Pathogens

33 pages, 8691 KB

Open AccessReview

Virulence and Resistance Mechanisms in Multidrug-Resistant Acinetobacter baumannii

by Priya Rajendran, Rameshkumar Marimuthu Ragavan, Renuka James, Bindu Dhanapal, Mullai Venkatachalam, Jeevarahini Reghupathy and Ramachandran Vignesh

Pathogens 2026, 15(8), 798; https://doi.org/10.3390/pathogens15080798 - 28 Jul 2026

Abstract

Acinetobacter baumannii, a Gram-negative opportunistic bacterium in the ESKAPE group (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, A. baumannii, Pseudomonas aeruginosa and Enterobacter spp.), has emerged as a leading cause of nosocomial infections worldwide. It is known to [...] Read more.

Acinetobacter baumannii, a Gram-negative opportunistic bacterium in the ESKAPE group (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, A. baumannii, Pseudomonas aeruginosa and Enterobacter spp.), has emerged as a leading cause of nosocomial infections worldwide. It is known to possess diverse virulence traits and antimicrobial resistance, making it a critical priority pathogen on the World Health Organization’s 2024 Bacterial Priority Pathogens List. Carbapenem-resistant A. baumannii (CRAB) is currently endemic across several continents, with global carbapenem resistance exceeding 70% in healthcare settings and multidrug-resistant infections being associated with alarming mortality rates. This review comprehensively discusses the molecular underpinnings of A. baumannii pathogenesis and virulence, detailing the array of factors coordinated by complex regulatory networks. The convergence of this pathogen’s virulence and antimicrobial resistance traits, resulting in multidrug resistance, leaves clinicians with only a handful of therapeutic options. The review also discusses upcoming therapeutic strategies, including phage therapy, antimicrobial peptides, monoclonal antibodies, photodynamic therapy, and vaccine candidates in the pipeline. While emerging therapeutics show promise, several challenges remain, and integrated approaches are warranted to efficiently combat A. baumannii’s virulence and resistance armamentarium. Full article

(This article belongs to the Special Issue Virulence and Resistance Mechanisms in Multidrug-Resistant Gram-Negative Bacteria: 2nd Edition)

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30 pages, 1463 KB

Open AccessArticle

Species Distribution of Candida Isolates in Amniotic Fluid, Vaginal Discharge, and Lochial Cultures in Obstetric and Gynecological Patients: A Retrospective Single-Center Study

by Silvia Gabriela Ionescu, Elena Teona Cosovanu, Cristina Daniela Dimitriu, Costin Damian, Diana Costin, Ancuta Goriuc, Elena Porumb Andrese, Demetra Gabriela Socolov, Mihaela Grigore, Luminita Smaranda Iancu, Irina Draga Caruntu and Ramona Gabriela Ursu

Pathogens 2026, 15(8), 797; https://doi.org/10.3390/pathogens15080797 - 28 Jul 2026

Abstract

(1) Background: Candida-positive cultures in pregnancy and the postpartum period are frequently identified in obstetric and gynecological practice. This study evaluated species distribution and associated maternal and obstetric variables in amniotic fluid, vaginal discharge, and lochial cultures. (2) Methods: A retrospective, single-center, [...] Read more.

(1) Background: Candida-positive cultures in pregnancy and the postpartum period are frequently identified in obstetric and gynecological practice. This study evaluated species distribution and associated maternal and obstetric variables in amniotic fluid, vaginal discharge, and lochial cultures. (2) Methods: A retrospective, single-center, cross-sectional comparative study was conducted at the “Cuza Vodă” Clinical Hospital of Obstetrics and Gynecology, Iași, including Candida-positive samples collected in 2024 (242 amniotic fluid, 2020 vaginal discharge, and 827 lochial isolates). Fungal identification was performed using Sabouraud and chromogenic media, with species-level identification in most cases and classification as Candida spp. when differentiation was not possible. (3) Results: Candida albicans predominated across all compartments (69.8%, 55.35%, and 66.9%, respectively), followed by non-albicans species, mainly Pichia kudriavzevii. Vaginal discharge cultures, obtained from both pregnant and non-pregnant women and analyzed after stratification by pregnancy status, showed multiple significant associations with maternal and obstetric variables; apparent associations with fetal variables were largely attributable to the inclusion of non-pregnant women and were not retained within the pregnant stratum. Amniotic fluid and lochial cultures demonstrated limited associations. Older maternal age was the only consistent independent correlate of non-albicans Candida isolation, whereas the remaining associated variables were compartment-specific. Multivariable models identified compartment-specific correlates of non-albicans Candida isolation but showed limited discriminatory performance, with low sensitivity in most strata. (4) Conclusions: Candida species distribution is compartment-dependent, with greater clinical heterogeneity in vaginal discharge and more limited patterns in amniotic fluid and lochia. These culture-positive findings support species-level identification for microbiological surveillance and provide perinatal microbiological context for future integrated maternal–neonatal studies; because antifungal susceptibility and neonatal outcomes were not assessed, no therapeutic or neonatal inferences can be drawn. Full article

(This article belongs to the Special Issue Advances in Fungal Pathogenesis and Antifungal Resistance)

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12 pages, 1010 KB

Open AccessArticle

District-Level Risk Mapping of Highly Pathogenic Avian Influenza in Poultry in Kazakhstan Using a Multi-Criteria Decision Model

by Aizada A. Mukhanbetkaliyeva, Irene Iglesias Martin, Fedor I. Korennoy, Alimzhan S. Kadyrov, Yersyn Y. Mukhanbetkaliyev, Saidulla I. Ruzmatov, Asem Zh. Abenova, Temirlan G. Bakishev, Andres M. Perez and Sarsenbay K. Abdrakhmanov

Pathogens 2026, 15(8), 796; https://doi.org/10.3390/pathogens15080796 - 27 Jul 2026

Abstract

Highly pathogenic avian influenza (HPAI) is a transboundary disease of birds with zoonotic potential. Since 2021, H5 HPAI virus variants have spread globally, with migratory waterfowl playing a key role in transcontinental dissemination. Kazakhstan lies at the intersection of major flyways, creating a [...] Read more.

Highly pathogenic avian influenza (HPAI) is a transboundary disease of birds with zoonotic potential. Since 2021, H5 HPAI virus variants have spread globally, with migratory waterfowl playing a key role in transcontinental dissemination. Kazakhstan lies at the intersection of major flyways, creating a persistent risk of virus introduction into domestic poultry. While HPAI risk in wild birds has been explored in Kazakhstan, the spatial risk for poultry has remained underassessed. Here, we present a risk map for HPAI in poultry across 174 administrative districts of Kazakhstan, using a multi-criteria decision analysis (TOPSIS) that integrates five quantitative risk indicators that relate to wild bird habitat, virus survival in the environment and poultry farm census. The obtained district-level risk index agreed well with historical outbreak data (2005–2025), with an area under the receiver operating characteristic (ROC) curve of 0.899 (95% CI: 0.846–0.952). Most districts (56%) were at negligible risk, whereas medium- and high-risk clusters occurred in the north, central, and southern regions, and near the Caspian Sea. These findings provide an operational, spatially explicit tool for targeting surveillance and biosecurity measures to high-risk areas, ultimately helping to mitigate the impact of one of the most devastating transboundary poultry diseases in Central Asia. Full article

(This article belongs to the Special Issue New Insights into Viral Infections of Domestic Animals)

36 pages, 1689 KB

Open AccessReview

Prevention and Treatment of Staphylococcus aureus Biofilms Using Promising Agr-QS-Targeting Anti-Biofilm Agents

by Salma Waheed Sheikh, Ahmad Ali, Asma Ahsan, Fei Shang, Ting Xue and Lauren Gollahon

Pathogens 2026, 15(8), 795; https://doi.org/10.3390/pathogens15080795 - 27 Jul 2026

Abstract

Staphylococcus aureus (S. aureus), a leading cause of nosocomial infections, contributes significantly to increased morbidity and mortality, especially when it forms biofilms on medical devices. This pathogen, specifically methicillin-resistant S. aureus (MRSA), remains a challenge to treat due to its ability [...] Read more.

Staphylococcus aureus (S. aureus), a leading cause of nosocomial infections, contributes significantly to increased morbidity and mortality, especially when it forms biofilms on medical devices. This pathogen, specifically methicillin-resistant S. aureus (MRSA), remains a challenge to treat due to its ability to form biofilms and rapidly develop resistance against antibiotics. Biofilm formation allows bacteria to adhere to biotic and abiotic surfaces, creating a protective matrix that shields them from immune responses and antibiotic therapies. The widespread prevalence of multidrug-resistant S. aureus biofilms poses a significant therapeutic challenge in clinical settings. Several novel therapeutic strategies have been developed to combat S. aureus biofilm-associated infections. Accumulating evidence suggests that natural plants and their derivatives possess antimicrobial and chemo preventive properties that can disrupt established biofilms. Several plant-derived compounds with anti-biofilm activities have been reported to target the regulatory proteins involved in the Agr quorum sensing (Agr-QS) system, underscoring their potential as therapeutic candidates for the prevention and treatment of biofilm-associated infections. However, despite these encouraging findings, clinical validation of these plant-based agents is essential to ensure their efficacy, safety, and optimal application in treating S. aureus biofilm infections. The continued exploration of natural biofilm inhibitors anticipates the urgent need for new treatments to combat biofilm-associated infections and multidrug-resistant pathogens like MRSA. This review provides a detailed overview of preventive and therapeutic interventions to eradicate biofilm-forming S. aureus infections. Full article

(This article belongs to the Section Bacterial Pathogens)

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10 pages, 414 KB

Open AccessArticle

Evaluation of an Automated Cartridge-Based PCR Assay for the Detection of Leishmania spp. DNA in Canine Lymph Node Samples

by Eva Spada, Francesca Di Gaudio, Germano Castelli, Federica Bruno, Roberta Perego, Luciana Baggiani, Vito Biondi, Fabrizio Vitale, Michela Tognoni and Daniela Proverbio

Pathogens 2026, 15(8), 794; https://doi.org/10.3390/pathogens15080794 - 27 Jul 2026

Abstract

An automated cartridge-based Vcheck M Canine Vector 8 Panel for qualitative detection of Leishmania spp. DNA in canine lymph node aspirates, an off-label specimen type, using laboratory qPCR as the comparator, was evaluated. Fifty-seven residual lymph node aspirate suspensions from dogs investigated for [...] Read more.

An automated cartridge-based Vcheck M Canine Vector 8 Panel for qualitative detection of Leishmania spp. DNA in canine lymph node aspirates, an off-label specimen type, using laboratory qPCR as the comparator, was evaluated. Fifty-seven residual lymph node aspirate suspensions from dogs investigated for suspected canine leishmaniosis (CanL) were tested. Reference qPCR detected L. infantum DNA in 29 samples. Vcheck M was positive in 22/29 qPCR-positive samples and negative in 28/28 qPCR-negative samples, corresponding to positive percent agreement/sensitivity of 75.9% (95% CI, 56.5–89.7) and negative percent agreement/specificity of 100.0% (95% CI, 87.7–100.0). Agreement was substantial (Cohen’s kappa, 0.76), and discordance was asymmetric (McNemar p = 0.016). Vcheck-negative/qPCR-positive results were mainly observed at low qPCR parasite loads: 6/7 discordant samples contained ≤30 parasites/mL, whereas all samples with ≥500 parasites/mL were Vcheck positive. Among Vcheck-positive clinical samples, Vcheck Ct correlated inversely with log10 qPCR parasite load (Spearman rho = −0.77; p < 0.001). In a single-run dilution series, the lowest instrument-positive L. infantum standard was 10³ parasites/mL. Purified L. major, L. braziliensis, and L. tropica DNA were also detected. Vcheck M showed high specificity as a rapid rule-in test for Leishmania spp. detection in canine lymph node aspirates. However, negative results should not exclude infection in symptomatic or strongly suspected dogs and should be confirmed by qPCR, particularly when low parasite burden is plausible or when the result is critical for diagnostic or therapeutic decision-making. Full article

(This article belongs to the Section Parasitic Pathogens)

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31 pages, 8239 KB

Open AccessArticle

Molecular Insights from Differential Proteomic Profiling of Premalignant Cervical Lesions and Cervical Cancer

by Diana Laura Gonzalez-Tolentino, Olga Lilia Garibay-Cerdenares, Sergio Encarnación-Guevara, Ángel Gabriel Martínez-Batallar, Ramiro Alonso-Bastida, Jeovanis Gil, Jorge Organista-Nava, Luz del Carmen Alarcón-Romero, Marco Antonio Leyva-Vázquez and Berenice Illades-Aguiar

Pathogens 2026, 15(8), 793; https://doi.org/10.3390/pathogens15080793 - 26 Jul 2026

Abstract

Cervical cancer (CC) affects women worldwide, and more than 95% of cases are caused by persistent infection with high-risk human papillomavirus (HR-HPV), such as type 16, which promotes the progression of precancerous lesions to cancer. This study aimed to identify differentially expressed proteins [...] Read more.

Cervical cancer (CC) affects women worldwide, and more than 95% of cases are caused by persistent infection with high-risk human papillomavirus (HR-HPV), such as type 16, which promotes the progression of precancerous lesions to cancer. This study aimed to identify differentially expressed proteins (DEPs) in biopsies from patients with HPV16+ low-grade squamous intraepithelial lesions (LSILs) and from patients with HPV16+ squamous cell carcinoma (SCC) compared with those from HPV-negative normal cervical tissue (NCT HPV−) controls. The samples were analyzed by high-performance liquid chromatography–tandem mass spectrometry (HPLC-MS/MS) using a data-independent acquisition (DIA) approach. Data processing and differential protein expression analysis were performed with the DIA-NN software (Data-Independent Acquisition Neural Networks), followed by bioinformatics analyses, including Venn diagrams, pathway enrichment, functional interactome, The Cancer Genome Atlas (TCGA)-SCC data integration, and Western blot detection. In total, 1607 DEPs associated with cell adhesion and extracellular matrix proteins were identified in LSILs, whereas 1516 DEPs associated with catalytic and transport activities were identified in SCC; the proteins overexpressed in LSILs (332) were enriched in processes such as metabolism, immune response activation, and stress and cell death responses. In contrast, proteins overexpressed in SCC (205) were associated with the cell cycle, DNA damage, drug metabolism, proteasome degradation, methylation, and immune response. Interaction analyses highlighted proteins related to early proteins 1,5,6 and 7 (E1, E5, E6, and E7). In terms of the two DEPs, S100 calcium binding protein A10 (S100A10/p11) and thymidine phosphorylase (TYMP) were detected in patients with LSIL, HSIL, and SCC at the protein level, consistent with their higher transcript levels in public datasets. Given the small, exploratory cohort, these findings are hypothesis-generating, and validation in a larger, balanced, independent cohort is required. In conclusion, this study identified DEPs associated with the progression of premalignant lesions to SCC that may represent candidate biomarkers and therapeutic targets warranting further investigation. Full article

(This article belongs to the Special Issue Recent Advances in Human Papillomavirus Research)

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14 pages, 3297 KB

Open AccessArticle

Leishmaniasis Incidence and Leishmania Species Distribution in the Apurímac, Ene and Mantaro River Valley, a High-Transmission Endemic Area of Peru

by Jime Rivera-Villar, Nyshon Rojas-Palomino, José Alarcón-Guerrero, Víctor Cárdenas-López, Rilder Gastelú-Quispe, Aide Sandoval-Juarez and Saúl Chuchón-Martínez

Pathogens 2026, 15(8), 792; https://doi.org/10.3390/pathogens15080792 - 25 Jul 2026

Abstract

Background: Leishmaniasis remains a major public health problem in Peru, and the Apurímac, Ene and Mantaro River Valley (VRAEM) is an endemic area where, despite its epidemiological relevance, information on incidence and circulating Leishmania species remains limited. Methods: We estimated district-level cumulative incidence [...] Read more.

Background: Leishmaniasis remains a major public health problem in Peru, and the Apurímac, Ene and Mantaro River Valley (VRAEM) is an endemic area where, despite its epidemiological relevance, information on incidence and circulating Leishmania species remains limited. Methods: We estimated district-level cumulative incidence of leishmaniasis in the 60 districts of the VRAEM during 2015–2024, using national surveillance data and census population figures. In addition, molecular species typing was performed on 112 Giemsa-stained tissue smears obtained in health facilities located in Ayacucho districts belonging to the VRAEM region. Results: A total of 3589 cases were reported, yielding an estimated annual incidence rate of 80.01 cases per 100,000 inhabitants, which is 4.3-fold higher than the national average. Marked spatial heterogeneity was observed, with districts such as Pangoa, Río Tambo, Llochegua, and Pichari reporting estimated annual incidence rates above 130 cases per 100,000 inhabitants. Furthermore, of 55 samples processed by High-Resolution Melting Analysis, Leishmania braziliensis was found in 54.6% of samples, followed by Leishmania guyanensis in 21.8%. Conclusions: The VRAEM constitutes a high-transmission endemic focus of tegumentary leishmaniasis with marked inter-district heterogeneity and circulation of multiple Leishmania species, with Leishmania braziliensis as the predominant species. These findings support the need for targeted surveillance and species-informed clinical management strategies. Full article

(This article belongs to the Special Issue Leishmania & Leishmaniasis)

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33 pages, 2809 KB

Open AccessArticle

Analytical Validation and Preliminary Diagnostic Performance Evaluation of GenoPATHX™ Multiplex qPCR for Quantitative Detection of Key Salmonella Serovars in Poultry Matrices

by Rejoice Nyarku, Emmanuel Kuufire, Viona Osei, Kingsley E. Bentum, Asmaa Elrefaey, Emmanuel Piiru, Tyric James, Yilkal Woube, Temesgen Samuel and Woubit Abebe

Pathogens 2026, 15(8), 791; https://doi.org/10.3390/pathogens15080791 - 25 Jul 2026

Abstract

Rapid detection and quantification of epidemiologically important Salmonella enterica serovars are critical for poultry surveillance, food safety monitoring, and risk-based intervention strategies. This study performed comprehensive analytical validation together with a preliminary field-based diagnostic performance evaluation of GenoPATHX™, a multiplex probe-based qPCR platform [...] Read more.

Rapid detection and quantification of epidemiologically important Salmonella enterica serovars are critical for poultry surveillance, food safety monitoring, and risk-based intervention strategies. This study performed comprehensive analytical validation together with a preliminary field-based diagnostic performance evaluation of GenoPATHX™, a multiplex probe-based qPCR platform designed for the simultaneous detection and quantification of priority Salmonella serovars in poultry-associated matrices. The platform consists of two multiplex panels, designated the Chicken Key Performance Indicator (CKPI) and Turkey Key Performance Indicator (TKPI), each designed to detect priority poultry-associated Salmonella serovars together with a genus-level S. enterica marker. Analytical performance was evaluated for amplification efficiency, linearity, limit of detection (LoD₉₅), limit of quantification (LoQ), repeatability, intermediate precision, analytical specificity (inclusivity/exclusivity), robustness, matrix effects, and performance in artificially inoculated matrices. Diagnostic performance was further assessed using naturally contaminated poultry environmental samples. The assay demonstrated robust amplification performance in both singleplex and multiplex formats, with high linearity (R² = 0.987–0.999) and LoD₉₅ values ranging from 60 to 545 genome equivalents per reaction. Complete analytical inclusivity and high exclusivity were achieved for the evaluated isolate panel. In field samples, the direct GenoPATHX™ workflow demonstrated 81.0% sensitivity, 91.3% specificity, and substantial agreement with the USDA-FSIS reference culture method (κ = 0.73). Overall, GenoPATHX™ exhibited robust analytical performance and enabled rapid, same-day quantitative detection of priority Salmonella serovars in poultry-associated matrices, supporting its application for poultry surveillance and food safety monitoring. Full article

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19 pages, 1041 KB

Open AccessArticle

Molecular Characterization and Prevalence of Trypanozoon Infection in Livestock on Small-Scale Farms in Thailand: An Integrative Control Approach

by Apiraya Rudeekiatthamrong, Pairpailin Jhaiaun, Giang Thi Nguyen, Wissanuwat Chimnoi, Darunwan Chuensaengarun, Chattraporn Rungchalermlak, Tanapat Mutchimadilok, Nipa Thammasonthijarern, Thawijit Phannithi, Tawanhathai Apichaimongkonkun, Laphatsararach Apinantanakorn, Kanittha Phetudomsinsuk, Tawin Inpankaew, Burin Nimsuphan, Ruttayaporn Ngasaman, Nuttapon Manojai, Jumnongjit Phasuk and Ketsarin Kamyingkird

Pathogens 2026, 15(8), 790; https://doi.org/10.3390/pathogens15080790 - 24 Jul 2026

Abstract

Trypanosomosis (Surra), caused by the hemoflagellate protozoan Trypanosoma evansi, has a negative impact on animal health and the livestock economy in many countries, including Thailand. Molecular epidemiological data and integrative control approaches for small-scale farms remain limited. This study determined the prevalence [...] Read more.

Trypanosomosis (Surra), caused by the hemoflagellate protozoan Trypanosoma evansi, has a negative impact on animal health and the livestock economy in many countries, including Thailand. Molecular epidemiological data and integrative control approaches for small-scale farms remain limited. This study determined the prevalence of Trypanozoon infection and its associated risk factors in Thai livestock under an integrative control approach. Molecular tools were used to characterize T. evansi typing. A total of 947 blood samples were collected from cattle and buffalo across 123 small-scale farms in 16 provinces covering four regions of Thailand, with diagnostic results and control recommendations communicated to farmers within 14 days. The ITS2-positive samples were then subjected to further molecular characterization, together with two dogs, seven horses, and an in vitro T. evansi isolate, comprising 40 Trypanozoon-positive samples. These were characterized using PCR targeting the TBR primer, maxicircle NADH5, VSG RoTat 1.2, and minicircle B, followed by ITS2-based phylogenetic analysis. Risk factors were assessed using Chi-square and Fisher’s exact tests, logistic regression, a generalized linear mixed model, and Firth’s penalized logistic regression. The overall prevalence of Trypanozoon infection was 3.17% (30/947), detected exclusively in beef cattle in the Northern region (15.30%; 30/196), specifically in Lampang (46.15%, 12/26), Tak (23.08%, 3/13), and Phrae (17.24%, 15/87) Provinces. No infections were reported in the Northeastern, Central, or Southern regions. Male sex, age under one year, and residence in the Northern region were significant individual-level risk factors, while farm-level lacrimation and the presence of Stomoxys were significantly associated with infection. Molecular characterization identified T. evansi type A and type non-A/B, with no type B or maxicircle-positive samples detected. Phylogenetic analysis grouped the Thai isolates with previously reported isolates from Thai deer and cattle, Iranian camels, and Colombian dogs. On follow-up, no Trypanozoon infection was detected in revisited farms. These findings confirmed that T. evansi, comprising types A and non-A/B, continues to circulate in Thai livestock, particularly in beef cattle in Northern Thailand. Data from this study can be used for surveillance and future development of more effective diagnostic systems. Full article

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23 pages, 6391 KB

Open AccessArticle

First Isolation, Identification, and Whole-Genome Sequencing of a Multidrug-Resistant Bovine-Derived Providencia stuartii in China

by Rong-Jun Gong, Xue-Li Ge, Jia-Min Ma, Yang-Sini Fu, Jia-Hao Chen, Man-Ting Li, Yong-Xiang Zhao, Liang Zhu, Qing-Hong Guo, Xin-Chao Liu and Wen-Chao Li

Pathogens 2026, 15(8), 789; https://doi.org/10.3390/pathogens15080789 - 24 Jul 2026

Abstract

Background: Providencia stuartii is an opportunistic pathogen associated with multidrug resistance. However, its occurrence, genomic characteristics, antimicrobial resistance profiles, and pathogenic potential in cattle-associated isolates remain poorly understood. Methods: A P. stuartii strain was isolated from a rectal swab of a diarrheic beef [...] Read more.

Background: Providencia stuartii is an opportunistic pathogen associated with multidrug resistance. However, its occurrence, genomic characteristics, antimicrobial resistance profiles, and pathogenic potential in cattle-associated isolates remain poorly understood. Methods: A P. stuartii strain was isolated from a rectal swab of a diarrheic beef cattle individual during bacterial investigation on a commercial farm in Anhui Province, China. The strain was identified by Gram staining, biochemical tests, 16S rRNA sequencing, and whole-genome sequencing. Antimicrobial susceptibility was assessed using the Kirby–Bauer method, and resistance and virulence genes were detected by PCR and genomic analysis. Pathogenicity was evaluated in a murine infection model. Results: The isolate was confirmed as P. stuartii, with a 4.22 Mb genome and 41.28% GC content. It showed multidrug resistance, including resistance to eight antimicrobial agents. Genomic analysis revealed multiple resistance and virulence determinants, genomic islands, prophages, and CRISPR regions. Under high-dose intraperitoneal challenge conditions, the isolate caused dose-dependent mortality, systemic recovery from major organs, and histopathological lesions in mice. Conclusions: This study reports the genomic and antimicrobial resistance characteristics of a multidrug-resistant P. stuartii strain recovered from a rectal swab of diarrheic beef cattle in China. High-dose intraperitoneal inoculation demonstrated that the isolate possessed pathogenic potential in mice under experimental conditions. However, the murine model did not reproduce natural exposure in cattle, and the findings do not establish bovine tissue invasion, clinical pathogenicity, or a causal role in diarrhea. Further epidemiological investigations and cattle-relevant experimental studies are required to determine the clinical significance of this organism in bovine populations. Full article

(This article belongs to the Section Bacterial Pathogens)

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18 pages, 5415 KB

Open AccessArticle

A Novel Real-Time SYBR Green PCR Assay for Integron Profiling in Pseudomonas aeruginosa: Unveiling the Resistance Nexus Between Food and Clinical Isolates

by Hend Emad, Mahmoud M. Amer, Munirah M. Alhammadi, Mona Alharbi, Yasmen F. Mahran, Attia A. Attia, Khalid Maniah and Azza SalahEldin El-Demerdash

Pathogens 2026, 15(8), 788; https://doi.org/10.3390/pathogens15080788 - 24 Jul 2026

Abstract

Background: The dissemination of extensively drug-resistant Pseudomonas aeruginosa strains across the human–food interface represents a critical threat to global public health, complicating empirical therapeutic choices and challenging traditional nosocomial restriction paradigms. Objective: This study investigated the prevalence, phenotypic resistome landscapes, and underlying integron-mediated [...] Read more.

Background: The dissemination of extensively drug-resistant Pseudomonas aeruginosa strains across the human–food interface represents a critical threat to global public health, complicating empirical therapeutic choices and challenging traditional nosocomial restriction paradigms. Objective: This study investigated the prevalence, phenotypic resistome landscapes, and underlying integron-mediated mobilization mechanics of P. aeruginosa across the One Health continuum using an optimized real-time quantitative PCR (qPCR) platform. Methods: A total of 116 samples comprising human clinical specimens (n = 61) and retail food matrices (n = 55) were screened. Phenotypic resistance was profiled against 16 antipseudomonal agents, with polymyxin and colistin resistance validated via reference broth microdilution. A novel real-time SYBR Green qPCR assay backed by derivative melting curve analysis was developed to track Class 1 (intI1) and Class 2 (intI2) integrons. Results: Pseudomonas aeruginosa was recovered from 36.2% (n = 42/116) of total samples, exhibiting a homogeneous distribution across clinical (32.8%) and retail food matrices (40.0%, p = 0.445). Antibiogram characterization revealed a critical resistance burden: 100% of isolates fell into extreme epidemiological tiers, with 71.4% (n = 30) exhibiting an extensively drug-resistant (XDR) phenotype and 28.6% (n = 12) reaching absolute pandrug-resistant (PDR) status. Absolute phenotypic resistance (100%) was recorded across both cohorts for meropenem, ceftazidime, aztreonam, tobramycin, polymyxins, and novel combination therapeutics (ceftazidime–avibactam and ceftolozane–tazobactam). Pairwise tracking demonstrated an exceptionally strong positive correlation in resistance distribution patterns between human and food isolates (Spearman’s rho = 0.948, p < 0.001). Analytical validation of the qPCR assay demonstrated tight, reproducible specific derivative melting peaks (T_m) at 83.2 °C for intI1 and 83.8 °C for intI2. Genotypic screening revealed universal conservation of plasmid-borne intI1 (100%), whereas plasmidic intI2 was variably distributed (61.9%). Crucially, intI2 carriage served as a definitive marker for the transition to complete pandrug resistance, being detected in 100% (n = 12/12) of PDR isolates compared to 46.7% (n = 14/30) of XDR strains (p = 0.001), supported by a strong positive correlation with the PDR phenotype (rho = 0.496, p = 0.0008). Conclusions: These findings reveal an overlapping, extreme resistome across the food-clinical interface and identify intI2 as a genetic tipping point for absolute pandrug resistance. The validated real-time assay provides a highly sensitive, proactive molecular surveillance framework capable of mapping high-risk mobile genetic platforms before they manifest as catastrophic empirical treatment failures. Full article

(This article belongs to the Section Bacterial Pathogens)

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9 pages, 597 KB

Open AccessCommunication

Establishment of an HSV-1 Mouse Model with Cutaneous Lesions

by Hye-Myung Ryu, Bushra Riaz and Seonghyang Sohn

Pathogens 2026, 15(8), 787; https://doi.org/10.3390/pathogens15080787 - 24 Jul 2026

Abstract

Herpes simplex virus type 1 (HSV-1) causes recurrent mucocutaneous lesions, yet existing animal models incompletely recapitulate characteristic skin manifestations. Here, we established a mouse model of cutaneous HSV-1 infection by combining epithelial barrier disruption with localized viral inoculation. Superficial scarification of the ear [...] Read more.

Herpes simplex virus type 1 (HSV-1) causes recurrent mucocutaneous lesions, yet existing animal models incompletely recapitulate characteristic skin manifestations. Here, we established a mouse model of cutaneous HSV-1 infection by combining epithelial barrier disruption with localized viral inoculation. Superficial scarification of the ear followed by HSV-1 exposure resulted in consistent lesion formation. Administration of hydrocortisone further increased the incidence of lesions, highlighting the critical roles of epithelial integrity and host immune regulation in the pathogenesis of HSV-1 infection. Using this model, we evaluated the antiviral efficacy of Acyclovir. Treatment significantly reduced lesion severity, lesion size, and viral gene expression, indicating partial suppression of viral replication. Notably, acyclovir treatment was associated with increased expression of T-bet and Foxp3 in lymphoid tissues, suggesting modulation of both effector and regulatory immune responses. Collectively, this model successfully reproduces localized HSV-1 skin lesions, serving as a useful platform for investigating viral pathogenesis and evaluating antiviral therapies. However, further studies, including direct viral quantification and histopathological analysis, are required. Full article

(This article belongs to the Special Issue Pathogen–Host Interactions: Death, Defense, and Disease)

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20 pages, 436 KB

Open AccessArticle

Real-World Use of Cefiderocol for Multidrug-Resistant Gram-Negative Infections in Critically Ill ICU Patients: A Single-Center Case Series

by Stelian Adrian Ritiu, Adelina Baloi, Marius Păpurică, Dorel Sandesc, Daiana Toma, Claudiu Rafael Bârsac, Sonia Elena Popovici, Madalina Butaș, Ana-Maria-Ionela Botoaca and Ovidiu Bedreag

Pathogens 2026, 15(8), 786; https://doi.org/10.3390/pathogens15080786 - 24 Jul 2026

Abstract

Background/Objectives: Carbapenem-resistant Gram-negative (CR-GN) infections are associated with high mortality in intensive care units (ICUs), with limited therapeutic options. We aimed to evaluate microbiological and clinical outcomes associated with cefiderocol in critically ill patients with multidrug-resistant (MDR) Gram-negative infections. Methods: This retrospective, single-center [...] Read more.

Background/Objectives: Carbapenem-resistant Gram-negative (CR-GN) infections are associated with high mortality in intensive care units (ICUs), with limited therapeutic options. We aimed to evaluate microbiological and clinical outcomes associated with cefiderocol in critically ill patients with multidrug-resistant (MDR) Gram-negative infections. Methods: This retrospective, single-center case series included 25 critically ill patients with carbapenem-resistant or multidrug-resistant Gram-negative infections treated with cefiderocol between May 2024 and October 2025 in a mixed ICU of a tertiary hospital in Romania. Microbiological cure was defined as eradication of the designated target pathogen at the end of therapy. Clinical evolution was assessed using the Acute Physiology and Chronic Health Evaluation II (APACHE II) and Sequential Organ Failure Assessment (SOFA) scores, as well as inflammatory biomarkers including white blood cell count (WBC), C-reactive protein (CRP), and procalcitonin (PCT). Results: Klebsiella pneumoniae was the predominant pathogen, identified in 23 of 25 patients (92%), followed by Acinetobacter baumannii (11/25, 44%) and Pseudomonas aeruginosa (6/25, 24%). As multiple pathogens were co-isolated in 16 patients (64%), percentages exceed 100% and are reported as proportions of patients rather than of total isolates. Microbiological cure was achieved in 68% of patients. Mortality was markedly higher in patients without microbiological eradication (88% vs. 35%). Higher baseline APACHE II (HR 1.18, 95% CI 1.02–1.37) and SOFA scores (HR 1.33, 95% CI 1.07–1.65) were associated with increased mortality. Early initiation of cefiderocol (≤10 days from ICU admission) was associated with improved microbiological and clinical outcomes compared to delayed treatment. Reductions in severity scores and inflammatory markers, including C-reactive protein (CRP) and procalcitonin (PCT), were observed during therapy. Pathogen type and combination therapy were not clearly associated with outcomes in this cohort. Conclusions: Cefiderocol was observed in association with clinically relevant rates of microbiological eradication and improvements in clinical parameters in critically ill patients with MDR Gram-negative infections. Outcomes appeared to be primarily determined by baseline disease severity, while earlier initiation of therapy was associated with more favourable microbiological and clinical outcomes, although causality cannot be established given the observational design and absence of a comparator group. These findings are hypothesis-generating and supportive of a potential role for cefiderocol as a salvage option in high-risk ICU populations, pending validation in larger, prospective, controlled studies. Full article

(This article belongs to the Special Issue Advanced Antimicrobial Agents: Combatting Multi-Drug Resistant Bacterial Infections)

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19 pages, 13586 KB

Open AccessArticle

Compact Gut Microbial Dysbiosis Signature Associated with Necrotizing Enterocolitis and Altered Early Microbiota Development

by Ying Xiang, Zhuoqi Zhao, Zhong Feng, Jialu Zhuang, Yu Chen, Yansong Chen, Shumin Feng, Liya Pan, Zhilong Yan and Li Hong

Pathogens 2026, 15(8), 785; https://doi.org/10.3390/pathogens15080785 - 24 Jul 2026

Abstract

Background: Necrotizing enterocolitis (NEC) is a life-threatening intestinal disorder in preterm infants and is strongly associated with gut microbial dysbiosis. However, whether recurrent genus-level dysbiosis patterns can be observed across heterogeneous NEC cohorts and summarized as a compact, interpretable microbial signature remains unclear. [...] Read more.

Background: Necrotizing enterocolitis (NEC) is a life-threatening intestinal disorder in preterm infants and is strongly associated with gut microbial dysbiosis. However, whether recurrent genus-level dysbiosis patterns can be observed across heterogeneous NEC cohorts and summarized as a compact, interpretable microbial signature remains unclear. Methods: We analyzed two public neonatal gut microbiome cohorts and an independent real-world cohort within a three-stage framework of discovery, contextual analysis, and external validation. Community composition, alpha diversity, beta diversity, and differential genera were evaluated using harmonized genus-level features within each cohort. Machine learning feature prioritization was used to derive a reduced NEC-associated microbial signature. A four-group cohort was then used to examine this signature in relation to disease status, antibiotic exposure, and early postnatal development. The reduced signature was finally examined in our collected samples. Results: NEC-related microbial alterations showed marked heterogeneity at the whole-community level across cohorts, whereas several genus-level directional patterns recurred across datasets. Across datasets, NEC was repeatedly associated with enrichment of several opportunistic genera, including Enterobacter, Klebsiella, Serratia, and Escherichia–Shigella, and depletion of commensal or probiotic-associated taxa such as Bifidobacterium, Lactobacillus, and Pediococcus. Feature prioritization yielded a compact microbial signature that improved discrimination over the unfiltered abundance profile in the discovery cohort (AUC 0.674 vs. 0.59). In the four-group cohort, the NEC_ABT group remained distinct from healthy controls, supporting partial modification rather than normalization of the NEC-associated pattern. In our collected samples, the signature retained directional consistency but showed only modest external discrimination (AUC 0.618). Conclusions: These findings identify recurrent genus-level dysbiosis features associated with NEC across clinically heterogeneous settings. The compact microbial signature may serve as an exploratory biomarker for longitudinal and mechanistic validation, but not as a standalone diagnostic tool. Full article

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21 pages, 35267 KB

Open AccessArticle

Arthrobotrys thaumasia and A. musiformis (Fungi: Orbiliales) as Natural Potential Enemies of Haemonchus contortus (Nematoda: Trichostrongylidae)

by Mariana Cervantes-Martínez, Rosa Isabel Higuera-Piedrahita, Héctor Alejandro de la Crúz-Crúz, Gustavo Pérez-Anzúrez, María Eugenia López-Arellano, Agustín Olmedo-Juárez, Edgar Jesús Delgado-Núñez, Ana Yuridia Ocampo-Gutiérrez and Pedro Mendoza-de Gives

Pathogens 2026, 15(8), 784; https://doi.org/10.3390/pathogens15080784 - 23 Jul 2026

Abstract

Haemonchus contortus (Hc) is a parasitic nematode affecting sheep. Arthrobotrys is a group of nematophagous fungi (NF) that capture nematodes with specialized traps. This study evaluated the in vitro predatory ability of Arthrobotrys thaumasia (At) and A. musiformis ( [...] Read more.

Haemonchus contortus (Hc) is a parasitic nematode affecting sheep. Arthrobotrys is a group of nematophagous fungi (NF) that capture nematodes with specialized traps. This study evaluated the in vitro predatory ability of Arthrobotrys thaumasia (At) and A. musiformis (Am), as well as the nematocidal activity of their liquid culture filtrates against Hc. NF isolates were obtained by sprinkling soil onto agar plates containing nematodes. Isolates were identified morphologically and molecularly. The predatory activity (PA) was tested on agar plates inoculated with nematodes and fungi; plates inoculated only with nematodes served as controls. After 10 days at 18 °C and 28 °C, larvae were counted. For nematocidal activity (NA) of LCFs, fungi were grown in Czapek–Dox (CzDB) and potato dextrose broths. Two concentrations (100 mg/mL and 130 mg/mL) were tested. Nematodes and LCFs were incubated for 48 or 72 h. PA was analyzed using unpaired Student’s t-tests, and LCFs were evaluated by ANOVA with Tukey’s post hoc test. Taxonomic procedures confirmed the isolates as Am and At. For PA, effectiveness was 74.64% for Am and 83.53% for At. The highest NA of LCF of Am in CzDB was 51% at 100 mg/mL and 76.7% at 130 mg/mL. That of the LCF of At was 22% at the highest concentration. Both isolates show strong potential as biocontrol agents against haemonchosis. Full article

(This article belongs to the Section Parasitic Pathogens)

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38 pages, 6310 KB

Open AccessReview

Clinical Guidelines for Hepatitis E Vaccination in India: An Expert Panel Consensus Report on the Recombinant Hepatitis E Vaccine, HEV 239

by Mohammad Sultan Khuroo and Naira S. Khuroo

Pathogens 2026, 15(8), 783; https://doi.org/10.3390/pathogens15080783 - 23 Jul 2026

Abstract

(1) Background: Hepatitis E remains a major public health challenge in India. (2) Methods: In August 2025, the recombinant HEV 239 vaccine was approved in India for adults aged 18 to 65 years. To establish clinical guidelines tailored to the Indian setting, an [...] Read more.

(1) Background: Hepatitis E remains a major public health challenge in India. (2) Methods: In August 2025, the recombinant HEV 239 vaccine was approved in India for adults aged 18 to 65 years. To establish clinical guidelines tailored to the Indian setting, an expert panel consensus was conducted using a modified Delphi process in accordance with the ACCORD reporting guidelines. (3) Results: A steering committee put forth 18 statements covering vaccine safety, efficacy, and clinical indications, which were independently evaluated by 33 senior Indian hepatologists and epidemiologists. Consensus was assessed using the GRADE framework for level of evidence, balance of benefits and harms, and strength of recommendations. Of the 18 statements, 12 reached the 70% consensus threshold. The panel concluded that the vaccine, which is administered on a standard three-dose schedule, is safe and highly effective in healthy adults, providing protection for up to 10 years. Targeted vaccination was recommended for five high-risk populations: outbreak-affected groups, hyperendemic pockets, women of childbearing age, patients with CLD, and solid organ transplant recipients. Although derived from HEV genotype 1, the vaccine demonstrated cross-protective efficacy against HEV genotype 4. (4) Conclusions: This consensus report provides a framework for deploying the HEV vaccine to mitigate disease burden in India while emphasizing the need for real-world effectiveness and safety data from India. Full article

(This article belongs to the Special Issue Hepatitis E: Virus, Disease and Vaccine)

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Graphical abstract

14 pages, 1047 KB

Open AccessArticle

Implementation of a Composite Reference Standard Methodology in the Greek National Malaria Reference Center, Insights from a Seven-Year Experience

by Nikolaos Tegos, Leonidas Georgalis, Anastasia Bimpa, Anastasia Panagopoulou, Stavroula Beleri, Vasilios Papavasilopoulos, Annita Vakali, Chrisovaladou Kefaloudi, Danai Pervanidou, Christos Hatzichristodoulou and Eleni Patsoula

Pathogens 2026, 15(8), 782; https://doi.org/10.3390/pathogens15080782 - 23 Jul 2026

Abstract

Background: Malaria is currently ranked as an important public health issue in Greece despite its initial eradication back in 1974. Given that the clinical picture is often nonspecific, the need for the implementation of targeted laboratory methodologies is imperative to ensure prompt Plasmodium [...] Read more.

Background: Malaria is currently ranked as an important public health issue in Greece despite its initial eradication back in 1974. Given that the clinical picture is often nonspecific, the need for the implementation of targeted laboratory methodologies is imperative to ensure prompt Plasmodium identification and diagnosis. Methods: A total of 780 clinical samples from 763 suspected malaria cases from the National Malaria Reference Center were analyzed within a seven-year period (2017–2023). A composite laboratory reference standard methodology, including rapid diagnostic testing, microscopy, and polymerase chain reactions, was implemented for the purposes set by the current study in a group of 532 samples. A multivariable statistical analysis was implemented for the interpretation of the extracted data. Results: Microscopy and PCR successfully identified P. falciparum (111 vs. 131) and P. vivax (95 vs. 95) clinical samples. In addition, microscopy successfully identified selected cases of P. ovale (16) and P. malariae (nine), with current cases being confirmed by targeted, species-level real-time PCR protocols. Conclusions: The current study highlighted the importance of microscopic examination in malaria diagnostic procedures for Plasmodium species identification and typing. The combination of microscopy and PCR ensures a credible laboratory approach for submicroscopic clinical cases and patients with prior antimalarial treatment. This composite laboratory reference standard methodology is recommended by the Greek Malaria Reference Center for prompt identification of Plasmodium species in suspected malaria clinical cases. Full article

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13 pages, 1308 KB

Open AccessArticle

Effects of Essential Oil Treatment on Clostridium perfringens Cells

by Safa Q. Alfattani and Mahfuzur R. Sarker

Pathogens 2026, 15(8), 781; https://doi.org/10.3390/pathogens15080781 - 23 Jul 2026

Abstract

Clostridium perfringens type F is a Gram-positive, spore-forming bacterium that causes food poisoning (FP) in humans. Our previous findings demonstrated that essential oils (EOs), including clove, rosemary, and peppermint oils, significantly inhibited the germination, outgrowth, and vegetative growth of C. perfringens spores. We [...] Read more.

Clostridium perfringens type F is a Gram-positive, spore-forming bacterium that causes food poisoning (FP) in humans. Our previous findings demonstrated that essential oils (EOs), including clove, rosemary, and peppermint oils, significantly inhibited the germination, outgrowth, and vegetative growth of C. perfringens spores. We hypothesized that cytoplasmic membrane damage could be one mechanism by which EOs exert their inhibitory effects against C. perfringens vegetative cells. To test this hypothesis, vegetative cells of two clinical FP strains of C. perfringens, SM101 and E13, were treated with EOs, and the leakage of intracellular molecules was compared between EO-treated and untreated cells. Our results demonstrated increased leakage of intracellular molecules, including ATP, 260-nm-absorbing materials, and proteins, from EO-treated cells relative to untreated cells. In addition, EO-treated cells exhibited greater crystal violet uptake than untreated cells. These results suggest that EOs induced cytoplasmic membrane damage, thereby promoting the leakage of intracellular molecules and the uptake of extracellular crystal violet. Scanning electron microscopy further confirmed cytoplasmic membrane damage by revealing substantially greater membrane disruption in EO-treated cells than in untreated cells. In conclusion, EOs may exert their anti-C. perfringens effects by damaging the cytoplasmic membrane, thereby disrupting membrane permeability and suppressing cell multiplication. Full article

(This article belongs to the Special Issue Fighting Pathogens with Natural Antimicrobials)

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14 pages, 1795 KB

Open AccessArticle

Epidemiological Trends, Statistical Correlations, and Forecasting of Notifiable Infectious Diseases in South Korea, 2001–2024: A Regression and Time Series Analysis with Projections to 2028

by Hyeran Jung and Minsun Jung

Pathogens 2026, 15(8), 780; https://doi.org/10.3390/pathogens15080780 - 23 Jul 2026

Abstract

Background: Notifiable infectious diseases impose a substantial, evolving burden on public health systems. This study characterized long-term epidemiological trends, inter-class associations, and age-stratified patterns of notifiable infectious diseases in South Korea and projected future burden through 2028. Methods: We analyzed aggregate national surveillance [...] Read more.

Background: Notifiable infectious diseases impose a substantial, evolving burden on public health systems. This study characterized long-term epidemiological trends, inter-class associations, and age-stratified patterns of notifiable infectious diseases in South Korea and projected future burden through 2028. Methods: We analyzed aggregate national surveillance data (2001–2024) from the Korea Disease Control and Prevention Agency (KDCA) via the Korean Statistical Information Service (KOSIS). Because the statutory classification changed in 2020 from a four-group (Class 1–4, gun) to a four-grade (geup) system, all inferential trend, correlation, and forecasting analyses were restricted to the internally consistent 2001–2019 group-system period, and 2020–2024 grade-system data were used descriptively. Secular trends were estimated with ordinary least squares and, for count-specific inference, negative-binomial log-linear models (annual percent change, APC); residual autocorrelation was assessed (Durbin–Watson, Ljung–Box). Structural breaks were estimated objectively (Bai–Perron least-squares). Inter-class associations were tested with Pearson/Spearman correlations and re-evaluated with partial correlation (controlling for calendar year) and first-difference detrending to guard against spurious co-trending. Forecasts used a random-walk ARIMA (identified by ADF stationarity testing, ACF/PACF, and AICc) and a damped Holt–Winters model on the H1N1-adjusted series, combined into an ensemble; performance was validated by rolling-origin cross-validation (RMSE, MAE, MAPE). Results: After handling the 2009 H1N1 pandemic outlier, total notifications rose significantly over 2001–2019 (slope = 8344 cases/year; 95% CI 6603–10,084; R² = 0.866; p < 0.001). Class 2 (respiratory/vaccine-preventable) diseases showed the strongest trend (APC = 17.2%/year; 95% CI 8.0–27.3), with an objectively estimated structural break at 2004 (supF = 10.5) separating an early variable phase from sustained growth (2005–2019: +7418 cases/year; R² = 0.94). Raw Class 1–Class 2 correlation (Pearson r = 0.569, p = 0.011) did not survive temporal adjustment (partial r = 0.13, p = 0.59; detrended r = −0.31, p = 0.21), indicating shared secular co-trending rather than a direct epidemiological association; only Class 2–Class 3 remained associated after detrending (r = 0.52, p = 0.03). Young working-age adults (30–49 years) showed the steepest Class 1 increases. Ensemble projections for the pre-COVID trajectory were 238,000 (2025) to 269,000 (2028) annual notifications, with wide prediction intervals reflecting substantial uncertainty. Conclusions: South Korean notifiable-disease epidemiology was dominated by a genuine upward Class 2 respiratory trend; several previously reported inter-class correlations were attributable to common temporal trends rather than shared transmission. Post-COVID normalization monitoring and respiratory-disease surveillance investment are priority actions. Full article

(This article belongs to the Special Issue Epidemiology of Vector-Borne Pathogens)

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