate. Although multiple compounds showed inhibitory effects in reporter assays, only elbasvir achieved sustained suppression of HEV growth in long-term culture, reducing HEV RNA levels to below the limit of detection. In a chronic infection co-culture model, elbasvir maintained antiviral activity at non-cytotoxic concentrations. Time-of-addition analysis demonstrated that elbasvir inhibits an early step in the viral life cycle, specifically viral internalization. Furthermore, combination with ribavirin enhanced antiviral efficacy, resulting in sustained viral suppression without detectable cytotoxicity and exhibiting an additive interaction. Collectively, these findings identify elbasvir as a promising candidate for repurposing as an anti-HEV drug and support a combination strategy targeting distinct steps of the viral life cycle.

Pathogens doi: 10.3390/pathogens15060606

Authors: Salvatore Pennisi Luca Nalbone Mattia Pino Filippa Lamberta Graziella Ziino Alessandro Giuffrida

This study investigated the behavior of Salmonella Typhimurium ATCC 14028, Escherichia coli O157:H7 and Listeria monocytogenes ATCC 13932 during the ripening of ‘Nduja, a traditional spreadable fermented sausage for which quantitative microbiological data remain limited. An experimental challenge test was conducted under pilot-plant conditions simulating artisanal production, with products inoculated with the three pathogens and monitored over a 28-day ripening period. Microbiological analyses were performed at defined time points, alongside pH and water activity measurements, and inactivation kinetics were modelled using linear and non-linear approaches. The results showed a pathogen-dependent response to the combined antimicrobial hurdles of the process. Salmonella Typhimurium was declined to levels below the detection limit by day 28 (–7.10 log CFU/g), while E. coli O157:H7 showed a progressive reduction (–3.61 log CFU/g) but persisted at detectable levels. L. monocytogenes exhibited the highest resistance, with only a limited reduction (–1.32 log CFU/g); however, no net growth was observed throughout the ripening period, indicating that the product environment did not support its growth. The ripening process was characterized by decreasing pH and water activity, driven by lactic acid bacteria growth, with no differences between inoculated and control samples. Non-linear models provided the best fit to the survival data, highlighting the presence of resistant subpopulations. Overall, the results suggest that ‘Nduja ripening creates conditions unfavorable for sustained pathogen proliferation, although the extent of microbial reduction differed among the investigated microorganisms. These findings provide useful data for the microbiological characterization of this traditional product and may support future risk assessment and process validation studies.

Pathogens doi: 10.3390/pathogens15060605

Authors: Chenfeng Li Yurong Zhang Yingchun Ke Yeyang Zhang Meijun Chen Xingru Tao Pengle Guo Jingliang Chen Xiaoping Tang Weiyin Lin Linghua Li

This study investigated the molecular epidemiological characteristics of Cryptococcus neoformans (C. neoformans) isolates from AIDS patients with cryptococcal meningitis (CM) in southern China and examined their associations with clinical features and outcomes. A total of 100 clinical isolates were identified by MALDI-TOF MS and genotyped by multilocus sequence typing (MLST). Antifungal susceptibility to five agents was assessed using the FUNGUS 3 system. Baseline demographic, clinical manifestations, radiological, and laboratory data were collected from the corresponding 100 patients, and outcomes were evaluated at weeks 4, 12, 24, and 48. Seven sequence types (STs) were identified: ST5 (83/100, 83.0%), ST4 (5/100, 5.0%), ST31 (3/100, 3.0%), ST43 (1/100, 1.0%), ST93 (4/100, 4.0%), ST395 (1/100, 1.0%), and a presumptive novel ST685 (3/100, 3.0%). Most patients were male (80.0%), and headache was the most common symptom (85.0%). Susceptibility rates for 5-flucytosine, amphotericin B, fluconazole, itraconazole, and voriconazole were 98.9% (94/95), 71.9% (69/96), 82.3% (79/96), 59.4% (41/69), and 86.8% (59/68), respectively. Cumulative mortality reached 16%, 33%, 37%, and 39% at weeks 4, 12, 24, and 48. No significant differences were observed between 83 patients infected with ST5 and 17 patients with non-ST5 in clinical presentations or antifungal susceptibility. However, patients infected with ST5 exhibited consistently better survival rates across all time points; the 12-week non-survival rates were highest for patients infected with ST93 and ST395. Accordingly, ST5 is the dominant sequence type of C. neoformans in HIV-associated CM in southern China; meanwhile, non-ST5 types could have worse prognosis, indicating ST sequence typing may act as a prognostic biomarker in AIDS patients with CM.

Pathogens doi: 10.3390/pathogens15060604

Authors: Coad Thomas Dow

The rising incidence of early-onset colorectal cancer (CRC) remains incompletely explained despite extensive investigation into genetic, environmental and metabolic factors. Emerging evidence suggests that infectious agents may contribute to colorectal carcinogenesis. A recent study by Tehrani et al. demonstrated the presence of Mycobacterium avium subspecies paratuberculosis (MAP) in a majority of colorectal cancer lesions and in approximately half of precancerous lesions, providing a critical epidemiologic anchor. MAP, a zoonotic intracellular pathogen long associated with Crohn’s disease, exhibits biological features consistent with inflammation-driven carcinogenesis, including persistence, immune modulation and systemic dissemination. Mechanistically, MAP infection may promote tumorigenesis through chronic mucosal inflammation, epithelial barrier disruption and activation of human endogenous retroviruses (HERVs) linking persistent infection to genomic instability. Detection of MAP in early lesions argues against secondary colonization and supports a potential initiating or promoting role. Within a One Health framework, MAP represents a plausible, pleiotropic and potentially modifiable contributor to CRC. While causality remains unproven, the convergence of epidemiologic association, mechanistic plausibility and early lesion involvement warrants rigorous investigation.

Pathogens doi: 10.3390/pathogens15060603

Authors: Amélie Dewitte Maurane Dégardin Ivan Nemazanyy Florent Sebbane Sébastien Bontemps-Gallo

The flea-borne transmission of Yersinia pestis relies on biofilm formation and metabolic adaptation within the insect gut. The pentose phosphate pathway (PPP) is central to these processes, yet the contribution of its non-oxidative branch remains poorly defined. Here, we investigated the roles of transketolase (TktA) and transaldolase (TalB) in plague bacillus physiology, metabolism, and flea colonization. TktA was essential for growth, preventing assessment of its role in biofilm formation and in vivo colonization. In contrast, TalB was dispensable for growth but required for optimal biofilm formation. In fleas, the ΔtalB mutant colonized the proventriculus but displayed a lower bacterial load than the wild-type strain at later time points, indicating a defect in sustained colonization. Metabolomic analyses revealed that disruption of tktA severely impairs PPP-associated metabolism, whereas loss of talB is associated with disruption of nucleotide homeostasis, carbon redistribution toward glycolysis, and a redox imbalance. These findings demonstrate a functional partitioning of the non-oxidative PPP and identify it as a key metabolic control node linking metabolism to colonization dynamics in Y. pestis.

Pathogens doi: 10.3390/pathogens15060602

Authors: Cunyi Qiu Zhiding Zhou Meilin Yang Huaxin Wang Xuezhao Li Zhihua Feng Yefei Zhou

Porcine epidemic diarrhea (PED), caused by the PED virus (PEDV), remains one of the most devastating diseases in the swine industry, with a mortality rate approaching 90–100% in suckling piglets due to severe dehydration and electrolyte imbalances. Passive immunization with egg yolk antibodies (IgY) represents a promising therapeutic strategy. In this study, we developed a novel recombinant adenovirus, rADM-IFN-G-ped, co-expressing selected antigenic regions of the PEDV S protein and chicken interferon-gamma (ChIFN-γ) as a molecular adjuvant. Laying hens were immunized with this construct to produce PEDV-specific IgY, which was subsequently purified from eggs using a polyethylene glycol (PEG-6000) precipitation method. The induced IgY demonstrated potent neutralizing activity against PEDV in vitro, with a neutralization titer (NT50) of 1:96, which was significantly higher than that of IgY derived from hens immunized with a commercial inactivated PEDV G2b vaccine (NT50 = 1:52). In a passive immunization and challenge trial, piglets treated with the rADM-IFN-G-ped-derived IgY exhibited significantly reduced fecal viral RNA shedding following challenge with the virulent PEDV-NX-2022 strain, compared to control groups. Crucially, while all piglets in the challenge control group succumbed to infection within 72 h, a 50% survival rate was achieved in the IgY-treated group. Histopathological examination of intestinal tissues further confirmed the protective efficacy, showing that IgY treatment markedly alleviated villous atrophy, epithelial necrosis, and inflammatory cell infiltration in the small intestine. These findings demonstrate that vaccination of laying hens with the rADM-IFN-G-ped recombinant adenovirus elicits a robust immune response, enabling the production of protective IgY. This proof-of-concept study establishes the viability of the multi-epitope adenoviral IgY platform as a passive immunization strategy against PEDV.

Pathogens doi: 10.3390/pathogens15060601

Authors: Pietro Hiram Guzzi Francesco Branda Fabio Scarpa Giancarlo Ceccarelli Massimo Ciccozzi Federico Manuel Giorgi Pierangelo Veltri

Hantaviruses are emerging zoonotic pathogens responsible for two severe clinical syndromes: (i) haemorrhagic fever with renal syndrome (HFRS) and (ii) hantavirus cardiopulmonary syndrome (HCPS), collectively causing more than 200,000 human cases annually worldwide. Despite their public-health importance, the molecular mechanisms governing the host response and the population-level dynamics of rodent-to-human spillover remain incompletely characterised. The timeliness of this framework is underscored by the April–May 2026 outbreak of Andes orthohantavirus aboard the MV Hondius cruise ship, the first such cluster in a maritime setting, with three deaths reported across multiple countries. This event revealed critical gaps in existing models that treat humans solely as dead-end spillover hosts. Our coupled Susceptible-Exposed-Infectious-Recovered-Dead (SEIRD) model assumes no human-to-human transmission and is therefore designed for hantavirus strains where spillover does not lead to secondary human cases, specifically Hantaan virus (HTNV), Puumala virus (PUUV), Sin Nombre virus (SNV), and Dobrava-Belgrade virus (DOBV). The Andes virus (ANDV) outbreak aboard the MV Hondius is used as a real-world case study to assess the boundaries of our model and to motivate future extensions, not as a direct validation target for its quantitative predictions. Here, we present an integrated computational study combining three complementary analyses. First, we performed a preliminary phylogenetic analysis of the viral sequence, identifying Orthohantavirus andesense as the likely etiological agent responsible for the vessel-associated outbreak. Second, we carried out a downstream transcriptomic analysis of Hantaan virus (HTNV)-infected human umbilical vein endothelial cells (HUVECs), using publicly available RNA-seq data (GEO accession GSE133751, n=3 per group). This analysis identified 184 upregulated and 19 downregulated genes, highlighting a transcriptional response dominated by interferon-stimulated genes (ISGs), including CXCL10, CXCL11, MX2, DDX58, IRF7, STAT1, OASL, and CMPK2. We then constructed a protein–protein interaction (PPI) network using STRING, comprising 176 nodes and 3210 edges, and applied a composite network centrality score to rank putative regulatory hubs. This analysis identified ISG15, IRF1, CXCL10, STAT1, and DDX58 as the most central nodes. Pathway enrichment analysis confirmed a strong activation of interferon signalling (Reactome, p=1.3×10−63), antiviral defence mechanisms (Gene Ontology, p=3.8×10−58), and NF-κB-related pathways, together with a concurrent suppression of ribosomal translation. Finally, we developed a coupled SEIRD epidemiological model that explicitly represents rodent-to-rodent and rodent-to-human transmission with logistic rodent population growth. Preliminary simulation analysis demonstrates that reducing human exposure to rodent excreta is substantially more effective than rodent population control alone for reducing human disease burden, and that rodent control in isolation can paradoxically increase human cases through a dilution-like effect. The integrated framework provides molecular and epidemiological insights relevant to hantavirus surveillance, therapeutic target identification, and public-health intervention design.

Pathogens doi: 10.3390/pathogens15060600

Authors: Andie Hach Anne Vandenburg-Carroll Douglas G. Marthaler Stephen Vadia Qirui Zhang Melanie Prarat Ailam Lim

This method comparison demonstrated equivalent analytical and diagnostic performance of IndiMix JOE with intype IC-RNA as an alternative real-time polymerase chain reaction (RT-PCR) chemistry compared to the National Animal Health Laboratory Network (NAHLN) influenza A virus (IAV) RT-PCR surveillance assay using AgPath-ID One-Step RT-PCR Reagents in avian swabs and tissues and bovine milk. In avian samples, IndiMix JOE with intype IC-RNA using a fast reduced-volume protocol had comparable results to the NAHLN reference RT-PCR with and without intype IC-RNA using standard NAHLN thermocycling conditions. A six-way comparison of RT-PCR chemistries and exogenous internal controls in milk samples illustrated equivalent mean CT values (ANOVA, p = 0.9938). Additional experiments in avian swabs and tissues, bovine milk and semen samples resulted in comparable analytical sensitivity in limits of detection (LOD), linearity (R2 > 0.977), and PCR efficiencies, and no significant differences in mean CT values (ANOVA, p > 0.05). Diagnostic performance showed 100% agreement across field and spiked sample matrices compared to the NAHLN reference method. The study supports the use of IndiMix JOE and intype IC-RNA as an alternative, with practical improvements relevant to surveillance workflows including enhanced testing flexibility, efficiency, outbreak response capacity, and reduced time to results.

Pathogens doi: 10.3390/pathogens15060599

Authors: Abirami Karthikeyan Aqib Javaid Grace Naa Ayorkor Charway Nazia Tabassum Tae-Hee Kim Young-Mog Kim Won-Kyo Jung Fazlurrahman Khan

Prophages are bacteriophage genomes that are part of bacterial chromosomes. They are not just dormant passengers; they actively shape pathogen biology. For example, in skin-infecting pathogens such as Staphylococcus aureus, Streptococcus pyogenes, and Pseudomonas aeruginosa, prophages carry important virulence factors, cytotoxins, superantigens, immune evasion clusters, and epigenetic regulators that directly affect the course of skin and soft tissue infections. This same prophage biology provides a therapeutic strategy: prophage-derived molecules, including endolysins, holins, spanins, and polysaccharide depolymerases, demonstrate potent antimicrobial and antibiofilm activity against drug-resistant skin pathogens, with several candidates now in clinical development. Engineered chimeric lysins, CRISPR-encoded prophage delivery systems, and the systematic mining of the skin microbiome phageome collectively enhance the translational potential of this biology. This review integrates mechanistic insights into prophage-mediated virulence. It assesses the translational landscape of prophage-derived therapeutics, delineating the conceptual and clinical frontiers that characterize the forthcoming chapter in this domain.

Pathogens doi: 10.3390/pathogens15060598

Authors: Marzena Korbecka-Paczkowska Tomasz M. Karpiński

Background: Streptococcus mutans is a key etiological agent of dental caries, and its ability to form biofilms significantly increases resistance to antimicrobial agents. This study evaluated the activity of selected oral antiseptics against both planktonic and biofilm forms of S. mutans. Methods: Seven pure antiseptic compounds and nine commercially available mouthwashes were tested. Minimal inhibitory concentrations (MICs) were determined using a microdilution method, and their clinical relevance was assessed using the Clinical Efficiency of MIC (CEMIC) index. Antibiofilm activity was evaluated using a crystal violet assay and by measuring biofilm thickness using 3D microscopy and the Biofilm Thickness Analyzer application. Results: The highest antibacterial and antibiofilm activity was observed for octenidine (OCT), chlorhexidine (CHX), and polyhexanide (PHMB), as well as for mouthwashes containing these agents, all showing high CEMIC values. Hydrogen peroxide (H2O2) and fluoride-containing formulations also demonstrated notable activity. In contrast, ethacridine lactate and boric acid showed variable effects, while sodium hypochlorite and potassium permanganate exhibited weak antibacterial activity and no antibiofilm effect. Conclusions: OCT, CHX, and PHMB were the most effective against both planktonic and biofilm forms of S. mutans. H2O2 and fluoride-containing formulations also showed promising activity. These findings support the role of selected antiseptics in reducing dental plaque formation, a key factor in the development of oral diseases.

Pathogens doi: 10.3390/pathogens15060597

Authors: Lisa Brizzolara Jari Intra Paola Faverio Alice Biffi Francesca Basta Cristina Delfini Nicoletta Novati Elisa Zucchetti Fabrizio Luppi Marialuisa Lavitrano Marco Casati

Background: The clinical burden of pulmonary diseases associated with the genus Aspergillus is increasing, although diagnostic focus remains largely on A. fumigatus. This study evaluated the diagnostic value of testing for A. flavus and A. niger-specific IgG in patients with chronic respiratory conditions. Methods: A retrospective study was conducted on 274 subjects (156 with chronic respiratory diseases, Bronchiectasis, Hypersensitivity Pneumonitis [HP], and Interstitial Lung Disease [ILD] non-HP, and 67 healthy controls). Cut-off values were established at the 97.5th percentile of the control group: 30 mg/L for A. fumigatus, 13 mg/L for A. flavus, and 8 mg/L for A. niger. Results: Among 109 patients who tested negative for A. fumigatus, 49.5% showed positivity to at least one other species, preventing a significant diagnostic gap in exposure detection. Notably, 40.4% of these patients exhibited simultaneous positivity for both A. flavus and A. niger. In the HP subgroup, 100% of patients who were A. fumigatus-positive also showed concurrent positivity to the two other species. Furthermore, the Bronchiectasis group showed the highest rate of isolated A. flavus at 11.8%. Conclusions: Testing solely for A. fumigatus IgG leads to substantial underestimation of Aspergillus exposure. Integrating A. flavus and A. niger into diagnostic panels is essential for a comprehensive immunological assessment of fungal interaction, particularly in HP and ILD, where identifying specific antigenic exposure is crucial for managing chronic inflammation and preventing disease progression.

Pathogens doi: 10.3390/pathogens15060596

Authors: Chen Zhang Chaozheng Xie Zhengtao Zhang Renjie Luo Fang Xu

Background: Sepsis, a life-threatening organ dysfunction caused by dysregulated host responses to infection, frequently involves impaired macrophage efferocytosis that leads to apoptotic cell accumulation, secondary necrosis, and persistent inflammation. Early prognostic stratification remains challenging, as current biomarkers lack sufficient specificity and sensitivity, underscoring the urgent need for novel prognosis-related indicators. Methods: We integrated bulk transcriptomic data from a discovery cohort (GSE205672) and an independent validation cohort (GSE133822) with single-cell RNA-seq profiles of early- and late-stage sepsis (GSE167363, GSE175453). WGCNA and five consensus machine-learning algorithms were combined to screen core efferocytosis-associated genes, and expression was validated via qPCR in PBMCs from sepsis patients and CLP-induced septic mice. Results: ADAP2 was identified as the core gene achieving strict consensus across all five algorithms, with early upregulation and late depletion in sepsis, predominant expression in monocytes/macrophages—particularly M1-like and IFN-responsive subsets—and a significant correlation with efferocytosis scores and immune cell infiltration. Its expression was negatively correlated with sepsis severity (SOFA score) and showed a trend toward worse survival in patients with low ADAP2 levels. Conclusions: This multi-dimensional transcriptomic study establishes ADAP2 as a candidate biomarker with potential prognostic value in sepsis, closely linked to macrophage efferocytosis. These findings may aid early risk stratification and inform macrophage-directed immunotherapies, although prospective validation and functional studies are required.

Pathogens doi: 10.3390/pathogens15060595

Authors: Ana Marija Radevska Bojana Chapkunovska Katerina Spasovska Fadil Cana Stefan Pandilov Verica Simin Pavle Banović Dejan Jakimovski Aleksandar Cvetkovikj

Background: Toxocara canis and Toxocara cati are zoonotic nematodes of dogs and cats that maintain a human infection risk through environmental contamination with highly resistant eggs. Data on toxocariasis in North Macedonia are limited, and ocular toxocariasis (OT) remains a clinically important but easily overlooked manifestation of human infection. Methods: This retrospective assessment combined coprological data from dogs and cats with a complementary clinical description of a human case of OT. Routine fecal samples from dogs and cats from January 2018 to March 2026 were morphologically examined. The human case of OT was a 13-year-old boy with unilateral ocular disease. Results: Of 465 samples, Toxocara spp. eggs were detected in 14, corresponding to an overall detection of 3.0%. Detection was 3.0% in dogs (11/371) and 3.2% in cats (3/94), with no significant difference between species. The human clinical component involved unilateral visual loss, strabismus, and posterior segment inflammatory changes. Conclusions: Dogs and cats in North Macedonia showed sporadic Toxocara spp. egg shedding, supporting ongoing zoonotic exposure potential. The clinically confirmed OT case complements the animal data and underscores the need for improved awareness among clinicians and veterinarians, as well as strengthened preventive measures aimed at reducing environmental contamination and zoonotic exposure.

Pathogens doi: 10.3390/pathogens15060594

Authors: Ially de Almeida Moura Antônio Wesley Oliveira da Silva Gabriel da Silva Correia Giancarlo Bomfim Ribeiro Mayara Macêdo Barrozo Thaís Almeida Corrêa Patrícia Silva Gôlo Isabele da Costa Ângelo Caio Márcio de Oliveira Monteiro Éverton Kort Kamp Fernandes Vânia Rita Elias Pinheiro Bittencourt Alexandre Dias Munhoz Wendell Marcelo de Souza Perinotto

The rise in anthelmintic resistance in small ruminants has driven the search for sustainable control alternatives. Among these, entomopathogenic fungi such as Metarhizium robertsii stand out for their potential to reduce the free-living stages of gastrointestinal nematodes (GINs). This study evaluated the field efficacy of M. robertsii (LCM S15) under climatic conditions in the Recôncavo region of Bahia, Brazil. The experiment was conducted between November 2022 and July 2023 in a completely randomized design with four groups (n = 8): aqueous control, oil control, aqueous suspension, and oil formulation of M. robertsii. Egg counts per gram of feces (EPG) and L3 larval recovery were assessed by coproculture and the Baermann technique. Efficacy ranged from 15.23% to 27.34%, with the oil formulation showing higher performance. Haemonchus sp. and Trichostrongylus sp. were the most prevalent genera. These findings suggest the potential of M. robertsii (LCM S15) as a biological control agent under field conditions.

Pathogens doi: 10.3390/pathogens15060593

Authors: Aysegul Yılmaz Seyhan Turk Umit Yavuz Malkan İbrahim Celalettin Haznedaroglu Safiye Gocer Sukru Volkan Ozguven Can Turk

The renin–angiotensin system (RAS) regulates inflammation, tissue homeostasis, and barrier integrity in lung and colon epithelial cells. Beyond classical pathways, non-canonical components including angiotensin-converting enzyme 2 (ACE2), epidermal growth factor receptor (EGFR), insulin-like growth factor 2 receptor (IGF2R) and aminopeptidase N (ANPEP) are implicated in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections and bacterial sepsis due to their roles in tissue repair and signaling. Despite their similar inflammatory and coagulopathic features, their impact on RAS-associated non-immune gene expression in epithelial tissues remains unclear. This study investigates the regulation of these targets in lung (BEAS-2B) and colon (CRL-1831) cells following exposure to recombinant SARS-CoV-2 spike protein N-terminal domain fragment (S1-NTD) and Pseudomonas aeruginosa-derived lipopolysaccharide (LPS). Cells were treated with 100 ng/mL of S1-NTD or LPS for 12–72 h. Viability was assessed via XTT assays, and molecular changes were analyzed through qRT-PCR and Western blotting. Both stimuli induced a time and dose-dependent decrease in metabolic activity. ACE2 was significantly downregulated in lung cells, while transient upregulation occurred in colon cells at 24 h. EGFR expression increased in colon cells following LPS exposure but decreased in lung cells after S1-NTD treatment. Both IGF2R and ANPEP were upregulated by S1-NTD in lung cells at 72 h, whereas colon cells showed earlier upregulation at 24–48 h. Our findings reveal that viral and bacterial stimuli elicit distinct, tissue-specific regulatory patterns in RAS-associated pathways. These alterations may contribute to epithelial barrier dysfunction and inflammation, highlighting these proteins as potential targets for managing secondary bacterial infections and inflammatory lung–gut complications in COVID-19.

Pathogens doi: 10.3390/pathogens15060592

Authors: Juned Ahmed Samuel Ajulo Smriti Shringi Bradd J. Haley Devendra H. Shah

Lactulose, a synthetic disaccharide widely used in human and veterinary medicine as a therapeutic and prebiotic, is generally assumed to be metabolized exclusively by commensal microbiota. We recently showed that non-typhoidal Salmonella (NTS) can also metabolize lactulose, but the genetic basis and functional consequences remain unclear. Here, we hypothesized that NTS encodes specific genetic determinants and regulatory networks for lactulose uptake, hydrolysis, and downstream metabolism. Using the globally disseminated NTS serotype S. Kentucky, we defined the lactulose-responsive transcriptome. Lactulose exposure induced a distinct gene network including candidate transporters, periplasmic binding proteins, and glycoside hydrolases predicted to cleave lactulose into galactose and fructose, with downstream metabolism via the mannitol cycle and Leloir-associated pathways. Notably, lactulose also triggered broad transcriptional remodeling that repressed genes encoding motility, fimbrial biosynthesis, and biofilm formation, resulting in significantly reduced motility and biofilm across NTS serotypes. These findings provide novel mechanistic insight into lactulose metabolism, suggesting that lactulose metabolism is concurrently associated with reduced expression of persistence-associated phenotypes in NTS under the conditions tested. This raises the possibility that lactulose exposure could influence NTS persistence in both the host-associated and environmental context. Follow-up studies are needed to validate the potential of these findings to reduce food safety and public health risks linked to persistence and transmission phenotypes.

Pathogens doi: 10.3390/pathogens15060591

Authors: Armelia Sari Widyarman Nadeeka S. Udawatte Swiluva Sigalovada Swilly Sumardy Ma Citra Fragrantia Theodorea Mario Richi Wiwiek Poedjiastoeti Chaminda Jayampath Seneviratne

This non-randomized, open-labelled, controlled pilot trial investigated the impact of stunting on oral health and the oral microbiome, and evaluated the effect of 14-day probiotic or essential oil mouthwash interventions in children aged 8–12 years. Thirty-six participants (18 stunted, 18 non-stunted) were randomized into three parallel arms: probiotic lozenges (Limosilactobacillus reuteri DSM 17938 + ATCC PTA 5289), essential oil mouthwash, or water control. D-25OH level was assessed with ELISA, OHI-S, and PBI were examined, and oral microbiome was analyzed using 16S metagenomic sequencing. Stunted children demonstrated significantly higher gingival inflammation (PBI, F = 10.57, p = 0.002), reduced microbial alpha diversity, reductions in commensal Streptococcus spp., and increases in pathobionts, including Parvimonas micra, Fusobacterium nucleatum, and Tannerella forsythia. Beta-diversity analysis revealed distinct microbial communities (p = 0.001), with network analysis identifying these anaerobes as keystone hubs in stunted individuals. Salivary vitamin D and oral hygiene indices (OHI-S) also differed by stunting status. Fourteen-day interventions produced only modest, non-significant improvements in clinical indices and failed to induce significant shifts in microbial diversity or composition. These findings indicate that nutritional stunting is independently associated with oral dysbiosis and gingival inflammation. Short-term antiseptic interventions appear insufficient to reverse established microbial shifts, highlighting the need for sustained, integrated nutritional—oral health strategies.

Pathogens doi: 10.3390/pathogens15060590

Authors: Camille E. Spears Divya Chandramohan Melinda B. Tanabe Nicholas C. Anstead Emily L. M. Turkily Lucas S. Blanton Thelma Akien Christopher L. Dayton James C. Saca Gregory M. Anstead

Typhus (FBT), caused by Rickettsia typhi, rarely causes neurological disease. Herein, we describe neurological involvement in two cases of FBT. In the first case, an adult presented with persistent fever which deteriorated into status epilepticus. He was successfully treated with doxycycline and made a complete recovery. In the second case, a patient suffered an ischemic stroke and had a protracted clinical course but ultimately made a near complete recovery. In addition to these cases, we conducted a comprehensive narrative review of 43 cases of neurologic involvement in FBT reported from 1989 to 2025. Cases were excluded if there were pathologic discrepancies with typical cases of FBT. Presentations ranged from cranial nerve palsies to meningitis and fulminant encephalitis. This review highlights the spectrum of CNS complications associated with FBT and underscores the importance of early recognition and treatment with doxycycline to improve outcomes. Given the potential severity of neurologic involvement, clinicians in endemic areas should maintain a high index of suspicion for FBT in patients presenting with a febrile illness and neurologic symptoms.

Pathogens doi: 10.3390/pathogens15060589

Authors: Jasmin Hawwash Hilke Oltmanns Andrea Vanessa Volk Jessica Meißner

Staphylococcus (S.) pseudintermedius, as a commensal of the skin and mucosa, leads to a variety of diseases in dogs, most commonly skin and ear infections. The development of methicillin-resistant S. pseudintermedius (MRSP) is an emerging risk for animals and humans. The aim of this study was to test cetirizine and N-acetylcysteine as synergistic substances with cephalexin for treating S. pseudintermedius infections. Each of the five methicillin-sensitive S. pseudintermedius (MSSP) isolates and five MRSP isolates, and one control strain were tested. The minimal inhibitory concentration (MIC) of the substances was tested by broth microdilution assay. In a checkerboard assay, the MIC of cefalexin alone was compared to the MIC of the substances combined. The determined dose reduction index (DRI) shows the influence each substance had on the efficacy of cefalexin. Furthermore, the minimal bactericide concentration (MBC) of N-acetylcysteine (NAC) was identified, and a time kill assay was performed to determine its time-related efficacy on selected isolates. Cetirizine showed no inhibition on bacterial growth or influence on antibiotic efficacy. NAC inhibited bacterial growth at 2 mg/mL. A significant synergistic influence was shown against the MRSP (p < 0.001) and MSSP isolates (p < 0.01). The MBC of the MSSP isolates and control strain was 12.8 and 25.6 mg/mL for the MRSP isolates. The time kill assay showed that NAC is bactericidal within 120 s at the prior determined MBC concentrations. NAC showed an antibacterial effect alone and a synergistic influence on cefalexin’s antibacterial properties. Thus, NAC shows promising efficacy in treating infections with S. pseudintermedius; according to the preliminary study conducted here, this effect may be independent of the resistance profile.

Pathogens doi: 10.3390/pathogens15060588

Authors: Ruku Shinohara Yukimi Misawa Shuichi Mizuno Saeka Honda Koki Kikuchi Rei Settsu Yosuke Kato Kaori Okayama Mizue Oda Mitsuaki Okodo

Cannonballs, compact aggregates of neutrophils observed in Papanicolaou (Pap) smears, are frequently associated with Trichomonas vaginalis (T. vaginalis) infection but are generally regarded as nonspecific inflammatory findings. To clarify their morphologic features, we analyzed cervicovaginal liquid-based cytology specimens from 29 cervicitis cases, including six positive for T. vaginalis. Cannonballs were evaluated using immunocytochemistry for T. vaginalis and cytokeratin, cell block analysis, and morphometric analysis, with negative cases as controls. All positive cases contained T. vaginalis-associated cannonballs, with a mean positivity rate of 57.4%. Parasites were intermingled with aggregated neutrophils, and cell block analysis demonstrated parasite-centered neutrophilic aggregates. Cytokeratin staining patterns differed morphologically between positive and negative cases. In addition, T. vaginalis-associated cannonballs were significantly smaller than adjacent squamous epithelial cells. These findings suggest that some cannonballs in trichomoniasis may represent parasite-associated neutrophilic structures and provide insight into host–parasite interactions in cervicovaginal inflammation.

Pathogens doi: 10.3390/pathogens15060587

Authors: Dragana Mijatović Ana Marija Radevska Dejan Jakimovski Lidija Popović-Dragonjić Biljana Popovska Jovičić Jagoda Gavrilović Siniša Sević Dajana Lendak Irina Stojanac Alejandro Cabezas-Cruz Andreas Pilz Tomás Cervantes Rincón Jasmine Oberti-Cantergiani Davide F. Robbiani Pavle Banović

Tick-borne encephalitis (TBE) is an emerging vector-borne disease in Europe, but its epidemiology remains poorly defined in Serbia. In orthoflavivirus-endemic settings, diagnostic challenges may contribute to underrecognition of TBE, particularly among patients with suspected West Nile virus (WNV) infection. We conducted a multicenter retrospective study including patients hospitalized between 2018 and 2023 with suspected WNV neuroinvasive disease or viral encephalitis of unknown etiology. Serum samples were tested for TBEV-neutralizing antibodies using a microneutralization assay. Among 79 patients, TBEV-neutralizing antibodies were detected in four (5.1%). Most reactive cases occurred in patients initially classified as having suspected WNV-associated meningoencephalitis, while TBE had not been considered in the differential diagnosis at admission. These findings suggest that TBE may be underrecognized in Serbia and highlight the importance of confirmatory testing in orthoflavivirus-endemic settings. Strengthening clinical awareness and surveillance will be essential to better define the burden of TBE and inform prevention strategies.

Pathogens doi: 10.3390/pathogens15060586

Authors: Shanta Barua Asep Gunawan Autchara Kayan Masa Tenaya Mehmet Ulas Cinar Made Kardena Syeda Hasina Akter Nurulfiza Mat Isa Henry Annandale Subir Sarker David T. Williams Sam Abraham Jasim M. Uddin

African swine fever virus (ASFV) is an evolving threat to global swine health and food security, driven by its complex epidemiology, multi-host transmission cycles, and ongoing spread across countries. This review summarizes the global scenario and transmission pathways of ASFV, highlighting the outbreaks associated with evolving risk patterns to support effective disease prevention and control. ASF has been reported in pig-producing regions across Africa, Europe, and, more recently, Asia, largely driven by the spread of genotype II strains. The virus is transmitted through direct contact with infected pigs or pig-products, indirectly via contaminated materials, and through soft ticks of the genus Ornithodoros spp., with epidemiological patterns varying according to wildlife reservoirs and regional factors. Control measures mainly rely on early detection, movement control, strict quarantine, robust biosecurity measures, and international trade regulations. Despite significant advances, persistent challenges, including the absence of a widely available commercial vaccine, long-term stability of the virus, human activities, and inconsistencies in global response capacities, continue to hinder disease eradication efforts. This review underscores the need for transnational strategies and policies that integrate economically sustainable disease management systems and reduce the long-term impact of ASFV.

Pathogens doi: 10.3390/pathogens15060585

Authors: Pietro Di Taranto Fiorenza Petruzzi Antonella Cristina Romano Mariateresa Toce Lucia Palazzo Alessandra Alessiani Loredana Capozzi Stefano Castellana Laura Del Sambro Adelia Donatiello Carmine Pedarra Gilda Occhiochiuso Giovanni Castelli Alessandra Barlaam Giovanni Normanno Antonio Parisi

Poultry is the main reservoir of Campylobacter spp. and most human cases result from consuming undercooked poultry or handling raw meat. In 2022, a total of 55 samples, including neck skin, cecal contents, and processing waters, were collected at two poultry slaughterhouses in Italy and analysed according to ISO 10272-2:2017 at the Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata laboratories. Overall, 51/55 (92.72%) samples tested positive for Campylobacter. Among the isolates, 64.71% were identified as C. coli, and 35.29% as C. jejuni. Phenotypic and genotypic analysis were performed to assess antimicrobial resistance and virulence characteristics. All C. jejuni isolates and 72.72% of C. coli showed resistance to fluoroquinolones. Resistances to tetracycline and carbapenem were observed in 60.78% and 45.09% of isolates, respectively. Genomic analysis confirmed the presence of the tet(O) gene, conferring tetracycline resistance. In addition, OXA-450 and OXA-466 genes, conferring beta-lactam resistance, were detected in 78.43% and 3.92% of isolates. Virulence-associated genes were detected. Specifically, the ciaB gene was found in 50/51 (98.04%) of isolates, whereas jlpA, cdtA, cdtB, and ctdC genes were exclusively identified in C. jejuni strains. The high prevalence of pathogenic and antimicrobial-resistant Campylobacter strains highlights the need for strengthened control measures along the poultry production chain.

Pathogens doi: 10.3390/pathogens15060584

Authors: Sveinung Wergeland Sørbye Bente Marie Falang Mona Antonsen Elin Richardsen

Background: Effective triage of HPV DNA-positive women is needed to reduce unnecessary colposcopies while maintaining cervical cancer prevention. We evaluated genotype-specific 7-type HPV E6/E7 mRNA triage in a real-world screening cohort. Methods: In this population-based single-centre study at the University Hospital of North Norway, 42,791 women underwent primary screening with the cobas HPV DNA assay during the period 2019–2024. Among 2370 HPV DNA-positive women, reflex cytology, 7-type HPV mRNA testing, and an ATHENA-derived triage strategy were compared using histologically confirmed CIN3+ through 31 December 2025 as the endpoint. Results: CIN3+ was detected in 60/2370 women (2.5%). Test positivity was 47.0% for cytology, 54.7% for ATHENA-derived triage, and 33.4% for HPV mRNA. Sensitivity was 78.3%, 86.7%, and 73.3%; specificity was 53.8%, 46.1%, and 67.7%; and PPV was 4.2%, 4.0%, and 5.6%, respectively. Colposcopies per CIN3+ detected were 23.7, 24.9, and 18.0. Conclusions: HPV mRNA triage improved referral precision and colposcopy efficiency, but with lower sensitivity than ATHENA-derived triage. These findings support 7-type HPV mRNA testing as a potentially useful molecular triage option where structured follow-up is feasible.

Pathogens doi: 10.3390/pathogens15060583

Authors: Sung Hun Jang Bo Kyeung Jung Jae-Sik Jeon Jeong Su Han Jae Kyung Kim

We aimed to characterize age-stratified C-reactive protein (CRP) patterns across respiratory virus infections, assess age-related CRP shifts in virus-not-detected-by-PCR episodes, and evaluate the independent associations of age and virus type with CRP levels. We retrospectively analyzed 19,002 test-level episodes with paired respiratory PCR and serum CRP results from a single tertiary-care center between 2008 and 2024. Episodes were classified as virus-not-detected-by-PCR or virus-positive according to multiplex PCR results; the former was not considered a healthy control and may include off-panel infections, bacterial/mixed infections, false-negative results, or non-infectious inflammation. Descriptive analyses and multivariable linear regression of log-transformed CRP were used to assess adjusted associations. Median CRP increased with age in both virus-positive and virus-not-detected-by-PCR episodes, rising from 0.38 to 7.42 mg/dL across age groups in the latter. Age showed the strongest association with CRP. Adenovirus showed a positive adjusted association, whereas influenza A/B, respiratory syncytial virus A/B, and parainfluenza virus types 1 and 3 showed selective negative associations. Overall, CRP variation was more strongly associated with age-related clinical background than with virus type, although selective virus-associated differences were observed, supporting the interpretation of CRP as a non-specific, composite host-response indicator within broader clinical contexts.

Pathogens doi: 10.3390/pathogens15060582

Authors: Chiara Antonelli Najwa Benfradj Anna Maria Vettraino

Phytophthora species are among the most destructive plant pathogens worldwide, causing severe losses in agricultural, forest, and natural ecosystems. In recent years, the management of Phytophthora diseases has increasingly shifted toward eco-sustainable strategies, with growing interest in plant-derived extracts, particularly essential oils, as low-risk alternatives to synthetic fungicides. In this study, a structured review was combined with new in vitro assays to assess the antifungal activity of essential oils from Thymus vulgaris (TV-EO) and T. serpyllum (TS-EO) against P. cinnamomi, P. drechsleri, P. cactorum, P. citrophthora, P. nicotianae, P. palmivora, and P. infestans. Literature searches were conducted in April 2025 using the Web of Science and Scopus databases, following PRISMA guidelines, with the search term “Thymus” or “Thyme” and “Phytophthora”. Twenty studies included in the review demonstrated that the activity of Thymus essential oils against Phytophthora species was highly variable and shaped by chemotype, Thymus species, pathogen, and experimental setup. Additional in vitro assays further confirmed a clear dose-dependent inhibitory effect for both TV-EO and TS-EO. TS-EO consistently exhibited stronger activity than TV-EO, likely reflecting its carvacrol-rich chemotype, while thymol-based TV-EO showed lower but still significant inhibition depending on the pathogen species. Overall, these results highlight the potential of Thymus essential oils as eco-friendly tools for the management of Phytophthora diseases. However, the strong dependence on chemotype, pathogen species, and assay conditions underscores the need for standardized testing, detailed chemical characterization, and in vivo validation.

Pathogens doi: 10.3390/pathogens15060581

Authors: Annabella Salvati Loredana Alessio Gloria Trombaccia Giovanni Cimmino Marisa De Feo Fausto Ferraro Stefania De Pascalis Nicola Coppola

A 61-year-old woman developed prosthetic valve infective endocarditis after osteomyelitis caused by extensively drug-resistant (XDR) Acinetobacter baumannii. Moreover, a systematic descriptive review of published case reports was performed to describe the main features, treatment, and outcomes of this condition. Methods: Apart from the description of our case, a PubMed and Embase literature review was conducted up to January 2026 using the terms “A. baumannii” AND (“infective endocarditis” OR “endocarditis” OR “valvular infection”). We included clinical cases of IE caused by A. baumannii published as full-text articles in English. Results: After orthopedic osteosynthesis surgery following a femur fracture, our patient developed osteomyelitis by XDR A. baumannii and was treated for a short period of time. Later, prosthetic aortic valve endocarditis was diagnosed. Despite treatment with cefiderocol and eravacycline, she died. An additional 18 clinical cases of IE by A. baumannii were identified from the literature, bringing the total to 19 cases. IE affected prosthetic valves in nine cases, native valves in nine and involved a right atrial mass in one. Twelve cases were caused by MDR or XDR A. baumannii. Mortality occurred more frequently in cases not treated with surgery (9/13, 69%) compared to those treated with surgery (1/6, 16.7%). However, given the very small sample size, these data should be interpreted with caution. Conclusion: This case, together with previously reported observations, highlights the severity of EI by A. baumannii and the need of multidisciplinary management.

Pathogens doi: 10.3390/pathogens15060580

Authors: Wenjing Zhang Yiming Guo Lijun Guan Lifang Si Zhanqin Zhao

Pasteurella multocida serotype A (P. multocida) is frequently associated with severe respiratory disease in swine (Sus scrofa), highlighting the need for effective preventive strategies. To identify protective antigens suitable for a subunit vaccine targeting porcine P. multocida infection, six recombinant proteins (rAspA, rLolA, rOmpP6, rOppA, rRps6, rSmpA) were expressed in a prokaryotic system, and their efficacy was evaluated in a Mus musculus (Kunming) mouse model. All proteins were purified using His-tag affinity chromatography, and SDS-PAGE analysis confirmed expression with bands at the expected molecular weights (61, 26, 21, 63, 19, and 17 kDa). Each protein, formulated with ISA 201 adjuvant, was administered to mice in two immunizations. Indirect ELISA of sera collected at multiple time points demonstrated that all vaccines induced high antigen-specific IgG levels. rOppA, rLolA, rOmpP6, and rRps6 were expressed in soluble form, whereas rAspA and rSmpA formed inclusion bodies. In a lethal challenge model, rLolA and rRps6 conferred the highest protection (60% each), followed by rAspA and rOmpP6 (30%), rOppA (20%), and rSmpA (10%). Under the conditions tested, the highest protection observed was 60%, and none of the six antigens achieved complete protection against homologous A7 challenge in mice. This first head-to-head comparison under identical conditions provides a reference framework for future antigen screening studies.

Pathogens doi: 10.3390/pathogens15060579

Authors: Floriana Bonura Arcangelo Pepe Dario Genovese Emanuele Amodio Chiara Filizzolo Fabio Campisi Mariangela Pizzo Emilia Palazzotto Simona De Grazia Giovanni M. Giammanco

Acute gastroenteritis (AGE) remains a leading cause of pediatric mortality and morbidity, with rotavirus as the leading cause of severe disease. Post-vaccine surveillance is essential to monitor circulating pathogens and assess vaccination impact. Sicily was the first Italian region to implement universal rotavirus vaccination in 2012. We retrospectively studied 693 children hospitalized for suspected viral AGE at the Children’s Hospital of Palermo (March 2017–February 2020), testing stool samples for viral and bacterial enteric pathogens. Rotavirus remained the most common agent (13.3%), followed by norovirus (12.1%), adenovirus (11.3%), Salmonella spp. (4.6%) and astrovirus (3.2%). The study population was categorized as rotavirus-associated AGE (RV-AGE) or other-cause AGE (O-AGE). Epidemiological, clinical and virological features were compared with the pre-vaccine period (2011–2012). At least one pathogen was detected in 47.5% of samples. RV-AGE cases were older than those with O-AGE (median 32.6 vs. 30.5 months; p < 0.01) and had greater clinical severity, with higher frequency of vomiting, fever and dehydration. Rotavirus infection was significantly associated with unvaccinated status. Compared with the pre-vaccine era, rotavirus prevalence declined (32.6% vs. 13.3%), seasonal patterns were attenuated and genotype distribution shifted toward G2P[4], G9P[8] and equine-like G3P[8] strains. Despite the decline in RV-AGE following vaccine introduction, rotavirus remains a relevant cause of pediatric AGE, underscoring the need for high vaccination coverage and continued surveillance.

Pathogens doi: 10.3390/pathogens15060578

Authors: Chenyu Zhan Mingyu Zhang Guijuan Hao Yue Zhang Fangkun Wang

Macrococcus is a genus of Gram-positive cocci in the Staphylococcaceae family and a close phylogenetic relative of Staphylococcus. It is not a significant human pathogen but is known to widely colonize different environments, including animal skin and food products. Phylogenetically, Macrococcus is distinct from yet closely related to Staphylococcus, particularly the sciuri group. The species is effectively identified through such molecular markers as hsp60 and 16S rDNA. A key biochemical feature is an identified FAD-dependent oleate hydratase in Macrococcus equipercicus (M. equipercicus). Critically, Macrococcus carries various mobile antibiotic-resistance genes, especially against β-lactams (e.g., mecB, mecD) and macrolides (e.g., mef(F), msr(G)); these genes are located on plasmids, SCCmec-like elements, or resistance islands (e.g., McRImecD), which facilitates their horizontal transfer. Surveillance confirms the widespread presence of methicillin-resistant Macrococcus, often with a multidrug-resistant phenotype, in food animals and their products. Although its own pathogenicity is low, Macrococcus acts as a reservoir and transmission platform for resistance genes: through horizontal gene transfer, it can potentially confer resistance to pathogenic Staphylococcus, thereby posing a threat to animal and public health. This review summarizes the basic biological characteristics and drug resistance-related research progress of the genus Macrococcus; it aims to provide a reference for subsequent studies as well as to establish technical support and a theoretical basis for the epidemiological investigation, drug-resistant strain identification, and clinical drug-resistance risk prevention and control of Macrococcus.

Pathogens doi: 10.3390/pathogens15060577

Authors: Kaja Hussain Jasmiminal Elango Ayyanar Chakravarthi Madda Vignesh Kumar Ramakrishnan Hari Kishan Raju Konuganti Manju Rahi

Ticks are major ectoparasites of livestock in India, causing substantial economic losses and transmitting a wide range of pathogens. Control strategies rely heavily on synthetic pyrethroid acaricides; however, resistance associated with mutations in the voltage-gated sodium channel (VGSC) gene has been widely reported. Information on tick species diversity and molecular resistance status in Puducherry remains limited. This study investigated tick diversity, host-associated infestation patterns, and the presence of key VGSC knockdown resistance (kdr) mutations (C190A and G215T). A cross-sectional survey was conducted across 15 villages in Puducherry, where ticks were collected from cattle, goats, and dogs and identified using standard morpho-taxonomic keys. A total of 3779 ticks representing eight ixodid species were identified, showing clear host-associated infestation patterns. Individual ticks were selected from stratified pools (based on village, host, tick species, and sex) and processed individually for genomic DNA extraction. A fragment of the VGSC gene was amplified by PCR and analysed using Sanger sequencing. Molecular analysis of 62 high-quality VGSC sequences representing all eight species across the surveyed villages revealed no mutations at the investigated loci (C190A and G215T) in the analysed samples. All sequences exhibited the wild-type genotype at these positions. These findings provide baseline molecular information on the screened VGSC loci in ixodid ticks from Puducherry and highlight the importance of continued surveillance, phenotypic validation, and integrated tick management strategies.

Pathogens doi: 10.3390/pathogens15060574

Authors: Sylvie Bambara Marie Claire Isingizwe Taofeek Tope Adegboyega Leon Mutesa

In Africa, the disease burden of diarrheal and respiratory diseases is amplified by limited surveillance capacity, diagnostic limitations, and socioeconomic inequalities. In rapidly urbanizing settings such as Kigali (Rwanda), integrating wastewater-based epidemiology (WBE) into existing surveillance systems offers a promising strategy for generating real-time epidemiological intelligence, identifying community-level hotspots, and addressing gaps in traditional reporting systems. Gastrointestinal and respiratory infections remain major causes of morbidity and mortality globally, particularly in low- and middle-income countries (LMICs), where traditional clinical surveillance systems frequently underreport the true disease burden. This systematic review synthesizes current evidence on the detection of gastrointestinal and respiratory pathogens in wastewater and evaluates the utility of WBE for early warning and public health action. A narrative review approach was used to identify peer-reviewed literature, global health reports, and surveillance studies focusing on the wastewater detection of gastrointestinal and respiratory pathogens. Databases including PubMed, Scopus, and Google Scholar were searched for studies published between 2000 and 2026. The search yielded 1247 records, of which 312 duplicates were removed. After title/abstract screening, 228 full-text articles were retrieved and assessed for eligibility. After a detailed evaluation, 108 studies were excluded for the following reasons: absence of pathogen-specific wastewater data (n = 46), a focus on environmental monitoring without public health relevance (n = 25), insufficient methodological description (n = 21), or other eligibility limitations such as a lack of primary data (n = 16). WBE provides a non-invasive, cost-effective approach for monitoring symptomatic and asymptomatic infections. Challenges involve variability in sampling, environmental factors affecting viral decay, and differences in laboratory workflows. WBE is a powerful complement to traditional infectious disease surveillance, offering early warning capabilities, population-level coverage, and real-time insights into pathogen circulation. Integrating WBE into surveillance programs, especially in LMICs such as Rwanda, can significantly strengthen epidemic preparedness, guide resource allocation, and improve outbreak response. Sustained investment in laboratory capacity, standardized protocols, and multisector collaboration is essential to fully leverage WBE for public health protection.

Pathogens doi: 10.3390/pathogens15060576

Authors: Eun-Seop Lee Chae-Min Kim Si-Heon Song Su-Bin Jeon Byeong-Su Kang Md Sekendar Ali Eon-Bee Lee

Marine natural products, including seaweeds, sponges, and marine microorganisms, have emerged as promising sources of bioactive compounds with diverse pharmacological properties. We investigated the antibacterial and antioxidant potential of ethanol extracts (30%, 60%, and 90%) from Saccharina japonica collected from two Korean coastal regions, Gijang and Wando, and evaluated their bioactive metabolites through integrated in vitro and in silico approaches. Among the extracts, the 60% ethanol fraction exhibited the highest total phenolic content and strongest 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging activity, indicating superior antioxidant capacity. Antibacterial assays revealed selective concentration-dependent inhibition against Staphylococcus aureus, while limited effects were observed against Escherichia coli. Kinetic analysis further demonstrated growth suppression of S. aureus at higher concentrations. Molecular docking was performed against multiple bacterial proteins, including DNA gyrase, topoisomerase IV, and tyrosyl-tRNA synthetase. Sterol compounds, particularly fucosterol and 24-methylene cholesterol, showed strong binding affinities across key targets, suggesting their potential role as multitarget antibacterial agents. ADMET predictions indicated favorable pharmacokinetic properties, although high lipophilicity and potential hERG II inhibition were noted. Overall, while the antibacterial effects observed were moderate, these findings suggest that marine-derived sterols from S. japonica may represent compounds of interest for further mechanistic investigation and optimization in complementary antibacterial strategies.

Pathogens doi: 10.3390/pathogens15060575

Authors: Jade Jansen Shirley Man Fenna Kootstra Ad C. van Nuenen Karel A. van Dort Claudio Zamperini Conraed Willem Houck Neeltje A. Kootstra Teunis B. H. Geijtenbeek

Antiretroviral therapy (ART) effectively suppresses HIV-1 replication but does not purge the latent HIV-1 reservoir. Strategies aimed at HIV-1 latency reversal and subsequent elimination of infected cells are being explored. Targeting the inhibitor of apoptosis proteins (IAP) and DEAD-box polypeptide 3 (DDX3) RNA helicase reduces the HIV-1 reservoir ex vivo. However, the mechanisms driving apoptosis of HIV-1 infected cells remain unclear. Here, we uncovered the mechanism regarding HIV-1 transcriptional activation and induction of apoptosis specific for HIV-1 infected cells using an acute in vitro infection model. Inhibition of IAP by second mitochondrial-derived activator of caspases mimetic (SMACm; AZD5582) resulted in activation of non-canonical NF-κB pathway (RelB/p52) that induced HIV-1 transcription, confirming previous reports, whereas inhibition of DDX3 sensitized HIV-1 infected cells for apoptosis (DDX3i; FH1321). Transcriptome analysis revealed that HIV-1 actively suppressed apoptosis-related genes in HIV-1 infected cells. SMACm treatment resulted in a broad induction of these genes irrespective of infection. Notably, DDX3 inhibition specifically restored the expression of the majority of HIV-1 suppressed genes, and when combined with SMACm, restored almost all HIV-1 downregulated genes, thereby rendering HIV-1 infected cells sensitive to apoptosis. Thus, our data strongly suggest that inhibition of host factors IAP and DDX3 not only induces activation of HIV-1 transcription but also restores HIV-1 suppressed apoptotic processes in infected cells.