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Pathogens
Volume 15
Issue 2
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Pathogens, Volume 15, Issue 2 (February 2026) – 115 articles

Cover Story (view full-size image): Urban forests provide important ecosystem services, but climate change is compromising their functionality by causing various stresses that lead to tree suffering and mortality. A three-year study at Parco Nord Milano (PNM) in Italy analysed the impact of thinning on the occurrence of fungal pathogens in declining forest stands. Various tree species showed clear signs of decline and a higher incidence of pathogens (Botryosphaeriaceae) as a result of thinning. Thinning therefore altered the microclimate and the balance of phytopathogenic microbes, increasing the vulnerability of the trees. These results call into question the benefits of this management practice in conditions of environmental stress. View this paper
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17 pages, 739 KB  
Article
Characterization of Fungi Communities in Organic Soybean Seeds Using DNA Sequencing: Effects of Cultivar and Location
by Hanna Olszak-Przybyś, Marcin Przybyś, Jolanta Bojarszczuk and Jerzy Księżak
Pathogens 2026, 15(2), 239; https://doi.org/10.3390/pathogens15020239 - 23 Feb 2026
Viewed by 478
Abstract
Organic soybean seeds are susceptible to colonization by numerous fungal pathogens, which can reduce their germination capacity and nutritional quality. This study evaluated fungi transmitted by seeds and their effects on selected seed quality parameters, as well as the influence of variety, location [...] Read more.
Organic soybean seeds are susceptible to colonization by numerous fungal pathogens, which can reduce their germination capacity and nutritional quality. This study evaluated fungi transmitted by seeds and their effects on selected seed quality parameters, as well as the influence of variety, location and growing season. In total, 471 fungal isolates belonging to 24 genera and 37 species were obtained from three soybean varieties (Erica, Es Commandor and Cerez PZO) cultivated at two locations during the 2022–2023 seasons. All obtained isolates were identified based on ITS sequencing, and Fusarium isolates were further characterized to the species level using TEF and RPB2 markers. Fusarium spp. was the most frequently isolated genus, accounting for 35.7% of all isolates, followed by Alternaria spp. (15.9%) and Aspergillus spp. (11.9%). Fungal frequency and species diversity differed significantly between years. Seed germination capacity was significantly lower in 2023 than in 2022 and coincided with higher fungal colonization, lower spring temperatures, and increased rainfall. A significant negative Pearson’s correlation (r = −0.58, p < 0.05) was found between fungi abundance and seed oil content, indicating a direct impact of fungal colonization on nutritional quality. These results highlight the role of environmental conditions in seed-borne pathogen communities and the need for monitoring and seed health management to ensure soybean seed quality. Full article
(This article belongs to the Section Fungal Pathogens)
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16 pages, 1768 KB  
Article
Prevalence, Antimicrobial Resistance Profiles, and Molecular Characteristics of Methicillin-Resistant Staphylococcus aureus Among School Children in Nha Trang, Central Vietnam
by Stephen Anyona Omae, Shah Mohammad Monir, Hien-Anh Thi Nguyen, Kim-Mai Huynh, Natsuki Ariyoshi, Lien Thuy Le, Dat Thanh Le, Trieu Bao Nguyen, Hoang Huy Le, Luong Dinh Nguyen, Miyuki Tsuruoka, Hirono Otomaru, Erik Koehne, Michiko Toizumi, Duc-Anh Dang, Hung Thai Do and Lay-Myint Yoshida
Pathogens 2026, 15(2), 238; https://doi.org/10.3390/pathogens15020238 - 22 Feb 2026
Viewed by 737
Abstract
Staphylococcus aureus (S. aureus), especially Methicillin-resistant Staphylococcus aureus (MRSA), remains a major public health concern both in hospital and community settings. The MRSA carriage situation among schoolchildren in Vietnam is limited. A cross-sectional study was conducted in March 2023 to assess [...] Read more.
Staphylococcus aureus (S. aureus), especially Methicillin-resistant Staphylococcus aureus (MRSA), remains a major public health concern both in hospital and community settings. The MRSA carriage situation among schoolchildren in Vietnam is limited. A cross-sectional study was conducted in March 2023 to assess the prevalence, antimicrobial resistance patterns, and molecular characteristics of MRSA among schoolchildren in Nha Trang, Vietnam. Schoolchildren from grades 1 to 12 (ages 6–18 yrs) were enrolled. Background epidemiological data and nasal swabs were collected. Nasal samples were processed at the Pasteur Institute in Nha Trang, Vietnam, for S. aureus culture. Out of 1210 participants enrolled, S. aureus prevalence was 18.3% (222/1210), of which 41% (91/222) were MRSA. Primary school children showed the highest prevalence of MRSA colonization (48%), 32.8% in secondary, and 27.8% in high school. Among MRSA isolates, high levels of resistance were detected against trimethoprim-sulfamethoxazole (100%), erythromycin (68.2%) and clindamycin (45.1%). Multidrug resistance (MDR) occurred in 30% (27/90) of MRSA isolates. Staphylococcal cassette chromosome mec (SCCmec) subtype IVa was dominant (66.0%), followed by type IV (7.0%) and type V (6.0%). MLST data revealed genetic diversity whereby ST45 dominated, followed by ST546 and ST188. Continuous MRSA surveillance is essential to monitor emerging strains in the communities. Full article
(This article belongs to the Section Bacterial Pathogens)
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16 pages, 1195 KB  
Article
Genomic and Clinical Profile of Hypervirulent Klebsiella pneumoniae (ST23-K1) Liver Abscesses: Experience with Patients at a Romanian University Hospital
by Dragoș Ștefan Lazăr, Elena Nedu, Adina-Alexandra Nanu, Maria-Irina Fediuc, Maria Alexandra Malciolu-Nica, Maria Nica, Mihaela Oprea, Oana-Claudia Albu, Laura-Ioana Popa, Daniela Andreea Blidaru and Simin Aysel Florescu
Pathogens 2026, 15(2), 237; https://doi.org/10.3390/pathogens15020237 - 20 Feb 2026
Viewed by 2533
Abstract
Introduction: Hypervirulent Klebsiella pneumoniae (hvKp) is an emerging global pathogen that causes severe liver abscesses and metastatic infections. Despite rising concerns regarding multidrug-resistant convergence, molecular data in Romania remain limited. This study characterizes the epidemiological, clinical, and genomic profiles of hvKp liver [...] Read more.
Introduction: Hypervirulent Klebsiella pneumoniae (hvKp) is an emerging global pathogen that causes severe liver abscesses and metastatic infections. Despite rising concerns regarding multidrug-resistant convergence, molecular data in Romania remain limited. This study characterizes the epidemiological, clinical, and genomic profiles of hvKp liver abscesses in a tertiary hospital, aiming to describe the local virulence landscape and inform clinical management. Results: This study characterizes 15 cases of hvKp liver abscesses in a tertiary hospital. The cohort was predominantly male, with high rates of type 2 diabetes. Although clinical presentation was severe, featuring complications like endogenous endophthalmitis, the outcomes following prolonged antibiotic therapy were generally favorable. Phenotypically, 93.3% of isolates retained a wild-type susceptible profile, while a single ESBL-positive case highlighted the risk of resistance convergence. Genomic sequencing confirmed the presence of the ST23-K1 pandemic lineage carrying key virulence determinants (rmpA, iuc, and peg-344). Crucially, cgMLST analysis revealed genetic heterogeneity, suggesting sporadic community acquisition rather than a clonal nosocomial outbreak. Conclusions: These findings represent the first detailed molecular description of hvKp in Romania, confirming the local circulation of high-risk ST23 clones and underscoring the necessity for early detection and surveillance. Full article
(This article belongs to the Section Emerging Pathogens)
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13 pages, 272 KB  
Review
Ehrlichia canis: Is It a Pathogen for Humans and Other Primates?
by Valentina Virginia Ebani
Pathogens 2026, 15(2), 236; https://doi.org/10.3390/pathogens15020236 - 20 Feb 2026
Viewed by 777
Abstract
Ehrlichioses and anaplasmosis are among the most commonly reported tick-borne diseases in humans and some animal species. Ehrlichia canis is the causative agent of canine monocytic ehrlichiosis; it primarily affects dogs and is usually transmitted by Rhipicephalus sanguineus ticks. Some reports suggest that [...] Read more.
Ehrlichioses and anaplasmosis are among the most commonly reported tick-borne diseases in humans and some animal species. Ehrlichia canis is the causative agent of canine monocytic ehrlichiosis; it primarily affects dogs and is usually transmitted by Rhipicephalus sanguineus ticks. Some reports suggest that this bacterium is a zoonotic pathogen capable of causing clinical symptoms consistent with human monocytotropic ehrlichiosis caused by Ehrlichia chaffeensis. Non-human primates seem to be susceptible to E. canis-infection, although it is not clear whether the bacterium can cause disease in these mammals. The number of cases of E. canis infections in human beings and other primates could be underestimated, mainly because inappropriate laboratory diagnoses are often carried out. Serological tests do not distinguish infection by E. canis from those due to other ehrlichial species; therefore, a correct diagnosis is possible only through molecular methods. Furthermore, E. canis is not usually recognized by veterinarians and clinicians as a possible pathogen of primates, and it is not included in the panel of tick-borne pathogens routinely investigated. Further studies are pivotal to verify the pathogenicity of E. canis in primates, and epidemiological investigations are needed to monitor its spread in animal and tick species not usually associated with this agent. Full article
(This article belongs to the Section Bacterial Pathogens)
18 pages, 1296 KB  
Article
Antifungal Susceptibility of Clinical Meyerozyma guillermondii Isolates Obtained Between 1994 and 2014: Original Research and Comparison with Published Data
by Aleksandra Górzyńska, Daria Konarska, Agnieszka Korzeniowska-Kowal, Anna Wzorek, Bartosz Pencakowski and Urszula Nawrot
Pathogens 2026, 15(2), 235; https://doi.org/10.3390/pathogens15020235 - 20 Feb 2026
Viewed by 780
Abstract
(1) Background: Meyerozyma guilliermondii is a yeast species widely distributed in the natural environment and one of the rare emerging pathogens capable of causing difficult to treat, severe infections. The species’ susceptibility profile is not fully defined; however, the species could be more [...] Read more.
(1) Background: Meyerozyma guilliermondii is a yeast species widely distributed in the natural environment and one of the rare emerging pathogens capable of causing difficult to treat, severe infections. The species’ susceptibility profile is not fully defined; however, the species could be more prone to develop resistance than other Candida species. The objective of this research was to determine the susceptibility of a local collection of Meyerozyma guilliermondii clinical isolates to classical antifungal drugs as well as a new one—manogepix. (2) Methods: The study included 20 clinical isolates identified using the MALDI–TOF method followed with sequencing of ITS1-2 region of DNA. Overall, the susceptibility to 12 antifungal drugs was tested. Nine drugs (amphotericin B, flucytosine, fluconazole, itraconazole, posaconazole, voriconazole, anidulafungin, caspofungin, and micafungin) were assessed using the MICRONAUT–AT test. The susceptibility to the new drug, manogepix, as well as isavuconazole, clotrimazole and anidulafungin, was determined using the microdilution method recommended by EUCAST. Additionally, anidulafungin and voriconazole MIC was also examined with commercial gradient tests. (3) Results: Overall, the isolates showed low MIC values for amphotericin B (0.125 to 1 mg/L) and for flucytosine (≤0.06 to 32 mg/L), with the exception of one isolate with a high MIC value. The MIC ranges for azoles were 2–64 mg/L (fluconazole), 0.008–0.5 mg/L (voriconazole), ≤0.03–≥4 mg/L (itraconazole) and 0.008–0.5 mg/L (posaconazole). One isolate showed non-WT phenotype to all tested azoles. For anidulafungin, the MIC values ranged from ≤0.06 to 0.25 mg/L; however, in the reference method, higher values were observed, but they did not exceed 2 mg/L (ECOFF value). For manogepix, the MIC values ranged from 0.002 to 0.125 mg/L. Finally, the comparison of the obtained and published susceptibility data was conducted. (4) Conclusions: The data obtained in this study are consistent with reports by other authors and indicate that resistance to azoles or 5-fluorocytosine among clinical isolates of Meyerozyma guilliermondii should be considered. The low MIC values of manogepix suggest the potentially good efficacy of this drug against Meyerozyma guilliermondii species. Full article
(This article belongs to the Section Fungal Pathogens)
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16 pages, 804 KB  
Article
The Clinical Utility of Serum Biomarkers in the Differentiation and Prognostic Assessment of Viral Meningitis
by Georgiana Enache-Leonte, Andrei Vâță, Maria Ioana Onofrei, Mihnea Eudoxiu Hurmuzache, Gabriela Rusu Zota, Dan Trofin, Ioana Alina Harja-Alexa and Mihaela Cătălina Luca
Pathogens 2026, 15(2), 234; https://doi.org/10.3390/pathogens15020234 - 20 Feb 2026
Viewed by 656
Abstract
Background and Objectives: Viral meningitis presents significant diagnostic challenges. The aim of this study was to identify predictive factors for meningitis etiology and clinical outcomes. Methods: This prospective, single center, comparative study enrolled patients meeting clinical, biological, and microbiological criteria for [...] Read more.
Background and Objectives: Viral meningitis presents significant diagnostic challenges. The aim of this study was to identify predictive factors for meningitis etiology and clinical outcomes. Methods: This prospective, single center, comparative study enrolled patients meeting clinical, biological, and microbiological criteria for bacterial (BM) or viral (VM) meningitis. Serum inflammatory markers, namely the neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), and systemic immune-inflammation index (SII), were quantified. In addition, the concentrations of oxidative stress biomarkers, glutathione peroxidase 3 (GPx3) and copper/zinc superoxide dismutase (Cu/Zn SOD), were also determined. Spearman correlation and logistic regression were used to evaluate associations between biomarkers and etiology, while receiver operating characteristic (ROC) curves assessed outcome correlations. Results: VM patients showed a significantly lower NLR (p = 0.007), PLR (p = 0.010), and SII (p = 0.016), with higher GPx3 (p < 0.0001) levels compared with BM patients. Cu/Zn SOD showed no significant difference (p = 0.442) between groups. Multivariate logistic regression identified the SII (OR = 1.015; 95% CI = 1.004–1.026) and GPx3 (OR = 0.847; 95% CI = 0.740–0.970) as etiology predictors. The SII was the strongest predictor of mortality in VM (AUC = 0.833). Conclusions: Serum markers of inflammation, including the systemic immune–inflammation index, along with antioxidant indicators such as GPx3, may serve as valuable additional tools for predicting meningitis etiology and forecasting patient outcomes. Full article
(This article belongs to the Special Issue From Acute Infection to Chronic Sequelae: A Research Trajectory in Viral Infections Disease Management)
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17 pages, 2824 KB  
Article
Persistent and Circulating Plasmodium falciparum dhfr and dhps Mutations in Busia County, Western Kenya
by Loise Ndung’u, Kelvin Thiong’o, Lewis Karani, Stephen Gitahi, Francis Kimani, Mathew Piero Ngugi and Daniel Kiboi
Pathogens 2026, 15(2), 233; https://doi.org/10.3390/pathogens15020233 - 20 Feb 2026
Viewed by 632
Abstract
Malaria in pregnancy remains a major driver of poor maternal and neonatal health outcomes in sub-Saharan Africa. For decades, intermittent preventive treatment in pregnancy (IPTp), with sulphadoxine-pyrimethamine (SP), has mitigated malaria-associated health risks, but concerns have been raised regarding accumulated Plasmodium falciparum dihydrofolate [...] Read more.
Malaria in pregnancy remains a major driver of poor maternal and neonatal health outcomes in sub-Saharan Africa. For decades, intermittent preventive treatment in pregnancy (IPTp), with sulphadoxine-pyrimethamine (SP), has mitigated malaria-associated health risks, but concerns have been raised regarding accumulated Plasmodium falciparum dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) mutations on the efficacy of SP. Western Kenya, including Busia County, is a high malaria transmission setting where molecular surveillance of dhfr and dhps mutations remains limited. This study assessed the prevalence and haplotype structure of dhfr and dhps mutations in P. falciparum isolates from Busia County, Kenya. A total of 66 samples of P. falciparum isolates collected from patients attending Matayos Sub-County Hospital between November 2024 and January 2025 were analysed. PCR amplification and Sanger sequencing targeted dhfr codons C50R, N51I, C59R, S108N/T, I164L, and dhps codons I431V, S436A/F, A437G, K540E, A581G, and A613S/T to determine mutation frequencies, haplotypes, and combined dhps and dhfr haplotype profiles. High frequencies of dhfr and dhps mutations were observed across the parasite isolates. The most common dhfr substitutions included N51I (85.2%) and C59R (75.4%), while S108N (32.8%) and S108T (19.7%) were detected at lower frequencies. Dhfr haplotypes identified included N51I + C59R, N51I + C59R + S108N, and a N51I + C59R + S108T + I164L variant. The I164L mutation was detected at a frequency of 18.0% and was observed exclusively on a non-canonical S108T background (19.7%). Dhps haplotypes were dominated by A437G (92.3%), K540E (40%) alone, and the A437G + K540E double mutant. Combined dhfr and dhps haplotype analyses revealed circulation of classical dhfr triple-mutant (N51I + C59R + S108N) backgrounds with dhps A437G. Quintuple haplotypes (dhfr N51I + C59R + S108T + I164L with dhps A437G) and rare complex haplotypes incorporating both I164L and K540E or I164L and S436F were also detected. These findings indicate the persistence and circulation of both canonical and non-canonical dhfr and dhps haplotypes in P. falciparum isolates from Busia County. This study highlights the need for continuous molecular and phenotypic surveillance to clarify the functional and epidemiological significance of parasites carrying S108T and I164L mutations, and to inform IPT policy. Full article
(This article belongs to the Special Issue Parasitic Diseases in the Contemporary World)
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30 pages, 2409 KB  
Review
Protease Inhibitors and Innate Immune Agonists as Antiviral Strategies Against Dengue and Zika Viruses
by Marianna Costa, Paola Trischitta, Federica Mastrolembo Barnà, Maria Teresa Sciortino and Rosamaria Pennisi
Pathogens 2026, 15(2), 232; https://doi.org/10.3390/pathogens15020232 - 19 Feb 2026
Viewed by 1246
Abstract
Emerging mosquito-borne flaviviruses, such as Dengue virus (DENV) and Zika virus (ZIKV), pose major global public health threats due to their geographic expansion, climate change, and the absence of effective antiviral therapies. Antiviral development against these pathogens has primarily focused on two complementary [...] Read more.
Emerging mosquito-borne flaviviruses, such as Dengue virus (DENV) and Zika virus (ZIKV), pose major global public health threats due to their geographic expansion, climate change, and the absence of effective antiviral therapies. Antiviral development against these pathogens has primarily focused on two complementary strategies. On the one hand, the blocking of viral replication by directly inhibiting essential viral enzymes, and on the other, enhancing the host’s innate immune defenses via targeted activation of intracellular antiviral pathways. Among the viral proteins required for replication, the NS2B–NS3 protease complex is one of the most conserved and druggable targets, prompting extensive efforts to design both covalent and non-covalent inhibitors. Covalent inhibitors, such as boronic acids, aldehydes, trifluoromethyl ketones, phenoxymethylphenyl derivatives, and α-ketoamides, form irreversible or slowly reversible bonds with the catalytic serine residue (Ser 135), producing long-lasting and high-affinity suppression of protease activity. In parallel, several classes of non-covalent, particularly allosteric, inhibitors have emerged as promising alternatives with improved specificity and reduced off-target reactivity. A complementary antiviral strategy involves the use of agonists of key innate immune sensors such as TLRs, RIG-I, and the cGAS–STING axis, which mediate the release of interferons (IFNs). This review brings together current knowledge on these two mechanistically distinct yet convergent approaches, highlighting how both can ultimately restrict flavivirus replication. Future opportunities involving modified peptide scaffolds, advanced delivery systems, and drug-repurposing strategies are finally discussed for the development of next-generation therapeutics against DENV and ZIKV. Full article
(This article belongs to the Special Issue Covalent Inhibitors of Pathogenic Bacterial, Protozoal, Viral, and Fungal Targets)
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15 pages, 487 KB  
Article
Declined Circulation and Seasonal Shifts of Human Coronavirus 229E in the Republic of Korea: Implications for Respiratory Virus Surveillance
by Mi-Ru Oh, Jeong Su Han, Sung Hun Jang, Ga-Yeon Kim and Jae Kyung Kim
Pathogens 2026, 15(2), 231; https://doi.org/10.3390/pathogens15020231 - 19 Feb 2026
Viewed by 520
Abstract
Human coronavirus 229E (HCoV-229E) is an alphacoronavirus that typically causes mild upper respiratory infections but remains understudied in terms of its long-term immuno-ecological behavior. Although the COVID-19 pandemic markedly altered human behavior and viral transmission, extended circulation patterns of HCoV-229E remain poorly defined. [...] Read more.
Human coronavirus 229E (HCoV-229E) is an alphacoronavirus that typically causes mild upper respiratory infections but remains understudied in terms of its long-term immuno-ecological behavior. Although the COVID-19 pandemic markedly altered human behavior and viral transmission, extended circulation patterns of HCoV-229E remain poorly defined. We analyzed annual, seasonal, and age-specific trends using real-time PCR–based respiratory virus surveillance data from Dankook University Hospital collected between 2007 and 2024. Among 23,284 nasopharyngeal swab specimens, 344 were positive for HCoV-229E (overall positivity, 1.43%). Positivity declined significantly over time (OR per year, 0.916; 95% CI, 0.894–0.939; p < 0.001). Compared with spring (1.04%), positivity was highest in winter (2.69%) and lowest in summer (0.29%) (both p < 0.001), whereas autumn (0.81%) showed no significant difference. Early childhood (1–5 years) demonstrated a higher likelihood of positivity than infants aged 0 years (aOR, 1.51; p = 0.007) and the highest crude positivity rate (1.89%). Although underlying mechanisms were not directly assessed, this long-term analysis documents a persistent decline and attenuation of seasonal dominance in HCoV-229E detection beyond the period of pandemic-related suppression. These findings underscore the value of sustained laboratory-based surveillance in identifying and tracking long-term changes in respiratory virus circulation patterns and in supporting public health monitoring aligned with Sustainable Development Goal 3 (SDG 3). Full article
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10 pages, 1112 KB  
Case Report
The Paucity of Typical Pathology: An Autopsy Series of Typhus Group Rickettsiosis-Associated Hemophagocytic Lymphohistiocytosis
by Joshua Klinnert, Vasily Ovechko, Michelle M. Felicella, April McDougal, Sarah E. Muir, Patricia A. Crocquet-Valdes, David H. Walker and Lucas S. Blanton
Pathogens 2026, 15(2), 230; https://doi.org/10.3390/pathogens15020230 - 19 Feb 2026
Viewed by 514
Abstract
Murine typhus (also called flea-borne or endemic typhus) is an undifferentiated febrile illness caused by the bacterium Rickettsia typhi. The disease, transmitted by rat and cat fleas, is endemic to seaboard regions worldwide. Recently, murine typhus has reemerged as an increasingly recognized [...] Read more.
Murine typhus (also called flea-borne or endemic typhus) is an undifferentiated febrile illness caused by the bacterium Rickettsia typhi. The disease, transmitted by rat and cat fleas, is endemic to seaboard regions worldwide. Recently, murine typhus has reemerged as an increasingly recognized cause of febrile illness in the United States, especially in Texas and Southern California. In addition to fever, manifestations often include headache, malaise, myalgias, and a maculopapular rash in approximately half of cases. Although usually considered a mild illness, when untreated, symptoms can last up to 3 weeks. Severe manifestations such as pneumonitis, acute kidney injury, and meningoencephalitis may occur. Historically, death has occurred in 0.4%, but in Southern California, the case fatality rate has been recently recorded at 1.8%. As murine typhus has reemerged, there have been growing reports that this infection has triggered hemophagocytic lymphohistiocytosis, a life-threatening hyperinflammatory syndrome. We herein report two fatal cases of hemophagocytic lymphohistiocytosis secondary to murine typhus. Autopsy revealed typhus group rickettsial antigen in tissues via immunohistochemistry, along with hemophagocytosis. Interestingly, the classic vascular and perivascular histopathologic findings associated with disseminated rickettsial infection were absent. These findings highlight an aberrant inflammatory cascade leading to hemophagocytic lymphohistiocytosis. Full article
(This article belongs to the Special Issue New Insights into Rickettsia and Related Organisms)
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20 pages, 1841 KB  
Article
Optimizing Lysis and Extraction Workflows for Enrichment-Free qPCR Detection of Salmonella enterica in Poultry Matrices
by Rejoice Nyarku, Emmanuel Kuufire, Kingsley E. Bentum, Viona Osei, Asmaa Elrefaey, Tyric James, Yilkal Woube, Evangelyn Alocilja, Temesgen Samuel and Woubit Abebe
Pathogens 2026, 15(2), 229; https://doi.org/10.3390/pathogens15020229 - 18 Feb 2026
Viewed by 699
Abstract
Salmonella remains a leading cause of foodborne illness worldwide, with poultry products representing a major source of human exposure, underscoring the need for rapid and reliable detection methods. Although qPCR offers sensitive and timely pathogen detection, assay performance is highly dependent on sample [...] Read more.
Salmonella remains a leading cause of foodborne illness worldwide, with poultry products representing a major source of human exposure, underscoring the need for rapid and reliable detection methods. Although qPCR offers sensitive and timely pathogen detection, assay performance is highly dependent on sample preparation efficiency and nucleic acid purity, particularly in complex food matrices. In this study, we systematically optimized the sample preparation workflow of a SYBR Green based qPCR assay for enrichment-free detection of Salmonella enterica in poultry. Multiple lysis chemistries, incubation times, DNA extraction methods, centrifugation strategies, inoculum sources, and magnetic nanoparticle (MNP) assisted workflows were evaluated using phosphate-buffered saline and chicken rinsate matrices. Among the conditions tested, lysis with 20 µL Proteinase K and 400 µL PrepMan™ for 20 min produced the lowest and most consistent Cq values. Although Promega Wizard® produced slightly lower mean Cq values than PrepMan™, statistical analysis showed no significant differences between extraction methods or centrifugation protocols, indicating comparable overall performance. Broth-derived inocula yielded earlier and more reproducible Cq values than colony-derived preparations. In contrast, inclusion of MNP processing resulted in higher Cq values in both matrices compared to the non-MNP workflow. Overall, these findings demonstrate that optimized lysis, extraction, and centrifugation workflows enhances the consistency and analytical reliability of direct qPCR detection of Salmonella in poultry matrices, supporting laboratory-based rapid detection applications. Full article
(This article belongs to the Section Bacterial Pathogens)
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21 pages, 3103 KB  
Article
Treatment and Cleaning of Contact Lenses with Plasma-Activated Solutions
by Stephanie Arndt, Julia Spörl, Petra Unger, Tim Maisch, Mark Berneburg and Sigrid Karrer
Pathogens 2026, 15(2), 228; https://doi.org/10.3390/pathogens15020228 - 18 Feb 2026
Viewed by 507
Abstract
The occurrence of contact lens complications caused by inadequate cleaning of the lenses using “All-in-One” contact lens cleaning solutions (CLCSs) represents a medically relevant problem worldwide. This study explores the potential of cold atmospheric plasma (CAP) to enhance the efficacy of CLCSs and [...] Read more.
The occurrence of contact lens complications caused by inadequate cleaning of the lenses using “All-in-One” contact lens cleaning solutions (CLCSs) represents a medically relevant problem worldwide. This study explores the potential of cold atmospheric plasma (CAP) to enhance the efficacy of CLCSs and address complications from inadequate lens hygiene. It was examined whether exposure to CAP for 1–24 h could boost the antibacterial effects of CLCSs and other solutions, including Milli-Q water (M-QW), physiological saline (NaCl), and Dulbecco’s Phosphate Buffered Saline (DPBS). Additionally, the stability of reactive oxygen and nitrogen species (RONS) and their impact on pH immediately after treatment and over 1–4 weeks was assessed. Furthermore, the cleaning efficacy of plasma-activated solutions (PASs) was tested on lipid-coated silicone hydrogel lenses. Results showed that CAP increased RONS concentrations immediately, with elevated levels persisting over time. While no significant improved antibacterial effect was observed against Escherichia coli in CLCSs, CAP treatment generated disinfectant properties in M-QW and NaCl solutions. Importantly, CAP-treated CLCSs significantly improved the cleaning performance on lipid-coated lenses, though M-QW’s cleaning ability worsened post-treatment. pH measurements indicated notable decreases in M-QW and NaCl after CAP, whereas buffered solutions like CLCSs and DPBS remained stable. Overall, CAP demonstrates promise for contact lens disinfection and surface modification; however, further research and pre-clinical trials are necessary before clinical application in ophthalmology. Full article
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12 pages, 632 KB  
Article
Helicobacter pylori and Non-Helicobacter pylori Helicobacter (NHPH) Zoonotic Infections: A Survey Among Greek Veterinarians Aiming to Enhance Communication
by Eirini Fragkiadaki, Francisco Cortez Nunes, Maria Linou, Beatriz Martinez-Gonzalez, Dionyssios N. Sgouras and Teresa Letra Mateus
Pathogens 2026, 15(2), 227; https://doi.org/10.3390/pathogens15020227 - 18 Feb 2026
Viewed by 738
Abstract
Helicobacter species affect humans and animals, mainly causing gastrointestinal but also extra-gastrointestinal pathologies. Besides Helicobacter pylori, which is the main human pathogen, Non-Helicobacter pylori Helicobacters (NHPH) are also associated with human diseases, thus raising concern about their zoonotic potential. Veterinarians are [...] Read more.
Helicobacter species affect humans and animals, mainly causing gastrointestinal but also extra-gastrointestinal pathologies. Besides Helicobacter pylori, which is the main human pathogen, Non-Helicobacter pylori Helicobacters (NHPH) are also associated with human diseases, thus raising concern about their zoonotic potential. Veterinarians are considered a risk group for NHPH infections and act as first-line communicators to animal owners about their prophylaxis. Therefore, we aimed to assess the knowledge and perception of veterinarians working in Greece about Helicobacter pylori and NHPH by asking them to participate anonymously in an online 34-question survey. The questionnaire consisted of three sections regarding environmental exposure to Helicobacter spp.; know-how about clinical signs in various species, including personal human experience; and willingness to get updated information about NHPH. Of the 111 respondents, 41.4% had not heard of H. suis (NHPH), and 35.0% were unaware of the species that could be affected. Almost 60.0% of companion animal veterinarians rarely suspect and 20.0% never suspect Helicobacter spp. infections in the case of gastritis. Nevertheless, 41.0% of respondents considered Helicobacter as zoonotic, and 87.0% wanted to receive information via professional channels and brochures. Despite the limited number of respondents and the exploratory nature of our study, as with similar data from Portugal, we emphasize the need to train veterinarians to have a more targeted focus on the zoonotic potential of Helicobacter within a One Health approach. Full article
(This article belongs to the Special Issue Emerging and Neglected Zoonotic Pathogens in Companion Animals)
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17 pages, 6742 KB  
Article
Combined Effects of Amikacin and Methylene Blue-Mediated Photodynamic Therapy on Pseudomonas aeruginosa Biofilms Mimicking Mono- and Polymicrobial Diabetic Foot Ulcer Infections
by Florencia Mariani, Celeste R. Costilla, Oscar J. Oppezzo and Estela M. Galvan
Pathogens 2026, 15(2), 226; https://doi.org/10.3390/pathogens15020226 - 18 Feb 2026
Cited by 1 | Viewed by 679
Abstract
Diabetic foot ulcer (DFU) infections frequently involve biofilm formation and exhibit limited responsiveness to conventional antibiotic therapy. In particular, Pseudomonas aeruginosa often participates in mono- and polymicrobial biofilms that display high tolerance to antimicrobial agents. This study evaluated the efficacy of methylene blue-mediated [...] Read more.
Diabetic foot ulcer (DFU) infections frequently involve biofilm formation and exhibit limited responsiveness to conventional antibiotic therapy. In particular, Pseudomonas aeruginosa often participates in mono- and polymicrobial biofilms that display high tolerance to antimicrobial agents. This study evaluated the efficacy of methylene blue-mediated antimicrobial photodynamic therapy (aPDT), alone and in combination with antibiotics, against P. aeruginosa biofilms formed either as single-species or in mixed communities with Enterococcus faecalis, under conditions mimicking DFU infections. Macrocolony biofilms were challenged with amikacin alone (for single-species biofilms) or amikacin plus ampicillin (for mixed biofilms), aPDT, or sequential combinations of these treatments, and bacterial viability was quantified by colony-forming unit enumeration. Antibiotic treatment alone produced only modest reductions in P. aeruginosa viability, even at high concentrations, while aPDT using methylene blue was effective only at high photosensitizer concentrations. In contrast, sequential treatment with antibiotics followed by aPDT and a second antibiotic challenge resulted in a marked reduction in P. aeruginosa viability in both mono- and polymicrobial biofilms. Scanning electron microscopy revealed extensive structural damage in P. aeruginosa cells following combined treatments, whereas E. faecalis remained unaffected. Overall, our findings demonstrate that combining aPDT with antibiotics significantly enhances antibiofilm activity against P. aeruginosa, highlighting this approach as a promising alternative for the management of biofilm-associated DFU infections. Full article
(This article belongs to the Section Bacterial Pathogens)
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8 pages, 466 KB  
Article
Comparative IgG-IgM Western Blot in the Diagnosis of Congenital Toxoplasmosis: A Valid Diagnostic Tool
by Alice Bonetti, Ambra Vola, Daniele Lilleri, Lucrezia Lo Grasso, Milena Furione, Alessia Arossa, Alessandra Ricciardi, Claudia Viganò, Alessia Bressan, Claudia Pavia, Annalisa De Silvestri, Giulia Gambini, Fausto Baldanti and Valeria Meroni
Pathogens 2026, 15(2), 225; https://doi.org/10.3390/pathogens15020225 - 17 Feb 2026
Viewed by 681
Abstract
Congenital toxoplasmosis (CT) results from vertical transmission of Toxoplasma gondii during maternal infection in pregnancy. Early diagnosis in newborns is crucial to initiate timely therapy and prevent long-term sequelae. The IgM Immunosorbent Agglutination Assay (ISAGA) has historically been considered an important diagnostic tool [...] Read more.
Congenital toxoplasmosis (CT) results from vertical transmission of Toxoplasma gondii during maternal infection in pregnancy. Early diagnosis in newborns is crucial to initiate timely therapy and prevent long-term sequelae. The IgM Immunosorbent Agglutination Assay (ISAGA) has historically been considered an important diagnostic tool for CT; however, its recent market withdrawal necessitates alternative approaches. We conducted a retrospective observational study at Fondazione IRCCS Policlinico San Matteo, Pavia, Italy, including 44 newborns born to mothers with confirmed toxoplasmosis between 2019 and 2022. Newborns were classified as CT (n = 19) or non-CT (n = 25) based on serological follow-up, comparative Western blot (CWB) and Interferon Gamma Release Assay (IGRA). Sensitivity and specificity of CWB, IgM Chemiluminescent Immunoassay (CLIA), and IgM ISAGA were assessed at birth and at one month. At birth, CWB demonstrated 88.9% sensitivity, significantly higher than IgM CLIA (52.6%) and IgM ISAGA (57.9%). Specificity was 100% at birth and 92% at one month. CWB retained high sensitivity at one month (81.8%). IGRA complemented CWB in confirming or excluding infection in cases with equivocal or false-negative serology. Comparative Western blot thus represents a robust diagnostic alternative for CT, ensuring early detection and timely treatment, particularly in the absence of IgM ISAGA. Full article
(This article belongs to the Special Issue Toxoplasmosis in Immunocompromised and Immunocompetent Host: Recent Updates About the Epidemiology, Diagnosis, Clinical Manifestations and Management)
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13 pages, 1338 KB  
Article
A Cross-Sectional Study of Rift Valley Fever Exposure in Humans and Livestock in Southwestern Uganda Using a One Health Approach: Evidence of Elevated Seroprevalence Outside Recognized Outbreak Periods
by Luke Nyakarahuka, Silvia Situma, Raymond Odinoh, Barnabas Bakamutumaho, Carolyne Nasimiyu, Jeanette Dawa, Justine Okello, Honest Kemiyondo, Alex Tumusiime, Mutesi Joanita, Jackson Kyondo, John Kayiwa, David Odongo, Deo Birungi Ndumu, Kariuki M. Njenga and Robert F. Breiman
Pathogens 2026, 15(2), 224; https://doi.org/10.3390/pathogens15020224 - 17 Feb 2026
Viewed by 1236
Abstract
Rift Valley fever (RVF) transmission has intensified in southwestern Uganda since 2016. To quantify human and livestock exposure and associated risks, we conducted a cross-sectional serosurvey in Isingiro, Kabale and Rubanda districts between October and November 2023. A total of 766 humans and [...] Read more.
Rift Valley fever (RVF) transmission has intensified in southwestern Uganda since 2016. To quantify human and livestock exposure and associated risks, we conducted a cross-sectional serosurvey in Isingiro, Kabale and Rubanda districts between October and November 2023. A total of 766 humans and 2383 livestock were sampled and tested for RVF antibodies using ELISA, with structured questionnaires capturing demographic, behavioral and environmental data. Human seroprevalence was 11.5% (88/766), varying by district (13.8% Isingiro, 11.8% Rubanda, 6.8% Kabale; p = 0.04). Independent predictors from the multivariate model included raw-meat consumption (aOR 6.11; 95% CI 1.16–27.80), cattle ownership (aOR 2.33; 95% CI 1.27–4.36), male sex (aOR 1.64; 95% CI 1.02–2.66) and younger age compared with ≥50 years (31–49 years: aOR 2.02; 95% CI 1.20–3.48; 18–30 years: aOR 2.37; 95% CI 1.04–5.14). Herd-level seroprevalence was 42.5% (204/480), associated with cattle presence (aOR 6.48; 95% CI 4.10–10.40), lack of carcass burial (aOR 15.70; 95% CI 4.23–63.60), on-farm slaughter (aOR 2.14; 95% CI 1.21–3.89) and increased mosquito activity (aOR 1.75; 95% CI 1.13–2.73). Animal-level seroprevalence was 14.6% (347/2383), highest in cattle (33.8%), with cattle having markedly higher odds than goats (aOR 6.73; 95% CI 4.96–9.14). These findings demonstrate substantial transmission and highlight cattle-centered interfaces as primary targets for control to humans. Full article
(This article belongs to the Special Issue Epidemiology of Vector-Borne Pathogens)
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12 pages, 1666 KB  
Article
Paramyxoviruses in Bats in Poland—The First Detection
by Anna Orłowska, Karol Stasiak, Jerzy Rola and Marcin Smreczak
Pathogens 2026, 15(2), 223; https://doi.org/10.3390/pathogens15020223 - 17 Feb 2026
Viewed by 698
Abstract
Bats are hosts to many diseases that emerge in humans and livestock. Knowledge about the diversity and circulation of paramyxoviruses in European bat populations, despite their recognized importance, remains limited. Here, we present data on the first detection of paramyxoviruses in Poland in [...] Read more.
Bats are hosts to many diseases that emerge in humans and livestock. Knowledge about the diversity and circulation of paramyxoviruses in European bat populations, despite their recognized importance, remains limited. Here, we present data on the first detection of paramyxoviruses in Poland in the new bat species of Cnephaeus serotinus and Cnephaeus nilsonii, which have never been previously recognized as paramyxovirus hosts, as well as in Myotis daubentonii and two unknown bat species. Viral RNA was detected in fecal and intestinal samples using the semi-nested RT-PCR protocol followed by Sanger sequencing. A widespread comprehensive phylogenetic study supported by haplotype network analyses of 376 nt sequences of paramyxoviruses detected in bats worldwide revealed that paramyxoviruses are closely related to the host and strongly correlate to the area of origin. Full article
(This article belongs to the Special Issue Laboratory Diagnostics in Epidemiological Diseases Caused by Animal Pathogens)
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15 pages, 1637 KB  
Article
Investigation of Gene Regions Responsible for Drug Resistance in Clinical Isolates of Mycobacterium tuberculosis Complex Resistant to at Least Two First-Line Anti-Tuberculosis Drugs
by Mahmut Ulger, Nurcihan Biltekin, Seda Tezcan Ulger and Gonul Aslan
Pathogens 2026, 15(2), 222; https://doi.org/10.3390/pathogens15020222 - 16 Feb 2026
Cited by 1 | Viewed by 669
Abstract
Early and rapid diagnosis of drug resistance in tuberculosis (TB) plays a key role in reducing the spread of resistance and enabling effective treatment. The aim of this study was to investigate mutations in drug resistance-associated gene regions of Mycobacterium tuberculosis complex (MTBC) [...] Read more.
Early and rapid diagnosis of drug resistance in tuberculosis (TB) plays a key role in reducing the spread of resistance and enabling effective treatment. The aim of this study was to investigate mutations in drug resistance-associated gene regions of Mycobacterium tuberculosis complex (MTBC) isolates resistant to at least two first-line anti-tuberculosis drugs through sequence analysis, in order to characterize the core molecular features of these strains in the region and to identify previously unreported, geographically distinct novel mutation sites. The drug susceptibility of 23 clinical isolates was assessed using the BACTEC MGIT 960 system, and resistance-associated point mutations were identified through DNA sequence analysis and comparison with GenBank reference sequences. AAG → AGG mutation was detected in the rpsL gene region at codon 43 (n = 7) and codon 88 (n = 1). Additionally, GAG → GCG point mutation was identified at codon 70 (n = 2), representing a new region not previously reported in the literature. The most frequent mutation was AGC → ACC at katG codon 315 (n = 10), followed by a C → T substitution at position −15 of the inhA promoter region (n = 4). Additionally, TCG → TTG at rpoB codon 531 (n = 4) and ATG → GTG at embB codon 306 (n = 1) were detected. The detection of resistance-associated mutations is essential for controlling drug-resistant tuberculosis. In this study, a novel rpsL mutation (GAG → GCG) at codon 70 and a previously unreported codon 88 mutation in our country were identified, contributing to the understanding of molecular resistance mechanisms and epidemiology. Full article
(This article belongs to the Section Bacterial Pathogens)
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16 pages, 2020 KB  
Article
Microbial Diversity and Composition Uncovered on Obturator Prosthesis Biofilms: Exploratory Findings from a Pilot Study
by Camila Vilela, Leonel Mendoza, Raquel Vilela, Francisca Daniele Moreira Jardilino, Cláudia Lopes Brilhante Bhering and Amalia Moreno
Pathogens 2026, 15(2), 221; https://doi.org/10.3390/pathogens15020221 - 16 Feb 2026
Viewed by 540
Abstract
Microbial communities on obturator prosthesis biofilms have yet to be investigated. This pilot study explores eukaryotes, prokaryotes, and viruses present on obturator prosthesis biofilms using metagenomics. The prostheses of the selected patients (n = 3) were collected and their biofilms were physically [...] Read more.
Microbial communities on obturator prosthesis biofilms have yet to be investigated. This pilot study explores eukaryotes, prokaryotes, and viruses present on obturator prosthesis biofilms using metagenomics. The prostheses of the selected patients (n = 3) were collected and their biofilms were physically removed. The total genomic DNA was extracted, followed by metagenomic analysis. The microbial diversity in each of the investigated biofilms was exceptionally abundant. Between 2616 to 3024 species were detected in the three biofilms. The highest percentage included prokaryotes and unclassified species, followed by low percentages of fungi, viruses, and archaea. Unusual pathogens rarely reported in oral biofilms, such as Mycobacterium and other species, were also found at very low percentages. Unigenes for functional pathways related to metabolism, cellular processes, human disease, and other microbial unigenes were abundant. In addition, unigenes for several antibiotic-resistance mechanisms were also detected. This study reveals, for the first time, that biofilm formation on obturator prostheses comprises a variety of dynamic microbial communities, suggesting a putative role in health and disease in patients following maxillofacial surgery. Full article
(This article belongs to the Special Issue Oral Microbiome and Human Systemic Health)
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13 pages, 953 KB  
Article
Differential Susceptibility to Antimony in Strains and Clinical Isolates of Leishmania amazonensis from Brazil: In Vitro and In Vivo Studies and Implications for Drug Response and Treatment Failure
by Victor de Sousa Agostino, Leonardo F. Geres, Stéphane de la Roca, Beatriz T. de Moraes, Juliana I. Aoki, Elizabeth M. Coser, Nilson Branco and Adriano C. Coelho
Pathogens 2026, 15(2), 220; https://doi.org/10.3390/pathogens15020220 - 15 Feb 2026
Viewed by 624
Abstract
In Brazil, cutaneous leishmaniasis is endemic and may be caused by Leishmania amazonensis. This species is the second most prevalent species in that country, and it is responsible for localized cutaneous and diffuse cutaneous leishmaniasis. Pentavalent antimony is still the first-line drug [...] Read more.
In Brazil, cutaneous leishmaniasis is endemic and may be caused by Leishmania amazonensis. This species is the second most prevalent species in that country, and it is responsible for localized cutaneous and diffuse cutaneous leishmaniasis. Pentavalent antimony is still the first-line drug for cutaneous leishmaniasis treatment in Brazil. In this study, we investigated the in vitro susceptibility to antimony of a panel of L. amazonensis strains and clinical isolates responsible for cutaneous and diffuse cutaneous leishmaniasis. There was a significant variation in susceptibility to antimony not only within these strains and isolates evaluated at either promastigote or intracellular amastigote stages, but also between the two parasite stages for some of these strains and isolates. Additionally, we investigated whether this in vitro susceptibility variation to antimony would affect the in vivo response to treatment, using an experimental BALB/c mouse model of cutaneous leishmaniasis infected with three strains differing in susceptibility. Despite antimony could mildly reduce the lesion size in mice infected with one of these strains, no significant reduction in the parasite burden was found in treated animals, and they were completely refractory to drug treatment. These findings indicate that antimony treatment, even at high dosages via the intraperitoneal route, was not effective against L. amazonensis infection in this animal model. Finally, this study provides a preclinical dataset of the activity of antimony against a panel of strains and isolates of a species responsible for localized cutaneous and diffuse cutaneous leishmaniasis in Brazil. Full article
(This article belongs to the Section Parasitic Pathogens)
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17 pages, 13158 KB  
Article
Respiratory Infection-Related Pathogens in the Pediatric Intensive Care Unit During 2019–2024 in Hubei, China
by Jiahui Chen, Ying Li, Dan Sun, Hebin Chen, Haizhou Liu, Wenqing Li, Yanli Wang, Feng Han, Jiali Xu, Xueru Liu, Hui Du, Youjing Liu, Qing Du, Yifei Zhang, Yan Li, Yi Yan, Di Liu and Xiaoxia Lu
Pathogens 2026, 15(2), 219; https://doi.org/10.3390/pathogens15020219 - 14 Feb 2026
Viewed by 914
Abstract
Respiratory infections are a leading cause of hospitalization and mortality in children, and the pediatric intensive care unit (PICU) is a critical setting for managing severe cases. However, the epidemiological patterns of respiratory pathogens in the PICU remain insufficiently characterized. In this retrospective [...] Read more.
Respiratory infections are a leading cause of hospitalization and mortality in children, and the pediatric intensive care unit (PICU) is a critical setting for managing severe cases. However, the epidemiological patterns of respiratory pathogens in the PICU remain insufficiently characterized. In this retrospective study, we analyzed respiratory pathogen testing results from 2126 pediatric patients admitted to the PICU of Wuhan Children’s Hospital between 2019 and 2024. The pathogen spectrum and epidemiological characteristics were evaluated across age groups and seasons. Respiratory syncytial virus (RSV, 18.06%) was the most frequently detected viral pathogen, while Streptococcus pneumoniae (6.96%) was the predominant non-viral pathogen. The overall infection burden was highest in children aged ≤ 1 year (53.75%) and 3 < age ≤ 6 years (54.70%), indicating that early childhood represents a high-risk period for severe respiratory infections requiring intensive care. Pathogen distribution varied significantly across age groups. Distinct seasonal patterns were observed for several respiratory pathogens, particularly among viral pathogens, whereas non-viral pathogens showed more variable seasonal distributions. Furthermore, screening for 10 common pathogens accounted for 75% of PICU respiratory infections, highlighting the clinical utility of multiplex molecular detection. This study delineates the pathogen spectrum of respiratory tract infections in the PICU and characterizes their age- and season-specific epidemiological patterns. This study defines the pathogen spectrum and age- and season-specific patterns of respiratory infections in the PICU, providing evidence to support targeted pathogen surveillance, optimized multiplex diagnostics, and risk-informed infection control strategies in pediatric critical care. Full article
(This article belongs to the Special Issue Advances in Molecular Epidemiology and Innovative Diagnostic Strategies for Infectious Disease Control)
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15 pages, 2437 KB  
Article
Genomic Insights into Chromosomal Colistin Resistance and Virulence–Resistance Convergence in MDR/XDR Klebsiella pneumoniae from Tertiary Hospitals in Peshawar, Pakistan
by Aiman Waheed, Sumera Afzal Khan, Sajjad Ahmad, Jody E. Phelan, Gulab Fatima Rani, Susana Campino, Taj Ali Khan and Taane G. Clark
Pathogens 2026, 15(2), 218; https://doi.org/10.3390/pathogens15020218 - 14 Feb 2026
Viewed by 806
Abstract
Background: Klebsiella pneumoniae is a World Health Organization-listed critical priority pathogen and a major cause of healthcare-associated infections, driven by the global emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) lineages and their alarming convergence with hypervirulence. Methods: In this study, [...] Read more.
Background: Klebsiella pneumoniae is a World Health Organization-listed critical priority pathogen and a major cause of healthcare-associated infections, driven by the global emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) lineages and their alarming convergence with hypervirulence. Methods: In this study, 152 clinical specimens, including urine, blood, pus, wound swabs, and respiratory samples, were collected from tertiary care hospitals in Peshawar, Pakistan. Standard microbiological and biochemical methods identified 55 K. pneumoniae isolates. Antimicrobial susceptibility testing (AST) was performed using the Kirby–Bauer disk diffusion and broth microdilution methods, with results interpreted according to Clinical and Laboratory Standards Institute (CLSI) guidelines. MDR and XDR phenotypes were defined based on European Centre for Disease Prevention and Control (ECDC) criteria. Whole-genome sequencing (WGS) was conducted on 16 phenotypically confirmed MDR/XDR isolates, followed by comprehensive bioinformatic analyses to characterize sequence types (STs), acquired antimicrobial resistance genes, resistance-associated chromosomal mutations, virulence determinants, plasmid replicons, and phylogenetic relationships. Results: Among 55 confirmed K. pneumoniae isolates, 19 (34.5%) were classified as MDR and 10 (18.2%) as XDR. WGS revealed substantial genomic heterogeneity, identifying 11 distinct STs, with ST39 being the most prevalent. Resistance to multiple antibiotic classes was mediated by the combined presence of plasmid-borne carbapenemases and extended-spectrum β-lactamases, alongside chromosomal mutations affecting outer membrane porins (OmpK35/OmpK36), fluoroquinolone targets (gyrA/parC), efflux regulation (ramR, marR), and lipid A modification pathways associated with colistin resistance (mgrB, pmrA/pmrB, arnC, crrB). IncF-family plasmids predominated and frequently co-occurred with additional resistance-associated replicons. Notably, one isolate exhibited an expanded virulence gene repertoire, including multiple siderophore systems and a complete type II secretion system, consistent with a hypervirulence-associated genomic profile. Phylogenetic analyses demonstrated close relatedness to international lineages from Asia, the Middle East, and Europe, indicating regional and transnational dissemination. Conclusions: This study highlights the complex interplay between plasmid-mediated gene acquisition and chromosomal adaptive mutations driving MDR and XDR phenotypes in K. pneumoniae circulating in Peshawar, Pakistan. The identification of hypervirulence-associated genetic features within an MDR background underscores the growing threat posed by convergent lineages and emphasizes the need for sustained WGS-based surveillance to inform infection control and antimicrobial stewardship strategies. Full article
(This article belongs to the Section Bacterial Pathogens)
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15 pages, 9648 KB  
Article
Ocular Involvement as a Key Marker of Systemic Disease in Dogs Naturally Infected with Leishmania infantum: Clinical, Laboratory, and Histopathological Insights
by Caroline Magalhães-Cunha, Ana Lúcia Abreu-Silva, Marcelo Pelajo-Machado, Celeste da Silva Freitas de Souza, Karen Lebreiro dos Santos, Lucas Almeida Zangirolami, Flávia de Oliveira Cardoso and Kátia da Silva Calabrese
Pathogens 2026, 15(2), 217; https://doi.org/10.3390/pathogens15020217 - 14 Feb 2026
Viewed by 697
Abstract
Canine visceral leishmaniasis (CVL), caused by Leishmania infantum, is a multisystemic disease in which ocular involvement is frequent but often underestimated. This study aimed to comprehensively evaluate the clinical, ophthalmological, parasitological, hematological, biochemical, and histopathological alterations in dogs naturally infected with L. [...] Read more.
Canine visceral leishmaniasis (CVL), caused by Leishmania infantum, is a multisystemic disease in which ocular involvement is frequent but often underestimated. This study aimed to comprehensively evaluate the clinical, ophthalmological, parasitological, hematological, biochemical, and histopathological alterations in dogs naturally infected with L. infantum from an endemic area of northeastern Brazil, with special emphasis on the relationship between ocular manifestations and systemic disease. Twenty-five symptomatic dogs were evaluated through clinical and ophthalmological examinations, parasitological culture, PCR, laboratory analyses, and histopathology of ocular and periocular tissues. Ocular alterations were observed in 80% of the animals, predominantly bilateral and frequently associated with multiple concurrent lesions, including ocular discharge, conjunctivitis, blepharitis, uveitis, and corneal opacity. Functional ophthalmological tests revealed keratoconjunctivitis sicca and corneal ulcers in a substantial proportion of dogs. Hematological abnormalities were highly prevalent, particularly anemia and thrombocytopenia. Comparative analysis demonstrated that dogs with ocular involvement exhibited significantly higher leukocyte counts and segmented neutrophils, as well as increased AST levels, indicating an enhanced systemic inflammatory response. Histopathological examination revealed intense plasmacytic inflammatory infiltrates and the presence of amastigote forms in ocular and periocular tissues, indicating that both immune-mediated and parasite-driven mechanisms could be involved in disease pathogenesis. Collectively, these findings underscore ocular involvement as a clinically relevant manifestation of CVL and reinforce the importance of routine ophthalmological evaluation in clinical management. Full article
(This article belongs to the Special Issue Leishmaniasis: Current Status and Future Perspectives)
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18 pages, 1473 KB  
Article
Cell Wall Proteome of Candida albicans Reveals Proteins Associated with Tolerance to Antibiofilm Activity of a Lippia graveolens Kunth Stem Extract
by Alejandra García-Núñez, Ana Lilia Martínez-Rocha, Carlos Antonio Alba-Fierro, Francisco Javier Ríos-Fránquez, Juan Pablo Cabral-Miramontes, María Estela Frías-Zepeda, Miguel Ángel Escobedo-Bretado and Estela Ruiz-Baca
Pathogens 2026, 15(2), 216; https://doi.org/10.3390/pathogens15020216 - 14 Feb 2026
Viewed by 774
Abstract
Candida albicans is an opportunistic fungus capable of forming biofilms that are resistant to conventional antifungal treatments. This study evaluated the antibiofilm activity of an ethanolic extract from Mexican oregano (Lippia graveolens Kunth) stem and its impact on the protein composition of [...] Read more.
Candida albicans is an opportunistic fungus capable of forming biofilms that are resistant to conventional antifungal treatments. This study evaluated the antibiofilm activity of an ethanolic extract from Mexican oregano (Lippia graveolens Kunth) stem and its impact on the protein composition of the C. albicans cell wall (CW). The proteomic analysis was restricted to the adherent cells that persisted after treatment, representing the more tolerant subpopulation. First, the biofilm-forming capacity of 18 clinical isolates was characterized. Subsequently, the effect of the Lippia graveolens Kunth stem extract on biofilm formation was assessed in clinical isolates of C. albicans with high and moderate biofilm-forming capacities. The results showed an MIC90 of 16 mg/mL against C. albicans isolates in planktonic growth. Furthermore, the extract exhibited an antibiofilm effect, showing a 77% inhibition in the highly biofilm-forming C. albicans 2400H strain at 1.6 mg/mL. To assess how the extract influences CW composition, we analyzed the CW proteome of the adherent biofilm cells of C. albicans that persisted after exposure to the stem extract. Mass spectrometry identified 1006 total proteins, where 156 were up-regulated, and 94 were down-regulated. Of the differentially expressed proteins, we identify 40 CW proteins (CWP’s) associated with dimorphic transition, adhesion, oxidative stress, and biofilm formation. These findings provide the potential of the L. graveolens Kunth stem as a natural antifungal agent against C. albicans biofilms. In addition, we identified CWP’s associated with tolerance to the extract’s antibiofilm activity, offering mechanistic insight into persistence and potential targets for improving antibiofilm interventions. Full article
(This article belongs to the Special Issue Candida albicans Virulence and Therapeutic Strategies)
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14 pages, 268 KB  
Article
Assessing Virulence Factor Genes in Pig-Derived Escherichia coli from the Region of Vojvodina Treated with Postbiotic Substance and Herbal Essential Oils
by Andrea Lauková, Jana Ščerbová, Valentína Focková, Igor Stojanov, Monika Pogány Simonová and Jasna Prodanov-Radulović
Pathogens 2026, 15(2), 215; https://doi.org/10.3390/pathogens15020215 - 13 Feb 2026
Viewed by 529
Abstract
Antibiotic-resistant, biofilm-forming Escherichia coli may constitute a reservoir of antibiotic resistance and other determinants that can be transmitted to pathogenic bacteria for animals and humans. Therefore, it is crucial to reduce the incidence of these types of E. coli. The aim of [...] Read more.
Antibiotic-resistant, biofilm-forming Escherichia coli may constitute a reservoir of antibiotic resistance and other determinants that can be transmitted to pathogenic bacteria for animals and humans. Therefore, it is crucial to reduce the incidence of these types of E. coli. The aim of this study was to determine whether essential oils from oregano, thyme, sage, and coriander, as well as the postbiotic substance PS412, can inhibit virulence factor genes possessing pig-derived E. coli. It aimed to find a new tool for the prevention and/or elimination of virulent E. coli. Altogether, 16 pig-derived E. coli from a pig farm in the region of Vojvodina (Serbia) were taxonomically identified using MALDI-TOF mass spectrometry; 14 strains (87.5%) with secure genus identification/probable species identification and 2 with highly probable genus identification. The fimA gene was detected in 62.5% of E. coli strains, and the crl gene in 87.5% of the strains. Ec3419/2 contained five analyzed genes. Five E. coli were found to form biofilm, as indicated by their growth on Congo red agar. The strains were mostly multi-resistant to antibiotics. Each E. coli strain produced the damaging enzyme, such as β-glucuronidase and/or α-chymotrypsin. However, they were susceptible to herbal essential oils (HEOs) with average inhibitory zones from 15 to 27 mm in diameter. They were also (6) susceptible to the PS412 (activity to 6400 AU/mL). The results contribute to the practical effectiveness of postbiotic substances, HEOs, and their combination as a novel approach to combating the virulence factors of E. coli. This insight also contributes to the strategy behind the One Health Concept. Full article
(This article belongs to the Special Issue Diagnosis, Immunopathogenesis and Control of Bacterial Infections)
20 pages, 2038 KB  
Article
In Vitro Evaluation of Colistin–Meropenem Activity Against XDR and PDR Carbapenemase-Producing Klebsiella pneumoniae and Acinetobacter baumannii
by Shahinda Rezk, Nada Younis Elwakeel, Abeer Ghazal, Amel Elsheredy, Daniel Baecker and Ahmed Noby Amer
Pathogens 2026, 15(2), 214; https://doi.org/10.3390/pathogens15020214 - 13 Feb 2026
Viewed by 624
Abstract
Carbapenem-resistant Klebsiella pneumoniae (CRKP) and Acinetobacter baumannii (CRAB) pose significant therapeutic challenges due to their high resistance and global spread. Combination therapy with colistin (COL) and meropenem (MEM) was used to enhance antimicrobial activity. This study evaluated the COL-MEM combination against CRKP and [...] Read more.
Carbapenem-resistant Klebsiella pneumoniae (CRKP) and Acinetobacter baumannii (CRAB) pose significant therapeutic challenges due to their high resistance and global spread. Combination therapy with colistin (COL) and meropenem (MEM) was used to enhance antimicrobial activity. This study evaluated the COL-MEM combination against CRKP and CRAB isolates with a high resistance profile. A total of 58 carbapenem-resistant clinical isolates (31 CRKP and 27 CRAB), including extensively resistant and pandrug-resistant strains, were collected over a period of 12 months. Synergy between COL and MEM was assessed by microdilution checkerboard (MCB) and time-kill (TKA) assays. Carbapenemase genes were detected using molecular methods. The results showed that the COL-MEM combination yielded synergy (35.5% and 40.7%, respectively) and additive effects (35.5% and 37.0%, respectively), while no antagonism was observed. TKA confirmed bactericidal activity, especially at doubled MCB-detected concentrations, indicating dose-dependent activity. The significant reduction in the minimum inhibitory concentration in the combination indicated its potential for dose optimization, minimizing COL-associated toxicities. Genotypic profiling showed that the expression of blaNDM and blaOxa-48 can reduce synergy. These findings, obtained with isolates of high resistance, support the efficacy of this combination therapy and could reduce the dose-related side effects of COL. However, they also highlight genotype-specific variations and COL resistance mechanisms as limiting variables. Full article
(This article belongs to the Special Issue Bacterial Resistance and Novel Therapeutic Approaches)
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47 pages, 694 KB  
Review
The Taxonomy of the Genus Entamoeba (Archamoebea: Endamoebidae): A Historical and Nomenclatural Review
by Lorena Esteban-Sánchez, Rafael Alberto Martínez-Díaz and Francisco Ponce-Gordo
Pathogens 2026, 15(2), 213; https://doi.org/10.3390/pathogens15020213 - 13 Feb 2026
Cited by 1 | Viewed by 766
Abstract
Throughout history, species within the genus Entamoeba have been described using criteria that were not always applied consistently, resulting in an often confusing and controversial taxonomy. Several factors contributed to this situation, including the limited number of morphological characters available for taxonomic studies, [...] Read more.
Throughout history, species within the genus Entamoeba have been described using criteria that were not always applied consistently, resulting in an often confusing and controversial taxonomy. Several factors contributed to this situation, including the limited number of morphological characters available for taxonomic studies, overlapping host ranges, mixed infections, and a cosmopolitan distribution associated with human and animal movements. The incorporation of genetic data as diagnostic and differential criteria during the second half of the twentieth century enabled the recognition of cryptic species and the proposal of new taxa; however, significant taxonomic issues remain unresolved. This review summarizes the historical development and major controversies in the taxonomy of Entamoeba, from its origins in the late nineteenth century, when morphology and host association were the available criteria, to the present day, in which molecular approaches provide a more realistic view of species diversity and interspecific relationships. Based on this analysis, general principles are proposed as a pragmatic synthesis to guide future taxonomic work on Entamoeba, emphasising lineage-based species delimitation, the central role of molecular evidence when diagnostic morphology is lacking, the contextual value of host data, and the need for nomenclatural decisions grounded in biological evidence and historical rigour. Full article
(This article belongs to the Section Parasitic Pathogens)
10 pages, 1361 KB  
Article
Development of a Multiplex Droplet Digital PCR Assay for Detection of Tick-Borne Pathogens
by Lijuan He, Lin Zhang, Like Duan, Xuexia Hou, Jingwen Li and Qin Hao
Pathogens 2026, 15(2), 212; https://doi.org/10.3390/pathogens15020212 - 13 Feb 2026
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Abstract
Tick-borne pathogens pose a significant threat to human health. In this study, a multiple droplet digital PCR (ddPCR) assay was developed to detect four tick-borne pathogens: Borrelia burgdorferi sensu lato (Bbsl), Coxiella burnetii (C. burnetii), spotted fever group Rickettsia [...] Read more.
Tick-borne pathogens pose a significant threat to human health. In this study, a multiple droplet digital PCR (ddPCR) assay was developed to detect four tick-borne pathogens: Borrelia burgdorferi sensu lato (Bbsl), Coxiella burnetii (C. burnetii), spotted fever group Rickettsia (SFGR), and Borrelia miyamotoi (B. miyamotoi). Based on the singleplex ddPCR reaction system of Bbsl, the primer probes of the other three species were incorporated to develop a multiplex ddPCR reaction system. The annealing temperature and the final concentration of the primer probes were then optimized for multiplex ddPCR. The multiplex ddPCR assay was assessed for its sensitivity, specificity, repeatability, and ability to detect simulated and actual samples. The developed multiplex ddPCR approach enables the simultaneous detection of Bbsl, C. burnetii, SFGR, and B. miyamotoi. The positive target microtitre clusters are closely grouped and distinctly separated from each other, with the multiplex ddPCR assay demonstrating a dynamic range of five orders of magnitude. The limits of detection (LOD) for the multiplex ddPCR assay were 4 copies/20 µL for Bbsl, 3 copies/20 µL for C. burnetii, 3 copies/20 µL for SFGR, and 2 copies/20 µL for B. miyamotoi. The assay demonstrated high specificity, with no observed cross-reactivity against non-target pathogens. Performance was validated using both spiked samples and field-collected clinical specimens. In the evaluation of 30 ticks and 30 serum samples, the ddPCR method (in both singleplex and multiplex formats) achieved higher positive detection rates for all four target pathogens compared to quantitative real-time PCR (qPCR). In addition, the detection proportions of multiplex and singleplex ddPCR were consistent. Multiplex ddPCR can detect low DNA concentrations in samples and enables the absolute quantification of Bbsl, C. burnetii, SFGR, and B. miyamotoi, providing a novel detection approach for the clinical diagnosis of tick-borne diseases. Full article
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13 pages, 974 KB  
Article
A Nested PCR-Based Point of Care Testing of Multiplex Pathogens Associated with Bloodstream Infection
by Shihao Jiao, Juntao Meng, Jianing Wu, Duoxiao Zhang, Xingyu Liu, Zhiqiang Han, Yuxin Wang, Shijue Gao, Zijin Zhao, Yujie Xiang, Junkai Ren, Qian Ma, Xinxin Li, Xinxin Shen, Xuejun Ma and Yanqing Tie
Pathogens 2026, 15(2), 211; https://doi.org/10.3390/pathogens15020211 - 13 Feb 2026
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Abstract
Bloodstream infections (BSI) carry high mortality, but traditional blood culture is too slow for urgent clinical needs. This study aims to develop a rapid point-of-care testing assay based on one-tube multiplex nested polymerase chain reaction (PCR) (OM-NPCR-POCT) for early diagnosis of three pathogens [...] Read more.
Bloodstream infections (BSI) carry high mortality, but traditional blood culture is too slow for urgent clinical needs. This study aims to develop a rapid point-of-care testing assay based on one-tube multiplex nested polymerase chain reaction (PCR) (OM-NPCR-POCT) for early diagnosis of three pathogens in bloodstream infection patients: Escherichia coli (ECO), Klebsiella pneumoniae (KPN), and Streptococcus pneumoniae (SPN). The analytical sensitivity of the one-tube multiplex nested PCR (OM-NPCR) was evaluated using recombinant bacterial plasmids. The analytical sensitivity of the OM-NPCR-POCT assay was assessed using simulated samples. The clinical performance was evaluated in 90 clinical blood samples, with results compared to conventional quantitative PCR (qPCR). Finally, the agreement between the two detection methods was assessed via Kappa analysis. The limits of detection (LODs), calculated from serial dilution experiments, were 4, 2, and 1 copies/μL for plasmids ECO, KPN, and SPN, respectively. The OM-NPCR-POCT assay achieved LODs of 20, 10, and 10 CFU/mL for ECO, KPN, and SPN in simulated samples, with a total testing time of approximately 2 h. The clinical evaluation of OM-NPCR-POCT demonstrates consistency with conventional qPCR while exhibiting higher sensitivity. This method has potential as a rapid diagnostic tool for early bloodstream infection detection. Full article
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16 pages, 310 KB  
Communication
Developing an Integrated Medical-Veterinary Data Framework for Investigating Human Toxoplasmosis: A One Health Perspective
by Anna Maria Fausta Marino, Renato Paolo Giunta, Antonio Salvaggio, Vincenzo Agozzino, Alessandra Aparo and Maurizio Percipalle
Pathogens 2026, 15(2), 210; https://doi.org/10.3390/pathogens15020210 - 13 Feb 2026
Viewed by 521
Abstract
Toxoplasmosis is an insidious globally distributed zoonosis accounting for approximately one-fifth of all foodborne illnesses in humans in Europe. It stands as a classic example of a disease transmitted through the close interconnection between humans, animals, and the environment. Accordingly, mitigation strategies and [...] Read more.
Toxoplasmosis is an insidious globally distributed zoonosis accounting for approximately one-fifth of all foodborne illnesses in humans in Europe. It stands as a classic example of a disease transmitted through the close interconnection between humans, animals, and the environment. Accordingly, mitigation strategies and health management protocols demand cross-sectoral involvement from medical, veterinary, environmental, and political actors, rendering the adoption of a ‘One Health’ perspective essential. Despite longstanding advocacy for One Health by the WHO, WOAH, and FAO, national health authorities have yet to establish the necessary operational infrastructure. Specifically, there is a lack of tools to enable information sharing among professionals, which is essential for the synergistic management of major health issues. A four-part epidemiological data collection tool specifically developed for human toxoplasmosis is proposed here to aid in the identification of risk factors and potential sources of infection. The proposed framework comprises four sequential sections intended to be completed through contribution from the patient, the attending physician, the involved veterinarian, and the national reference laboratory for toxoplasmosis. This questionnaire serves as a conceptual, non-validated tool designed to support a One Health–oriented epidemiological investigation process. Its practical performance, feasibility, and potential usefulness in routine surveillance or prevention strategies have not yet been assessed and will require validation in future studies. Nonetheless, the framework may serve as a model for the development of similar integrative tools applicable to other zoonotic diseases. Full article
(This article belongs to the Section Epidemiology of Infectious Diseases)
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