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Volume 14
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Pathogens, Volume 14, Issue 5 (May 2025) – 33 articles

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12 pages, 2216 KiB  
Article
Isolation, Identification, and In Vitro Fungicide Screening of the Pathogen Associated with Pear Dry Blight
by Xin Wang, Cai He, Peng Zhang, Lianxin Zhao, Wei Liu, Na Jin and Yanlan Guo
Pathogens 2025, 14(5), 432; https://doi.org/10.3390/pathogens14050432 - 29 Apr 2025
Abstract
Pear (Pyrus spp.) is a globally important fruit crop, with China leading in the production and cultivation area. Pear dry blight, a destructive fungal disease, has emerged as a significant threat to pear orchards in Wuwei, Gansu Province, China. This study aimed [...] Read more.
Pear (Pyrus spp.) is a globally important fruit crop, with China leading in the production and cultivation area. Pear dry blight, a destructive fungal disease, has emerged as a significant threat to pear orchards in Wuwei, Gansu Province, China. This study aimed to identify the causal pathogen, evaluate its pathogenicity, and assess the efficacy of commonly used fungicides. A total of 276 fungal isolates were obtained from symptomatic Pyrus bretschneideri stems and characterised through morphological and molecular analyses. Diaporthe fukushii was identified as the causal pathogen. Pathogenicity assays on Zaosu pear branches and Huangguan pear fruits resulted in 82% and 100% disease incidence, respectively, fulfilling Koch’s postulates. In vitro fungicide evaluations demonstrated that thiophanate-methyl and difenoconazole + propiconazole exhibited the strongest inhibitory effects, followed by mancozeb, metalaxyl-mancozeb, and carbendazim, whereas chloroisobromine cyanuric acid and dimethomorph were the least effective. These findings are critical for developing effective management strategies to mitigate the impact of pear dry blight on pear production. Full article
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16 pages, 1435 KiB  
Article
Programmed Cell Death-1 Expression in T-Cell Subsets in Chickens Infected with Marek’s Disease Virus
by Jumpei Sato, Yoshinosuke Motai, Shunsuke Yamagami, Shwe Yee Win, Fumiya Horio, Hikaru Saeki, Naoya Maekawa, Tomohiro Okagawa, Satoru Konnai, Kazuhiko Ohashi and Shiro Murata
Pathogens 2025, 14(5), 431; https://doi.org/10.3390/pathogens14050431 - 29 Apr 2025
Abstract
Marek’s disease virus (MDV) causes Marek’s disease (MD) in chickens, characterized by malignant lymphomas and immunosuppression. Sporadic MD outbreaks continue to occur even among vaccinated flocks in certain regions due to the increased virulence of the field strains. However, the mechanisms of tumorigenesis [...] Read more.
Marek’s disease virus (MDV) causes Marek’s disease (MD) in chickens, characterized by malignant lymphomas and immunosuppression. Sporadic MD outbreaks continue to occur even among vaccinated flocks in certain regions due to the increased virulence of the field strains. However, the mechanisms of tumorigenesis and immunosuppression caused by MDV remain to be fully elucidated. We previously reported that the mRNA expression of programmed cell death 1 (PD-1), an immune checkpoint molecule, was increased in tumor lesions caused by MDV, and its expression was positively correlated with the mRNA expression of Meq, an MDV-specific oncogene. In this study, we characterized PD-1-expressing T-cell subsets in the spleen and tissues of chickens that developed tumors to investigate the association between PD-1 expression and immunosuppression. Flow cytometric analysis revealed that the proportion of PD-1-expressing CD4+ T-cells, which are targets of MDV tumorigenesis, increased in the spleen and tumor tissues of chickens with MD. The proportion of PD-1+ CD4+ T-cells was higher in Meq-expressing cells than in those that were not. In the spleens of chickens with MD, the proportions of PD-1-expressing cells were increased in CD8+ and γδ T-cells, which play pivotal roles in defense against MD pathogenesis, relative to those of spleens from uninfected chickens. Moreover, the proportion of PD-1+ CD8+ T-cells expressing interferon (IFN)-γ did not increase in the spleen of chickens with MD. Additionally, no difference in the proportion of IFN-γ+ γδ T-cells expressing and not expressing PD-1 was observed in the spleens of chickens with MD, although the proportion of IFN-γ+ γδ T-cells expressing PD-1 in the spleens of uninfected chickens was higher. The function of PD-1-expressing CD8+ and γδ T-cells in chickens may be impaired after developing MD, which may cause MDV-induced immunosuppression. Full article
(This article belongs to the Section Viral Pathogens)
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18 pages, 5245 KiB  
Article
Detectability and Persistence of Cyclospora cayetanensis Oocysts in Artificially Contaminated Soil and Fresh Herbs Grown Under Controlled Climatic Conditions
by Ellie L. Rogers, Joseph Arida, John Grocholl, Joyce Njoroge and Sonia Almeria
Pathogens 2025, 14(5), 430; https://doi.org/10.3390/pathogens14050430 - 28 Apr 2025
Abstract
Cyclospora oocysts are thought to be highly resistant in the environment but the climatic factors which determine the presence/persistence of Cyclospora oocysts are currently unknown. The main objective of this study was to determine the effects of temperature, water content, and soil texture [...] Read more.
Cyclospora oocysts are thought to be highly resistant in the environment but the climatic factors which determine the presence/persistence of Cyclospora oocysts are currently unknown. The main objective of this study was to determine the effects of temperature, water content, and soil texture on C. cayetanensis detection/persistence in artificially contaminated soil and herbs grown under controlled environmental conditions. Soil and leaves of three potted herbs (cilantro, parsley, and basil) grown in growth chambers and inoculated with C. cayetanensis oocysts were collected at 7, 14, 21, 28–31, 35–38, 42–45, 49–52, and 56 days post inoculation (dpi). Under wet watering conditions, independent of temperature, positive C. cayetanensis detection was observed at each sampling collection in both soil and herb leaves. Additionally, all three herbs were found to be positive for the parasite throughout the study duration in arid watering conditions. Conversely, short-lived persistence in soil was observed under arid conditions independent of temperature in Sandy Clay Loam soil (up to 14 dpi) and in Silt Loam soil (up to 21 dpi). Our results on the effect of desiccation on the presence and persistence of oocysts may provide useful insights for the proper cleaning and sanitizing of utensils or food contact surfaces to help control the persistence of the parasite. Full article
(This article belongs to the Section Parasitic Pathogens)
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12 pages, 239 KiB  
Article
Evaluation of the Mycobacterium RealTime Kit Vircell (MRTVircell) Assay for Detecting Mycobacterium Species in Clinical Specimens
by Maria Aguilera Franco, Antonio Sampedro Padilla, Juan Francisco Gutiérrez-Bautista, Adrián González Martínez, Fernando Cobo, José Gutiérrez-Fernández, Juan Antonio Reguera, Jose María Navarro Mari and Javier Rodríguez-Granger
Pathogens 2025, 14(5), 429; https://doi.org/10.3390/pathogens14050429 - 28 Apr 2025
Viewed by 35
Abstract
Rapid and accurate diagnosis of mycobacterial infections is crucial for guiding therapeutic decisions. This study presents the first evaluation of a novel molecular assay, the Mycobacterium RealTime PCR Kit Vircell (MRTVircell), a real-time PCR-based test designed for the specific detection of Mycobacterium tuberculosis complex [...] Read more.
Rapid and accurate diagnosis of mycobacterial infections is crucial for guiding therapeutic decisions. This study presents the first evaluation of a novel molecular assay, the Mycobacterium RealTime PCR Kit Vircell (MRTVircell), a real-time PCR-based test designed for the specific detection of Mycobacterium tuberculosis complex (MTBC), Mycobacterium avium complex (MAC), Mycobacterium abscessus complex (MABC), and other nontuberculous mycobacteria (NTM) in both respiratory and non-respiratory samples. The evaluation was conducted under routine workflow conditions using 721 clinical specimens, including 559 respiratory and 162 non-respiratory samples. Among these, 5.69% were smear-positive, 6.38% were culture-positive for MTBC, and 9.85% were culture-positive for NTM. The performance of the MRTVircell was compared to both culture results and the Anyplex MTB/NTM real-time PCR assay. The two PCR systems demonstrated a 96.95% overall concordance rate for the detection of MTBC, NTM, and negative specimens. Based on culture as the reference method, the sensitivity, specificity, positive predictive value, and negative predictive value of the MRTVircell for MTBC detection were 80.43%, 99.64%, 94.87%, and 98.41%, respectively, while for Anyplex MTB/NTM (Seegene), these values were 76.09%, 99.64%, 94.59%, and 98.06%, respectively. For NTM detection, the sensitivity, specificity, positive predictive value, and negative predictive values were 28.17%, 99.29%, 83.33% and 91.63% for MRTVircell and 21.13%, 99.11%, 75%, and 91.67% for Anyplex MTB/NTM, respectively. MRTVircell is a rapid and reliable tool for the detection and differentiation of MTBC, MAC, MABC, and other NTM in clinical samples. Full article
18 pages, 2474 KiB  
Article
Kalmusia variispora (Didymosphaeriaceae, Dothideomycetes) Associated with the Grapevine Trunk Disease Complex in Cyprus
by Georgios Makris, Marcelo Sandoval-Denis, Pedro W. Crous and Loukas I. Kanetis
Pathogens 2025, 14(5), 428; https://doi.org/10.3390/pathogens14050428 - 28 Apr 2025
Viewed by 38
Abstract
Grapevine trunk diseases (GTDs) are widespread worldwide, causing serious economic losses to the vitiviniculture industry. The etiology of the complex pathogenic mycobiome associated with this group of diseases is critical to implementing appropriate management strategies. Diseased grapevines exhibiting typical GTD symptoms were collected [...] Read more.
Grapevine trunk diseases (GTDs) are widespread worldwide, causing serious economic losses to the vitiviniculture industry. The etiology of the complex pathogenic mycobiome associated with this group of diseases is critical to implementing appropriate management strategies. Diseased grapevines exhibiting typical GTD symptoms were collected from vineyards in different provinces of Cyprus, resulting in 19 pycnidial isolates. A subsequent multilocus sequence analysis of six genetic loci (ITS, LSU, SSU, b-tub, tef1-a, and rpb2) identified them as Kalmusia variispora, and twelve representative isolates are included in the phylogenetic analyses. According to pathogenicity trials on two-year-old potted vines (cv. Mavro), all tested isolates were pathogenic, exhibiting light to dark brown discoloration and lesions of varying levels, ranging from 4 to 12.3 cm long. The capacity of K. variispora isolates to produce cell-wall-degrading exoenzymes was qualitatively estimated on solid media. Cellulase, pectinase, and laccase production were evident for all the tested isolates, except isolate CBS 151329, where the latter enzyme was undetected. The severity of the symptoms was consistent with the laccase-producing capacity. The present study confirmed the association of K. variispora with grapevines as a pathogen and represents the first description of this ascomycete as a GTD causal agent in Cyprus. This highly virulent species may play a significant role in the GTD complex, and its biological cycle and epidemiology should be further investigated. Full article
(This article belongs to the Special Issue Filamentous Fungal Pathogens: 2nd Edition)
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10 pages, 3529 KiB  
Communication
Seroprevalence of Toxoplasma gondii in Wild European and American Mink (Mustela lutreola and Neogale vison) from Spain
by María Eugenia Lebrero, José Villora, María Asunción Gómez, Madis Podra, María del Carmen Aranda, Sergio Villanueva-Saz, Antonio Fernández, Patricia Lizarraga, Pablo Quilez, Álex Gómez and Diana Marteles
Pathogens 2025, 14(5), 427; https://doi.org/10.3390/pathogens14050427 - 28 Apr 2025
Viewed by 99
Abstract
Toxoplasmosis, caused by the intracellular parasite Toxoplasma gondii, affects a wide range of warm-blooded animals, including humans. Domestic and wild felines serve as definitive hosts, excreting oocysts that contaminate the environment. Intermediate hosts, such as the endangered European mink (Mustela lutreola [...] Read more.
Toxoplasmosis, caused by the intracellular parasite Toxoplasma gondii, affects a wide range of warm-blooded animals, including humans. Domestic and wild felines serve as definitive hosts, excreting oocysts that contaminate the environment. Intermediate hosts, such as the endangered European mink (Mustela lutreola) and the non-native American mink (Neogale vison), can become infected primarily through the ingestion of tissue cysts present in prey, while the ingestion of oocysts from contaminated soil or water plays a secondary role. This study analyzed the seroprevalence of T. gondii in 179 mink specimens (137 American mink and 42 European mink) collected in northern Spain from 2014 to 2020. Using an optimized indirect immunofluorescence assay, antibodies against T. gondii were detected in 47 samples (37 American mink and 10 European mink). Seroprevalence was higher in the Ebro basin than in the Cantabrian region, although the difference was not statistically significant. No significant associations were observed between seropositivity and species, sex, or habitat. These findings suggest environmental contamination by T. gondii oocysts in northern Spain and underscore the potential value of invasive American mink as sentinel species for monitoring public health risks associated with this parasite. The study also highlights the importance of wildlife surveillance in assessing environmental contamination and understanding transmission dynamics of infectious diseases in ecosystems. Full article
(This article belongs to the Special Issue Epidemiology of Infectious Diseases in Wild Animals)
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10 pages, 3832 KiB  
Case Report
First Case of Human Ocular Dirofilariasis in the Aosta Valley Region: Clinical Management and Morphological-Molecular Confirmation
by Erik Mus, Annalisa Viani, Lorenzo Domenis, Fabio Maradei, Antonio Valastro, Gianluca Marucci, Claudio Giuseppe Giacomazzi, Silvia Carla Maria Magnani, Roberto Imparato, Annie Cometto, Adriano Casulli, Riccardo Orusa and Luca Ventre
Pathogens 2025, 14(5), 423; https://doi.org/10.3390/pathogens14050423 - 28 Apr 2025
Viewed by 162
Abstract
Purpose: Dirofilariasis is a zoonotic infectious disease caused by a species belonging to the Dirofilaria genus. Human dirofilariasis cases have increased in Europe in the last few decades. Dogs and wild canids represent the definitive hosts and principal reservoirs of Dirofilaria repens, while [...] Read more.
Purpose: Dirofilariasis is a zoonotic infectious disease caused by a species belonging to the Dirofilaria genus. Human dirofilariasis cases have increased in Europe in the last few decades. Dogs and wild canids represent the definitive hosts and principal reservoirs of Dirofilaria repens, while mosquito species are biological vectors. Humans act as accidental hosts, and clinical manifestations depend on the location of the worm in the organs or tissues. We described the first case of ocular dirofilariasis in the Aosta Valley region (Italy). Case description: a 62-year-old Italian woman complained of recurrent ocular redness, pain and discomfort, accompanied by itching and foreign body sensation in the right eye. The slit lamp biomicroscopic examination revealed conjunctival congestion on the temporal region of bulbar conjunctiva, and a long whitish vermiform mobile mass was detected under the conjunctiva. The anterior chamber showed no flare or cells in either eye, and the dilated fundus examination was normal. The worm was immediately surgically removed to prevent further migration, and was diagnosed morphologically and molecularly as D. repens. Following surgical removal, the symptoms resolved completely and rapidly, with no recurrence of ocular symptoms recorded during 12-month follow-up visits. Conclusions: Ocular dirofilariasis can lead to misdiagnosis due to its rare ocular manifestations, and it is considered an emergent zoonosis in European countries. Accurate diagnosis and control of ocular dirofilariasis by D. repens require a multidisciplinary approach under the One Health framework to effectively address this emergent zoonosis. Full article
(This article belongs to the Special Issue One Health and Neglected Zoonotic Diseases)
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13 pages, 1138 KiB  
Article
Systemic IL-10 and IFN-γ Levels in Respiratory Syncytial Virus- and Rhinovirus-Infected Bulgarian Children with Acute Bronchiolitis and Their Impact on Clinical Manifestation
by Emiliya Kostadinova, Svetla Angelova, Tsvetana Tsonkova-Popova, Dima Zlateva, Rozalina Yordanova and Spaska Stanilova
Pathogens 2025, 14(5), 426; https://doi.org/10.3390/pathogens14050426 - 27 Apr 2025
Viewed by 67
Abstract
Respiratory syncytial virus (RSV) and rhinovirus (RV)—the two primary causative viruses of bronchiolitis in children—have been studied extensively in terms of their associations with disease severity and serious late disease outcomes. In this study, we explored the differences in the clinical values of [...] Read more.
Respiratory syncytial virus (RSV) and rhinovirus (RV)—the two primary causative viruses of bronchiolitis in children—have been studied extensively in terms of their associations with disease severity and serious late disease outcomes. In this study, we explored the differences in the clinical values of IFN-γ and IL-10 serum levels in RSV and RV bronchiolitis in the Bulgarian childhood population. Eighty-eight children with acute bronchiolitis, aged two months to two years, who were admitted to the General Pediatrics Clinic of University Hospital “Prof. St. Kirkovich”, Stara Zagora, Bulgaria served as this study’s subjects. The degree of wheezing and respiratory failure were classified. Naso-pharyngeal swabs were collected from all participants, and molecular identification of viruses was performed using real-time PCR. Serum samples were used to determine IFN-γ and IL-10 quantities using ELISA kits, and data are presented as the median IQR (25–75%). The total serum IL-10 levels were significantly enhanced in RSV-infected children compared to those infected with RV (14.4 (12.2–24.0) vs. 8.9 (7.2–12.5); p < 0.001) and the other viral bronchiolitis groups (14.4 (12.2–24.0) vs. 6.65 (3.9–15.3); p = 0.003). The highest IL-10 levels (14.4 (12.8–27.9)) were found in RSV-positive patients with first-degree respiratory failure. Almost identical serum IFN-γ levels were determined for RSV- and RV-positive bronchiolitis patients (3.2 (1.6–6.8) and 2.8 (1.1–7.3); p = 0.781). Variance analysis of IL-10 serum levels revealed statistically significant differences among the patient groups depending on the type of viral infection, concerning respiratory failure (p = 0.005) and wheezing severity (p = 0.017). Our findings show that the IL-10 levels and the type of virus have a combined effect on disease severity. These data might contribute to patients’ personalized/individualized therapy and the prevention of recurrent wheezing later in life. Full article
(This article belongs to the Section Viral Pathogens)
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17 pages, 4883 KiB  
Article
Prevalence of Blastocystis spp. and Other Gastrointestinal Pathogens Among Patients Admitted to Research Hospitals in Campania Region, Italy
by Marianna Ascierto, Annalisa Chianese, Francesco Foglia, Emiliana Finamore, Luciana Petrullo, Carla Zannella, Anna De Filippis, Maria Grazia Coppola and Massimiliano Galdiero
Pathogens 2025, 14(5), 425; https://doi.org/10.3390/pathogens14050425 - 27 Apr 2025
Viewed by 73
Abstract
Background. Blastocystis spp. is a common protozoan found in the gastrointestinal tract, typically existing as a non-pathogenic organism in humans and other animals. However, it can become pathogenic when the immune system is compromised due to bacterial, viral, fungal, or other parasitic infections, [...] Read more.
Background. Blastocystis spp. is a common protozoan found in the gastrointestinal tract, typically existing as a non-pathogenic organism in humans and other animals. However, it can become pathogenic when the immune system is compromised due to bacterial, viral, fungal, or other parasitic infections, as well as systemic conditions, leading to symptomatic blastocystosis. Methods. Fecal samples were collected from patients at the University Hospital of Campania “Luigi Vanvitelli” and Cotugno Hospital in Naples. Among these samples, those that tested positive for Blastocystis spp. and were associated with other microbial infections were further analyzed. Bacterial co-infections were identified using immunochromatographic tests (ICTs) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Viral infections were detected using chemiluminescent immunoassay (CLIA), while fungal infections were diagnosed through microscopic examination and molecular biology techniques. Additionally, co-infections with other parasites were identified through microscopic analysis after Ridley’s concentration and Giemsa staining (O&P). Results. Out of the 2050 stool samples collected, 121 were positive for Blastocystis spp., of which 75 were associated with other infections. We identified the vacuolar form in patients co-infected with bacteria (n = 22), viruses (n = 30), fungi (n = 3), and other parasites (n = 20). Conclusions. Our findings indicated a higher incidence of the vacuolar form of Blastocystis spp. in symptomatic and immunocompromised patients, suggesting that a weakened immune system may increase the risk of contracting Blastocystis and other microbial infections. Full article
(This article belongs to the Special Issue Parasites Series: Parasitic Infection, Characterisation and Host Health, Parasitic Diversity and Host Range)
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12 pages, 253 KiB  
Case Report
Rapid Clinical Resolution and Differential Diagnosis of a Neurological Case of Feline Infectious Peritonitis (FIP) Using GS-441524
by Amy Huynh, Pamela Moraguez, Logan M. Watkins, Jonathan H. Wood, Ximena A. Olarte-Castillo and Gary R. Whittaker
Pathogens 2025, 14(5), 424; https://doi.org/10.3390/pathogens14050424 - 27 Apr 2025
Viewed by 74
Abstract
Case summary: A 2-year-old male neutered domestic shorthair cat was presented with a progressive history of tetraparesis, ataxia, and inappetence over 4 days. A physical exam revealed mucopurulent nasal discharge and stertor. A neurologic exam revealed a multifocal neurolocalization. The cat was non-ambulatory [...] Read more.
Case summary: A 2-year-old male neutered domestic shorthair cat was presented with a progressive history of tetraparesis, ataxia, and inappetence over 4 days. A physical exam revealed mucopurulent nasal discharge and stertor. A neurologic exam revealed a multifocal neurolocalization. The cat was non-ambulatory tetraparetic and developed seizures while in hospital. Hematologic assessment revealed anemia, hypoalbuminemia and hyperglobulinemia. Magnetic resonance imaging (MRI) of the brain revealed multifocal meningeal contrast enhancement in the brainstem and cervical spine, as well as mandibular and retropharyngeal lymphadenopathy. Cerebrospinal fluid revealed marked neutrophilic pleocytosis; no infectious organisms were seen. Toxoplasma IgG/IgM and Cryptococcus antigen latex agglutination were negative. Mandibular and abdominal lymph nodes were aspirated, and cytology revealed mixed inflammation. The cat was suspected to have feline infectious peritonitis, and to aid in clinical diagnosis he was enrolled in research study—with targeted Nanopore-based sequencing specifically identifying and characterizing FCoV-1 RNA in spinal fluid and anal swab, but not in urine. The cat was treated with anticonvulsants (phenobarbital and levetiracetam), an antibiotic (ampicillin/clavulanic acid), and GS-441524. Neurologic signs did not improve on an antibiotic alone but improved significantly after two subcutaneous injections of GS-441524. The cat received an 84-day course of GS-441524 and, at the time of manuscript preparation (over 12 months after diagnosis), remains ambulatory and seizure-free without recurrence of neurologic signs and no detectable viral shedding in feces. Full article
(This article belongs to the Special Issue Feline Coronavirus Infections)
7 pages, 3654 KiB  
Communication
Syzygium aromaticum Essential Oil as a Safe Natural Solution to Control Bacteria in Hatching Eggs
by Gabriel da Silva Oliveira, Concepta McManus and Vinícius Machado dos Santos
Pathogens 2025, 14(5), 422; https://doi.org/10.3390/pathogens14050422 - 27 Apr 2025
Viewed by 94
Abstract
The bacterial contamination of eggshells may be responsible for embryonic mortality, as may the contamination of chicks and broilers. Poor incubation results may be related to high levels of contamination that overcome the antibacterial defenses of the eggshell structure and the poultry’s immune [...] Read more.
The bacterial contamination of eggshells may be responsible for embryonic mortality, as may the contamination of chicks and broilers. Poor incubation results may be related to high levels of contamination that overcome the antibacterial defenses of the eggshell structure and the poultry’s immune system. Although synthetic antibacterial formulations have demonstrated efficacy in controlling eggshell bacteria, they have also demonstrated toxicity to the embryo. In this study, we aimed to establish a safe and antibacterial topical protocol using Syzygium aromaticum essential oil (SAEO) to sanitize hatching chicken eggs. We evaluated the antibacterial capacity of this essential oil and used the ‘hen’s egg test on chorioallantoic membrane’ (HET-CAM) model to measure its toxicity. Our results demonstrated the high effectiveness of SAEO in reducing the bacterial load on eggshells, suggesting that this natural compound is a promising egg sanitizer. However, although the HET-CAM model did not indicate signs of toxicity for pure SAEO, we recommend that its application, following dilution in grain alcohol, be carried out on the surface of eggshells and never directly in the internal embryonic compartment, owing to the toxicity of alcohol. Full article
(This article belongs to the Section Bacterial Pathogens)
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23 pages, 5321 KiB  
Article
Experimental Infection of Ferrets with Bartonella henselae: In Search of a Novel Animal Model for Zoonotic Bartonellosis
by Erin Lashnits, Cynthia Robveille, Pradeep Neupane, Toni Richardson, Keith Linder, Gabriel McKeon, Ricardo Maggi and Edward B. Breitschwerdt
Pathogens 2025, 14(5), 421; https://doi.org/10.3390/pathogens14050421 - 26 Apr 2025
Viewed by 192
Abstract
Bartonella henselae is an important zoonotic pathogen with a wide range of clinical manifestations in humans. Despite advances in understanding its pathogenesis, there is no broadly applicable laboratory animal model for bartonellosis. This study aimed to assess the potential utility of an experimental [...] Read more.
Bartonella henselae is an important zoonotic pathogen with a wide range of clinical manifestations in humans. Despite advances in understanding its pathogenesis, there is no broadly applicable laboratory animal model for bartonellosis. This study aimed to assess the potential utility of an experimental model of chronic B. henselae infection using ferrets, a species previously utilized in various human pathogen studies. Six ferrets (n = 6) were divided into three groups: a control group (n = 2), a low-dose infection group (n = 2), and a high-dose infection group (n = 2). The two infection groups were inoculated intradermally with 105 (low dose) and 109 (high dose) CFU/mL B. henselae, respectively. Clinical signs, serological responses, and bacteriological findings were monitored over seven weeks; ferrets were then euthanized and tissues were examined histologically. Only minimal or transient systemic clinical signs and laboratory abnormalities developed in B. henselae inoculated ferrets. The high-dose group seroconverted to B. henselae antigen within two weeks, maintaining elevated titers throughout the study. Histopathological examination revealed that four B. henselae-infected ferrets had notable microscopic inflammatory lesions in the liver parenchyma (3/4), heart (1/4), and brain (1/4); similar lesions were not observed in the tissues of the two control ferrets. Despite the presence of microscopic lesions and seroconversion in the high-dose group, bacteremia was not documented and B. henselae DNA was not successfully amplified by quantitative PCR from lesional organs. This pilot study demonstrated that ferrets may serve as a promising model for investigating B. henselae pathogenesis, diagnosis, treatment, and prevention. Full article
(This article belongs to the Topic Zoonotic Vector-Borne Diseases of Companion Animals)
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8 pages, 928 KiB  
Case Report
Chronic Hepatitis E Virus Infection Without Liver Injury in a Patient with Chronic Kidney Disease
by Oliver Viera-Segura, Ilsy X. Duarte-López, Isidro Loera-Robles, Norberto Singh-Ríos, Arturo Calderón-Flores, Edgar D. Copado-Villagrana and Nora A. Fierro
Pathogens 2025, 14(5), 420; https://doi.org/10.3390/pathogens14050420 - 26 Apr 2025
Viewed by 131
Abstract
Hepatitis E virus (HEV), the causative agent of hepatitis E, is the leading cause of acute viral hepatitis worldwide; under immunosuppression, infection can lead to chronic liver disease. Furthermore, extrahepatic manifestations, particularly renal manifestations, are frequently associated with infection. This is important considering [...] Read more.
Hepatitis E virus (HEV), the causative agent of hepatitis E, is the leading cause of acute viral hepatitis worldwide; under immunosuppression, infection can lead to chronic liver disease. Furthermore, extrahepatic manifestations, particularly renal manifestations, are frequently associated with infection. This is important considering the global burden of chronic kidney disease (CKD). However, the study of chronic hepatitis E has been limited to liver disease, and its definition with respect to renal disease is still incomplete. Recently, through a protocol aimed at identifying HEV seroprevalence in a cohort of patients on hemodialysis, we incidentally identified HEV RNA in a patient with a history of alcoholism, diabetes mellitus, and essential systemic hypertension. In this study, we aimed to follow up this case to characterize hepatitis E in the context of CKD. Notably, we identified the development of chronic HEV genotype 3 infection without seroconversion or evidence of liver damage. Moreover, apparent immunocompetence was identified in the patient. Considering that HEV is still neglected in numerous countries and that it is not included in the differential diagnosis of kidney disease, our findings support the need to consider HEV infection in patients with renal disease, even in the absence of liver deterioration. Full article
(This article belongs to the Special Issue Genomic Epidemiology of High-Consequence Viruses)
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12 pages, 5539 KiB  
Article
Antiparasitic Activity of Chalepensin and Graveoline Isolated from Ruta chalepensis L.: In Vitro Evaluation Against Strongyloides venezuelensis
by Nancy E. Rodríguez-Garza, Miguel Marín, Javier Sánchez-Montejo, Joel H. Elizondo-Luévano, Aldo F. Bazaldúa-Rodríguez, Ramiro Quintanilla-Licea, César I. Romo-Sáenz, Rafael Peláez, Antonio Muro and Julio López-Abán
Pathogens 2025, 14(5), 419; https://doi.org/10.3390/pathogens14050419 - 25 Apr 2025
Viewed by 92
Abstract
Parasitic diseases constitute a significant challenge to global public health, with Strongyloides stercoralis ranking among the most prevalent and clinically significant parasites. The limitations of current nematocidal therapies highlight an urgent need for novel treatment strategies. In this study, the nematocidal activity of [...] Read more.
Parasitic diseases constitute a significant challenge to global public health, with Strongyloides stercoralis ranking among the most prevalent and clinically significant parasites. The limitations of current nematocidal therapies highlight an urgent need for novel treatment strategies. In this study, the nematocidal activity of chalepensin and graveoline, two compounds isolated from Ruta chalepensis, was evaluated against larval and adult stages of Strongyloides venezuelensis (model for S. stercoralis). The in vitro efficacy of these compounds was assessed on third-stage infective larvae (L3) and adult parthenogenetic females at various time points, while cytotoxicity was determined using Vero cells to calculate selectivity indices (SI). Both compounds showed good antiparasitic activity, but chalepensin exhibited superior nematocidal activity compared to graveoline, with an LC50 of 3.9 µg/mL and an SI of 990 for L3, and an LC50 of 16.8 µg/mL and an SI of 200 for adult females at 72 h. Morphological analysis via scanning electron microscopy in adult females revealed that graveoline induced mostly cuticle detachment, while chalepensin caused protuberances across the parasite body. These findings suggest that both compounds possess promising antiparasitic potential, with chalepensin emerging as a particularly potent candidate for further exploration. Full article
(This article belongs to the Special Issue Natural Products Chemistry: Innovation and New Frontiers in Antiparasitic Therapies)
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18 pages, 750 KiB  
Article
Green Antimicrobials: Innovative Applications of Hops Extracts as Biocontrol Agents
by Ana I. Paniagua-García, Ana Ibáñez and Rebeca Díez-Antolínez
Pathogens 2025, 14(5), 418; https://doi.org/10.3390/pathogens14050418 - 25 Apr 2025
Viewed by 77
Abstract
Natural compounds represent a fundamental source of antimicrobial agents with applications in numerous industries. This study investigates the antimicrobial properties of different fractions of extracts obtained from six hop varieties, as well as of certain compounds contained in hops and other plants. The [...] Read more.
Natural compounds represent a fundamental source of antimicrobial agents with applications in numerous industries. This study investigates the antimicrobial properties of different fractions of extracts obtained from six hop varieties, as well as of certain compounds contained in hops and other plants. The results indicate that soft resins exhibit the strongest antibacterial activity among the hop-derived fractions evaluated, reaching a minimum MIC90 value of 25 µg/mL (Fuggle variety) against Gram-positive bacteria (S. aureus) and 50 µg/mL (Chinook variety) against Gram-negative bacteria (E. coli). Furthermore, the composition of hops varies among varieties, resulting in divergent antimicrobial patterns, indicating the necessity for further research to elucidate the origins of these activities. Additionally, while hop-derived fractions exhibited noteworthy antibacterial properties, their antifungal activity against A. niger was found to be negligible. In addition, natural compounds such as carvacrol and thymol demonstrated the lowest MIC90 values against E. coli (130 and 250 µg/mL, respectively) and S. aureus (280 and 250 µg/mL, respectively). Moreover, xanthohumol exhibited a better MIC90 value against S. aureus (3 µg/mL), while no inhibitory effects were observed against E. coli. These insights highlight the necessity for further exploration of natural extracts in the development of new antimicrobial agents. Full article
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15 pages, 2442 KiB  
Communication
Agriculturally Sourced Multidrug-Resistant Escherichia coli for Use as Control Strains
by James E. Wells, Lisa M. Durso, Abasiofiok M. Ibekwe, Jonathan G. Frye, Manan Sharma, Clinton F. Williams and Md Shamimuzzaman
Pathogens 2025, 14(5), 417; https://doi.org/10.3390/pathogens14050417 (registering DOI) - 25 Apr 2025
Viewed by 78
Abstract
Bacteriological control strains with known characteristics ensure consistency and reproducibility of assay performance across different laboratories and are an important cornerstone of quality control in the microbiology laboratory. Ideally, control strains should be representative of the assay target and be widely available from [...] Read more.
Bacteriological control strains with known characteristics ensure consistency and reproducibility of assay performance across different laboratories and are an important cornerstone of quality control in the microbiology laboratory. Ideally, control strains should be representative of the assay target and be widely available from reputable sources. However, for work involving antibiotic resistance most controls come from human and veterinary clinical sources and are not optimized for work in agriculturally impacted environments or not widely available. The objective of this work was to identify and make widely available two E. coli isolates sourced from agricultural production settings that could be used as external controls supporting method development, research and environmental monitoring for extended spectrum β-lactamase producing (ESBL) and tetracycline resistant Escherichia coli. Previously collected E. coli suspects were screened based on antimicrobial susceptibility testing data, then confirmed as E. coli and characterized both phenotypically and genotypically. The positive control strain, ARS-C301 was ESBL positive and contained the CTX-M-55 and tet(A) genes, and the negative control strain, ARS-C101 was negative for both targets. Here we introduce two agriculturally sourced, fully characterized, and genetically sequenced control strains for use as laboratory controls in research involving extended-spectrum β-lactamase producing (ESBL) and tetracycline-resistant Escherichia coli isolated from the environment, available via publicly accessible culture collections, and commercially as a quantitative pellet. Full article
(This article belongs to the Section Bacterial Pathogens)
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18 pages, 1053 KiB  
Review
The Main Arboviruses and Virus Detection Methods in Vectors: Current Approaches and Future Perspectives
by Amanda Montezano Cintra, Nathália Mayumi Noda-Nicolau, Milena Leite de Oliveira Soman, Pedro Henrique de Andrade Affonso, Guilherme Targino Valente and Rejane Maria Tommasini Grotto
Pathogens 2025, 14(5), 416; https://doi.org/10.3390/pathogens14050416 - 25 Apr 2025
Viewed by 373
Abstract
Arthropod-borne viruses (arboviruses) represent a growing concern for global public and veterinary health, with cases reported across all continents. This review presents a broad overview of the geographic distribution of arboviruses transmitted by insect vectors, emphasizing the importance of early viral detection as [...] Read more.
Arthropod-borne viruses (arboviruses) represent a growing concern for global public and veterinary health, with cases reported across all continents. This review presents a broad overview of the geographic distribution of arboviruses transmitted by insect vectors, emphasizing the importance of early viral detection as a cornerstone of surveillance and outbreak preparedness. Special attention is given to the phenomenon of zoonotic spillover, where viruses maintained in natural transmission cycles often involving wildlife reservoirs and arthropod vectors cross into human populations, triggering emergent or re-emergent outbreaks. This article discusses key arboviral families of medical and veterinary significance, including Togaviridae, Flaviviridae, Nairoviridae, Phenuiviridae, Peribunyaviridae, and Orthomyxoviridae, highlighting their molecular and structural characteristics. These features are essential for guiding the development and implementation of specific and sensitive detection strategies. In addition, this work provides a comparative analysis of diverse laboratory methodologies for viral detection in vectors. From serological assays and viral isolation to advanced molecular tools and next-generation sequencing, we explore their principles, practical applications, and context-dependent advantages and limitations. By compiling this information, we aim to support researchers and public health professionals in selecting the most appropriate tools for vector surveillance, ultimately contributing to improved response strategies in the face of arboviral threats. Full article
(This article belongs to the Section Viral Pathogens)
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14 pages, 1662 KiB  
Article
Evaluating SARS-CoV-2 T Cell Immunity in COVID-19-Naive Vaccinated Individuals with and Without Spike Protein IgG Antibodies
by Vassiliki C. Pitiriga, Myrto Papamentzelopoulou, Dimitris Nikoloudis, Chrysa Saldari, Kanella E. Konstantinakou, Irene V. Vasileiou and Athanasios Tsakris
Pathogens 2025, 14(5), 415; https://doi.org/10.3390/pathogens14050415 - 25 Apr 2025
Viewed by 108
Abstract
Background: The effective management of vaccination schedules requires thorough knowledge of an individual’s immunoprotection level, including the interaction and persistence of immune responses at both the humoral and cellular levels following SARS-CoV-2 vaccination. This study aimed to investigate the potential relationship between the [...] Read more.
Background: The effective management of vaccination schedules requires thorough knowledge of an individual’s immunoprotection level, including the interaction and persistence of immune responses at both the humoral and cellular levels following SARS-CoV-2 vaccination. This study aimed to investigate the potential relationship between the levels and duration of the SARS-CoV-2 T cell response and IgG measurements in a cohort of COVID-19-naive individuals who had received the SARS-CoV-2 vaccine. Methods: We performed a retrospective descriptive analysis utilizing data retrieved from the electronic medical records of consecutive COVID-19-naive and vaccinated adult individuals who underwent COVID-19 immunity screening at a private healthcare center from September 2021 to September 2022. T cell response was evaluated using the IGRA methodology T-SPOT®.COVID (Oxford Immunotec, Abingdon, Oxfordshire, UK). Results: A retrospective analysis was conducted on a cohort of 262 individuals, comprising 148 females (56.5%) and 114 males (43.5%), with ages ranging from 17 to 92 years (mean age: 59.47 ± 15.5 years). Among the participants, 216/262 (82.4%) tested negative for SARS-CoV-2 IgG antibodies (group A), while 46/262 (17.6%) tested positive (group B). No significant difference was observed between the two groups in the time period post vaccination, with the mean times after vaccination being 136.38 ± 78.68 days in group A and 140.6 ± 79.5 days in group B (T-test, p = 0.74). Among the two groups, a positive T cell reaction against the S antigen was reported in 132/216 (61.1%) participants in group A and 39/46 (84.8%) in group B (X2 test, p = 0.002). Additionally, individuals with a positive antibody response demonstrated statistically significant higher T SPOT results compared to those with undetectable antibody levels, with a mean SFC count of 125.70 for group A and 158.73 for group B (Mann–Whitney test, p = 0.006). Conclusions: Our findings suggest that T cell immunity may persist even when antibodies are undetectable, highlighting the potential role of cellular immunity in providing protection against COVID-19 over time. Additionally, this study demonstrates a significant correlation between humoral and cellular immune response levels to SARS-CoV-2, suggesting that the activation of humoral immunity following SARS-CoV-2 vaccination is associated with higher levels of cellular immunity compared to individuals with undetectable antibody levels. Full article
(This article belongs to the Section Viral Pathogens)
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12 pages, 3232 KiB  
Article
Evidence Suggesting Possible Exposure to Influenza A Virus in Neotropical Bats from Mexico
by Brenda Aline Maya-Badillo, Guillermo Orta-Pineda, Gerardo Suzán, Karen Elizabeth Rivera-Rosas, Diego Zavala-Vasco, Adrián Uribe-Jacinto, Andrea Chaves, Alfredo Grande-Cano, René Segura-Velazquez and José Iván Sánchez-Betancourt
Pathogens 2025, 14(5), 414; https://doi.org/10.3390/pathogens14050414 - 25 Apr 2025
Viewed by 126
Abstract
With the first evidence of the association between bats and influenza A viruses, various studies have begun to emerge to understand this interesting and important association among bats conservation, animal health, and public health. This study aimed to verify the presence of anti-influenza [...] Read more.
With the first evidence of the association between bats and influenza A viruses, various studies have begun to emerge to understand this interesting and important association among bats conservation, animal health, and public health. This study aimed to verify the presence of anti-influenza A vipothesrus antibodies, as well as the molecular identification of these viruses in bats distributed in forest fragments located in southeastern Mexico. Blood samples were obtained from 600 bats belonging to 24 different species, using an enzyme immunoassay to detect antibodies against the nucleoprotein antigen of the avian influenza A virus. Likewise, oropharyngeal swabs, rectal swabs and organs were taken for quantitative reverse transcription PCR (qRT-PCR) of these viruses. A total of six bats (1%) tested positive either by serology or molecular methods, not both simultaneously. Although this suggests a very low prevalence of influenza A viruses in Mexican bats, it is the first study to address this association and, following the precautionary principle, we consider it necessary to establish systematic monitoring of the presence of influenza A in bats, since they are known to harbor infectious agents with zoonotic potential. Furthermore, it is possible that the association of influenza A viruses circulating in Latin American bats has an important co-evolutionary component with some bat species with exclusive distribution in the American continent. Full article
(This article belongs to the Special Issue Zoonotic Pathogens in the Tropics: From the Forest to the Cities)
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13 pages, 803 KiB  
Article
Test Performance Study on qPCR Assays for Detection of Phyllosticta citricarpa
by Tjaša Jakomin, Janja Zajc Žunič and Polona Kogovšek
Pathogens 2025, 14(5), 413; https://doi.org/10.3390/pathogens14050413 - 24 Apr 2025
Viewed by 92
Abstract
Citrus black spot (CBS), caused by the fungus Phyllosticta citricarpa, significantly affects citrus fruit marketability and can lead to premature fruit drop. Accurate and reliable detection of this quarantine pathogen is crucial, particularly for asymptomatic plant material. This study evaluated two qPCR [...] Read more.
Citrus black spot (CBS), caused by the fungus Phyllosticta citricarpa, significantly affects citrus fruit marketability and can lead to premature fruit drop. Accurate and reliable detection of this quarantine pathogen is crucial, particularly for asymptomatic plant material. This study evaluated two qPCR assays, the EPPO recommended assay PC and assay Pc-TEF1, based on TEF region, for detecting P. citricarpa through a collaborative test performance study (TPS). DNA from the isolates of Phyllosticta spp. and other fungi was spiked into citrus fruit peel extracts (lemon, orange, and pomelo) and distributed among 13 laboratories. Sample and qPCR assay stability under typical transport conditions was confirmed, although prolonged storage affected Pc-TEF1 assay performance. The assays were assessed based on sensitivity, specificity, reproducibility, and repeatability. Both assays demonstrated high performance, with repeatability and reproducibility exceeding 95%. The PC assay, as expected, detected different related Phyllosticta species, while Pc-TEF1 showed higher specificity for P. citricarpa included in the TPS alone. Additionally, inhibitory effects were observed specifically in the pomelo peel samples, suggesting matrix-dependent variability. This TPS confirms that both PC and Pc-TEF1 qPCR assays are robust. Further evaluation of the qPCR assays would support the selection of the most reliable assays for the detection of P. citricarpa, contributing to the effective management of CBS disease in citrus production and trade. Full article
(This article belongs to the Section Fungal Pathogens)
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20 pages, 1442 KiB  
Article
Inhibition of Biofilm Production and Determination of In Vitro Time-Kill Thymus vulgaris L. Essential Oil (TEO) for the Control of Mastitis in Small Ruminants
by Michela Galgano, Francesco Pellegrini, Daniela Mrenoshki, Luciana Addante, Alessio Sposato, Laura Del Sambro, Loredana Capozzi, Elisabetta Catalano, Marianna Solito, Francesco D’Amico, Davide Messina, Antonio Parisi, Annamaria Pratelli and Paolo Capozza
Pathogens 2025, 14(5), 412; https://doi.org/10.3390/pathogens14050412 - 24 Apr 2025
Viewed by 192
Abstract
Staphylococcus aureus and coagulase-negative staphylococci (CNS) are the main causative agents of mastitis in sheep. Their ability to form biofilms in vivo is considered an important virulence factor underlying mastitis outbreaks refractory to antibiotic treatments. Furthermore, pre- and postdipping immersion during milking in [...] Read more.
Staphylococcus aureus and coagulase-negative staphylococci (CNS) are the main causative agents of mastitis in sheep. Their ability to form biofilms in vivo is considered an important virulence factor underlying mastitis outbreaks refractory to antibiotic treatments. Furthermore, pre- and postdipping immersion during milking in iodine substances could determine the presence of residues in milk and therefore represent a health risk factor for consumers. The aim of this study was to evaluate the antibacterial and biofilm inhibitory activity of Thymus vulgaris L. essential oil (TEO) against staphylococci strains isolated from ovine clinical mastitis. In particular, 3 reference strains (S. aureus 25923 and 11623 and S. epidermidis 12228) and 12 clinical isolates (6 S. aureus and 6 CNS) were used. TEO solutions, from a concentration of 1% (v/v) to 1.25% (v/v), corresponding to 9.28–2.32 mg/mL, were obtained after solubilization in 10% dimethyl sulfoxide (DMSO) and used to evaluate the bacterial time-kill compared to that of an iodine-based solution. Antibacterial efficacy was then assessed by the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), while biofilm inhibition was assessed by minimum biofilm inhibitory concentration (MBIC) using a spectrophotometer at a wavelength of 570 nm. Additionally, biofilm-associated genes (icaA and icaD) were evaluated in all tested strains by PCR. The tested TEO concentrations were able to significantly and prominently reduce bacterial growth compared to controls, as demonstrated by bacterial time-kills. The MIC value was obtained at a concentration of 0.50% (v/v) for a single coagulation-positive isolate (S. aureus (f)) and at a concentration of 0.25% (v/v) for all other isolates. TEO showed effective bactericidal action with a 99.9% reduction in CFU/mL of all isolates in the MBC test at a concentration of 0.25% (v/v) for most of the tested strains. Furthermore, a marked inhibition in biofilm formation at all tested concentrations was observed, with MBIC value of 0.25%. All S. aureus tested were biofilm-producing strains and positive for icaA and icaD genes, while two CNS biofilm-producing strains were negative for both genes. These preliminary results suggest that TEO could be a promising alternative as an udder disinfectant during milking practices. Although in vivo studies are needed to confirm the efficacy and safety of TEO as an adjuvant in the prevention and treatment of udder infections, TEO could help counteract the emergence of antimicrobial resistance and reduce the potential risk of iodine residues in milk. Full article
(This article belongs to the Section Bacterial Pathogens)
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15 pages, 1258 KiB  
Article
Seasonal Dynamics and Legionellosis-Associated Hospitalization in Spain: A Retrospective Study
by Enrique Gea-Izquierdo, Rossana Ruiz-Urbaez, Valentín Hernández-Barrera and Ángel Gil-de-Miguel
Pathogens 2025, 14(5), 411; https://doi.org/10.3390/pathogens14050411 - 24 Apr 2025
Viewed by 76
Abstract
Legionellosis is a serious respiratory disease with a high mortality rate, particularly if it is untreated or occurs in the immunocompromised. Legionellosis must be reported in the Spanish Epidemiological Surveillance System. To optimize the epidemiologic knowledge of legionellosis and improve prevention, we have [...] Read more.
Legionellosis is a serious respiratory disease with a high mortality rate, particularly if it is untreated or occurs in the immunocompromised. Legionellosis must be reported in the Spanish Epidemiological Surveillance System. To optimize the epidemiologic knowledge of legionellosis and improve prevention, we have investigated whether the disease is associated with seasonality. This study has described legionellosis cases, the temporal trend by seasonality, hospitalization rate, case fatality rate, and costs by autonomous community and season. We retrospectively reviewed cases of legionellosis, documented patient and clinical characteristics, diagnostics, and seasonality of infection. This study combined national legionellosis notification and hospital discharge data that were linked via the Spanish National Health Service to provide a dataset of hospitalized cases occurring between 2002 and 2021 in Spain. There was a significant increase in the number of legionellosis cases due to the season of the year in Spain. An association between legionellosis and factors related to seasonality is suggested. An increasing trend in case fatality rate, seasonality, and regionality and a decrease in legionellosis hospitalization in Spain were identified. The characterization of changes in legionellosis trend and seasonality and timely synchronization and harmonization of hospitalization records are essential to strengthen disease monitoring and inform potential interventions in an epidemiological way. Full article
(This article belongs to the Special Issue Legionella and Waterborne Disease)
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16 pages, 1430 KiB  
Article
Plasma-Activated Water Against Carbapenem-Resistant Klebsiella pneumoniae and Vancomycin-Resistant Enterococcus faecalis
by Dragana Vuković, Maja Miletić, Boško Toljić, Nikola Milojević, Olivera Jovanović, Jovana Kuzmanović Pfićer, Nikola Škoro and Nevena Puač
Pathogens 2025, 14(5), 410; https://doi.org/10.3390/pathogens14050410 - 24 Apr 2025
Viewed by 191
Abstract
The scope of the antibacterial effects of plasma-activated water (PAW) is not yet fully comprehended. We investigated the activity of PAW produced by the in-house 3-pin atmospheric pressure plasma jet against carbapenem-resistant Klebsiella pneumoniae and vancomycin-resistant Enterococcus faecalis, with a focus on [...] Read more.
The scope of the antibacterial effects of plasma-activated water (PAW) is not yet fully comprehended. We investigated the activity of PAW produced by the in-house 3-pin atmospheric pressure plasma jet against carbapenem-resistant Klebsiella pneumoniae and vancomycin-resistant Enterococcus faecalis, with a focus on PAW’s potential to promote susceptibility to conventional antibiotics in these bacteria. Bacterial inactivation was determined by the colony count after 15 and 60 min PAW treatments. Minimum inhibitory concentrations (MICs) measured following repeated exposures to PAW across multiple generations of bacteria enabled the assessment of changes in susceptibility to antibiotics. The PAW’s efficacy was also analyzed through the detection of intracellular reactive oxygen and nitrogen species in treated bacteria. Time-dependent significant inactivation efficiency against K. pneumoniae was observed (log reduction 6.92 ± 0.24 after 60 min exposure), while effects on E. faecalis were limited. PAW demonstrated potential to decrease the MICs of crucial antibiotics. Namely, a 50 to 62.5% decrease in the MICs of colistin against K. pneumoniae and a 25% reduction in the MICs of vancomycin against enterococci were recorded. We found a significant increase in the superoxide anion concentration in K. pneumoniae and E. faecalis cells after PAW treatments. This study indicates that PAW’s inactivating efficacy coupled with the capacity for the potentiation of antibiotic effects is a promising combination against multidrug-resistant bacteria. Full article
(This article belongs to the Section Bacterial Pathogens)
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15 pages, 2353 KiB  
Article
Earthworms Significantly Alter the Composition, Diversity, Abundance and Pathogen Load of Fungal Communities in Sewage Sludge from Different Urban Wastewater Treatment Plants
by Manuel Aira, Ana Gómez-Roel and Jorge Domínguez
Pathogens 2025, 14(5), 409; https://doi.org/10.3390/pathogens14050409 - 24 Apr 2025
Viewed by 143
Abstract
Management of sewage sludge is of ongoing concern because this waste product is generated continuously and contains high levels of harmful constituents. Among these constituents, fungal pathogens are of increasing concern. Vermicomposting can reduce the amounts of bacterial pathogens in sewage sludge; however, [...] Read more.
Management of sewage sludge is of ongoing concern because this waste product is generated continuously and contains high levels of harmful constituents. Among these constituents, fungal pathogens are of increasing concern. Vermicomposting can reduce the amounts of bacterial pathogens in sewage sludge; however, information about the effects of earthworms on fungal pathogens is limited or non-existent. We therefore aimed to determine whether vermicomposting can control fungal pathogens present in sewage sludge. Using next-generation sequencing techniques, we characterized fungal communities in sewage sludge from eight wastewater treatment plants (WWTPs) and in casts (feces) of earthworms feeding on sewage sludge. Fungal communities in earthworm casts primarily included taxa that were absent from sewage sludges, indicating a significant change in fungal composition. Changes in fungal diversity depended on the source of sewage sludge (WWTP). All of the sewage sludges contained low levels of fungal pathogens, most of which were significantly reduced or eliminated by earthworms, such as Armillaria, Cystobasidium, Exophiala and Ophiosthoma. Moreover, earthworm gut transit enhanced beneficial (saprotrophic) fungi like Arthrobotrys, Aseroe, Crepidotus and Trichurus. Overall, digestion of sewage sludge by earthworms alone generated a mainly pathogen-free fungal community with a high proportion of saprotrophic taxa, which would enhance nutrient cycling rates. Full article
(This article belongs to the Section Fungal Pathogens)
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14 pages, 1166 KiB  
Article
Unravelling the Persistence of the Rare Serovar Salmonella Mikawasima in a Hospital Setting: A Whole-Genome Sequencing Study
by Ivana Ferencak, Ana Gveric Grginic, Tajana Juzbasic, Irena Tabain, Marija Tonkic, Ivana Goic-Barisic, Dragan Juric, Hrvojka Jankovic, Luka Katic and Anita Novak
Pathogens 2025, 14(5), 408; https://doi.org/10.3390/pathogens14050408 - 24 Apr 2025
Viewed by 197
Abstract
Salmonella Mikawasima is a rare, mainly environmental serovar. In 2020, an outbreak was observed in neonatal and maternal wards of the University Hospital of Split and was established as an endemic until the end of 2024. Using whole-genome sequencing, this study aimed to [...] Read more.
Salmonella Mikawasima is a rare, mainly environmental serovar. In 2020, an outbreak was observed in neonatal and maternal wards of the University Hospital of Split and was established as an endemic until the end of 2024. Using whole-genome sequencing, this study aimed to analyse the phenotypic and genotypic characteristics of S. Mikawasima isolates and to elaborate whether the spread of the same clone occurred. Sequenced isolates were classified as ST2030, with the presence of aminoglycoside and extended spectrum beta-lactam resistance genes. Ten percent of the sequenced isolates exhibit multi-drug resistance. Identified virulence factors that include biofilm formation genes suggest the potential persistence of S. Mikawasima in the hospital environment, while spatial and temporal analysis reveal clonal expansion and possible horizontal transmission between different hospital wards. This study provides a deep understanding of the genomic composition of S. Mikawasima and emphasises the need for more stringent infection prevention measures, especially in vulnerable neonatal and postpartum settings, to mitigate the risk of healthcare-associated infections, and it should be followed by further microbiological and epidemiological investigations to identify the source of infection. Full article
(This article belongs to the Special Issue Emerging and Neglected Pathogens in the Balkans)
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17 pages, 3572 KiB  
Review
The Relevance of the Predominant Clonal Evolution (PCE) Model for the Molecular Epidemiology and Subspecific Taxonomy of Trypanosoma cruzi
by Michel Tibayrenc
Pathogens 2025, 14(5), 407; https://doi.org/10.3390/pathogens14050407 - 24 Apr 2025
Viewed by 177
Abstract
The predominant clonal evolution (PCE) model is often misunderstood. Contrary to common belief, it is not restricted to strict mitotic clonality. Instead, it encompasses processes such as selfing, strong homogamy, and various forms of parthenogenesis, as widely acknowledged by researchers studying clonality. Moreover, [...] Read more.
The predominant clonal evolution (PCE) model is often misunderstood. Contrary to common belief, it is not restricted to strict mitotic clonality. Instead, it encompasses processes such as selfing, strong homogamy, and various forms of parthenogenesis, as widely acknowledged by researchers studying clonality. Moreover, the PCE model does not claim that genetic recombination is entirely absent or devoid of epidemiological and evolutionary significance. In this review, I will explore the reciprocal relationship between PCE and molecular epidemiology (strain typing) and discuss the implications of PCE for revising the subspecific nomenclature of Trypanosoma cruzi. Full article
(This article belongs to the Special Issue Chagas Disease: Celebrating the 115th Anniversary of the Discovery of Trypanosoma cruzi)
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14 pages, 1941 KiB  
Article
Molecular Characterization of Clinical Isolates of Methicillin-Resistant Staphylococcus aureus from Chonburi, Thailand
by Patcharawalai Wassanarungroj, Panida Nobthai, Sirigade Ruekit, Apichai Srijan, Prawet Sukhchat, Oralak Serichantalergs, John M. Crawford, Brett E. Swierczewski, Sidhartha Chaudhury and Paphavee Lertsethtakarn
Pathogens 2025, 14(5), 406; https://doi.org/10.3390/pathogens14050406 - 24 Apr 2025
Viewed by 157
Abstract
Staphylococcus aureus (S. aureus), especially methicillin-resistant S. aureus (MRSA), remains a major public health concern. This study reports the antimicrobial resistance profiles and molecular characteristics of 31 S. aureus isolated during 2017–2018 from inpatient and outpatient clinical specimens from Queen Sirikit [...] Read more.
Staphylococcus aureus (S. aureus), especially methicillin-resistant S. aureus (MRSA), remains a major public health concern. This study reports the antimicrobial resistance profiles and molecular characteristics of 31 S. aureus isolated during 2017–2018 from inpatient and outpatient clinical specimens from Queen Sirikit Naval Hospital (QSH) in Chonburi province, Thailand. All isolates were tested for antimicrobial susceptibility. Staphylococcal cassette chromosome mec (SCCmec) typing, Panton–Valentine leukocidin (pvl) toxin, pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and staphylococcal protein A (spa) typing were performed. Twenty-seven isolates were confirmed to be MRSA and exhibited resistance to up to seven antibiotics classes. The main MLST type was SCCmec type II (51.9%) and ST764 (55.6%). Five spa types were identified with t045 (55.6%) as the major type. All 31 S. aureus isolates were grouped into seven types using PFGE with the SCCmecII-ST764-t045 clone being the most prevalent. Overall, our findings reveal that the S. aureus isolates in this study differ from previous reports in Thailand, indicating a potential shift in local strains, highlighting the need for ongoing molecular surveillance of multidrug resistance patterns of MRSA in Southeast Asia. Full article
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12 pages, 228 KiB  
Article
Determination of Minimum Inhibitory Concentrations of Selected Antibiotics Against Trueperella pyogenes Originated from Bovine Clinical Endometritis
by Ottó Szenci, Ákos Jerzsele, Zoltán Somogyi, Ádám Kerek, Attila Répási, Lea Lénárt and László Makrai
Pathogens 2025, 14(5), 405; https://doi.org/10.3390/pathogens14050405 - 24 Apr 2025
Viewed by 183
Abstract
Bacteriological examination of uterine secretions provides essential information for the prevalence of bovine uterine pathogens and their influence on fertility. The objective of the present study was to determine the uterine pathogens in cases of clinical endometritis in two Holstein-Friesian dairy farms between [...] Read more.
Bacteriological examination of uterine secretions provides essential information for the prevalence of bovine uterine pathogens and their influence on fertility. The objective of the present study was to determine the uterine pathogens in cases of clinical endometritis in two Holstein-Friesian dairy farms between 21 and 27 days after calving and the minimum inhibitory concentration (MIC) of 14 antimicrobials for Trueperella pyogenes (T. pyogenes). Overall, the prevalence of T. pyogenes (Farms A and B) was 46.3% and 22% (p < 0.01), respectively. In contrast, Farm B had significantly more cases (p < 0.001) of Escherichia coli, but the distribution of uterine pathogens was similar. Regarding the prevalence of any bacteria, Farm B also had significantly more bacteria (p < 0.001) than Farm A. T. pyogenes isolates were highly susceptible to amoxicillin, amoxicillin/clavulanic acid, tylosin, and cephalosporins, such as ceftiofur, cefquinome, and cephalexin with MIC90 of ≤2 μg/mL. At the same time, MIC90 of tulathromycin, lincomycin, and florfenicol were between 4 and 8 μg/mL and of doxycycline, enrofloxacin, oxytetracycline, and gentamicin, were between 16 and 32 μg/mL, respectively. Meanwhile, sulfamethoxazole/trimethoprim showed the highest MIC90 (>32 μg/mL). In summary, T. pyogenes with high MIC90 against oxytetracycline, gentamicin, and sulfamethoxazole/trimethoprim were found, which calls attention to the prudent use of antibiotics. Full article
(This article belongs to the Special Issue Current Progress on Bacterial Antimicrobial Resistance)
13 pages, 3898 KiB  
Article
Enhanced Biofilm Disruption in Methicillin-Resistant Staphylococcus aureus Using Rifampin and Fluoroquinolone Combinations
by Yu Ri Kang, Joo-Young Park, Doo Ryeon Chung, Minhee Kang, Jae-Hoon Ko, Kyungmin Huh, Sun Young Cho, Cheol-In Kang and Kyong Ran Peck
Pathogens 2025, 14(5), 404; https://doi.org/10.3390/pathogens14050404 - 23 Apr 2025
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Abstract
Staphylococcus aureus biofilms complicate the treatment of device-related infections. We hypothesized that combining rifampin with fluoroquinolones could eradicate biofilms even in antimicrobial-resistant S. aureus strains. We determined the synergistic interactions of these combinations in a biofilm model. Thirty methicillin-resistant S. aureus (MRSA) isolates [...] Read more.
Staphylococcus aureus biofilms complicate the treatment of device-related infections. We hypothesized that combining rifampin with fluoroquinolones could eradicate biofilms even in antimicrobial-resistant S. aureus strains. We determined the synergistic interactions of these combinations in a biofilm model. Thirty methicillin-resistant S. aureus (MRSA) isolates with varying susceptibility profiles were evaluated. Minimum biofilm eradication concentrations (MBECs) were determined using the Calgary Biofilm Device, and the synergy was assessed using the fractional biofilm eradication concentration (FBEC) index. Scanning electron microscopy (SEM) was performed on one strain, and confocal laser scanning microscopy (CLSM) was conducted on four strains for visualizing and evaluating the biofilm viability. The MBEC90 for rifampin and levofloxacin were 512 mg/L and 256 mg/L, respectively, and exceeded 1024 mg/L for ciprofloxacin. Synergy was observed in 56.7% of strains for both the rifampin + ciprofloxacin and rifampin + levofloxacin combinations, with no difference between the combinations. A higher ciprofloxacin MBEC (≥16 mg/L) increased the likelihood of synergy with rifampin by 18-fold. SEM and CLSM analyses in a subset of strains confirmed the enhanced biofilm disruption with rifampin + ciprofloxacin compared to ciprofloxacin alone. Our findings suggest that rifampin combined with ciprofloxacin or levofloxacin may synergistically eradicate MRSA biofilms, offering a potential treatment option for device-related infections when alternatives are limited. Full article
(This article belongs to the Special Issue Antibiotics, Antibiotic Alternatives, and Combination Antimicrobial Therapy (Volume 2))
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Brief Report
Molecular Differentiation and Detection of AMR Genes from Nosocomial Staphylococcus spp.
by Isabelle Carneiro, Wellington Luiz Pontes da Silva, Debora Ribeiro de Souza Santos and Ivano de Filippis
Pathogens 2025, 14(5), 403; https://doi.org/10.3390/pathogens14050403 - 23 Apr 2025
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Abstract
Staphylococcus spp. is a major nosocomial pathogen, particularly affecting immunocompromised patients and infants. It is associated with bacteremia, endocarditis, and co-infections. Methicillin-resistant Staphylococci (MRS) carry the mecA gene, encoding PBP2a, which confers resistance to beta-lactam antibiotics. The aim of this study is to [...] Read more.
Staphylococcus spp. is a major nosocomial pathogen, particularly affecting immunocompromised patients and infants. It is associated with bacteremia, endocarditis, and co-infections. Methicillin-resistant Staphylococci (MRS) carry the mecA gene, encoding PBP2a, which confers resistance to beta-lactam antibiotics. The aim of this study is to investigate resistance profiles and develop a molecular method to identify nosocomial Staphylococcus spp. strains. A total of 64 strains from public hospitals in Rio de Janeiro were analyzed using phenotypic and molecular methods, with 17 classified as MDR. Different melting temperatures (Tm) were obtained through qPCR-HRM analysis, to identify S. aureus- (70.4 °C), S. haemolyticus- (79 °C), S. epidermidis- (74.1 °C) and mecA (70.5 °C)-positive strains (MRS). The mecA gene was detected in 51 strains, with 22 showing SCCmec type IV. The spread of MRSA and MDR Staphylococci, particularly MDR S. haemolyticus, is a growing concern. In our study, among 64 Staphylococci strains, only 11 were susceptible to methicillin, showing the continuous emergence of resistant strains. qPCR-HRM is a cost-effective, sensitive and fast method for rapid Staphylococcus spp. identification, aiding in nosocomial infection control. Full article
(This article belongs to the Special Issue Nosocomial Infections and Antimicrobial Resistance: Challenges and Solutions)
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