Pathogens

Advances in the knowledge of the pathogenesis of SARS-CoV-2 allowed the survival of COVID-19 patients in intensive care units. However, due to the clinical characteristics of severe patients, they resulted in the appearance of colonization events. Therefore, we speculate that strains of Candida spp. isolated from COVID-19 patients have virulent genetic and phenotypic backgrounds involved in clinical worsening of patients. The aim of this work was to virutype Candida spp. strains isolated from colonized COVID-19 patients, analyze their genomic diversity, and establish clonal dispersion in care areas. The virulent potential of Candida spp. strains isolated from colonized COVID-19 patients was determined through adhesion tests and the search for genes involved with adherence and invasion. Clonal association was done by analysis of intergenic spacer regions. Six species of Candida were involved as colonizing pathogens in COVID-19 patients. The genotype analysis revealed the presence of adherent and invasive backgrounds. The distribution of clones was identified in the COVID-19 care areas, where C. albicans was the predominant species. Evidence shows that Candida spp. have the necessary genetic tools to be able colonize the lungs, and could be a possible causal agent of coinfections in COVID-19 patients. The detection of dispersion of opportunistic pathogens can be unnoticed by classical epidemiology. Epidemiological surveillance against opportunistic fungal pathogens in COVID-19 patients is an immediate need, since the findings presented demonstrate the potential virulence of Candida spp. Full article

Diagnosing neurocysticercosis (NCC) is difficult due to its variable clinical presentations and the different imaging techniques used to detect brain damage. This study aimed to evaluate the use of cerebrospinal fluid serology and PCR for diagnosing biological neurocysticercosis in a non-endemic country. We tested samples from patients living in France with suspected NCC and confirmed that 45 of the patients presented with the disease. A total of 89% of patients had previously traveled to countries where the disease was endemic. The sensitivity of Western blots compared to ELISA was not significantly different (80% vs. 60%) (p > 0.05), and neither was the sensitivity of Western blots vs. PCR (78% vs. 56%) (p > 0.05). However, the PCR sensitivity was higher in definitive NCC (81%) than in probable NCC (44%; p < 0.05). PCR tests using cerebrospinal fluid should be considered as a diagnostic criterion for identifying NCC. Full article

Human Lyme borreliosis (LB) represents a multisystem disorder that can progress in stages. The causative agents are transmitted by hard ticks of the Ixodes ricinus complex that have been infected with the spirochete Borrelia burgdorferi sensu lato. Today, LB is considered the most important human tick-borne illness in the Northern Hemisphere. The causative agent was identified and successfully isolated in 1982 and, shortly thereafter, antibiotic treatment was found to be safe and efficacious. Since then, various in vitro studies have been conducted in order to improve our knowledge of the activity of antimicrobial agents against B. burgdorferi s. l. The full spectrum of in vitro antibiotic susceptibility has still not been defined for some of the more recently developed compounds. Moreover, our current understanding of the in vitro interactions between B. burgdorferi s. l. and antimicrobial agents, and their possible mechanisms of resistance remains very limited and is largely based on in vitro susceptibility experiments on only a few isolates of Borrelia. Even less is known about the possible mechanisms of the in vitro persistence of spirochetes exposed to antimicrobial agents in the presence of human and animal cell lines. Only a relatively small number of laboratory studies and cell culture experiments have been conducted. This review summarizes what is and what is not known about the in vitro susceptibility of B. burgdorferi s. l. It aims to shed light on the known unknowns that continue to fuel current debates on possible treatment resistance and mechanisms of persistence of Lyme disease spirochetes in the presence of antimicrobial agents. Full article

Human papillomaviruses are small DNA tumor viruses that infect cutaneous and mucosal epithelia. The viral lifecycle is linked to the differentiation status of the epithelium. During initial viral infection, the genomes replicate at a low copy number but the mechanism(s) the virus uses to control the copy number during this stage is not known. In this study, we demonstrate that the tyrosine kinase focal adhesion kinase (FAK) binds to and phosphorylates the high-risk viral E2 protein, the key regulator of HPV replication. The depletion of FAK with a specific PROTAC had no effect on viral DNA content in keratinocytes that already maintain HPV-16 and HPV-31 episomes. In contrast, the depletion of FAK significantly increased HPV-16 DNA content in keratinocytes infected with HPV-16 quasiviruses. These data imply that FAK prevents the over-replication of the HPV genome after infection through the interaction and phosphorylation of the E2 protein. Full article

Clostridium perfringens is a versatile pathogen, inducing diseases in the skin, intestine (such as chicken necrotic enteritis (NE)), and other organs. The classical sign of NE is the foul smell gas in the ballooned small intestine. We hypothesized that deoxycholic acid (DCA) reduced NE by inhibiting C. perfringens virulence signaling pathways. To evaluate the hypothesis, C. perfringens strains CP1 and wild-type (WT) HN13 and its mutants were cultured with different bile acids, including DCA and isoallolithocholic acid (isoalloLCA). Growth, hydrogen sulfide (H₂S) production, and virulence gene expression were measured. Notably, isoalloLCA was more potent in reducing growth, H₂S production, and virulence gene expression in CP1 and WT HN13 compared to DCA, while other bile acids were less potent compared to DCA. Interestingly, there was a slightly different impact between DCA and isoalloLCA on the growth, H₂S production, and virulence gene expression in the three HN13 mutants, suggesting possibly different signaling pathways modulated by the two bile acids. In conclusion, DCA and isoalloLCA reduced C. perfringens virulence by transcriptionally modulating the pathogen signaling pathways. The findings could be used to design new strategies to prevent and treat C. perfringens-induced diseases. Full article

To explore and profile the level of cytokines in the sera of patients infected with Schistosoma japonicum to explore the helper T-cell response of patients either at the chronic or advanced stage of the disease. We randomly selected 58 subjects from several areas endemic for schistosomiasis japonica in China and collected serum samples to be tested for 18 different cytokines secreted by (1) Th1/Th2 cells (GM-CSF, IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-12p70, IL-10, IL-13, IL-18 and TNF-α) and (2) Th9/Th17/Th22/Treg cells (IL-9, IL-17A, IL-21, IL-22, IL-23 and IL-27). The Th1/Th2 cytokines in chronic patients were not significantly different from those in healthy people, while patients with advanced schistosomiasis had higher levels of IL-2, IL-23 and IL-27 and lower levels of IL-18 and IFN-γ. With respect to the Th9/Th17/Th22/Treg cell cytokines, there were higher levels of IL-23. Thus, a limited variation of the cytokine response between the three patient groups was evident, but only in those with advanced infection, while there was no difference between chronic schistosomiasis infection and healthy subjects in this respect. The cytokine expression should be followed in patients with advanced schistosomiasis who show a cytokine pattern of a weakened Th1 cell response and an increased Th17 response. Full article

A two-year longitudinal cohort study was conducted on a total of 407 purebred Chios and Lacaune ewes from four intensive dairy sheep farms to assess potential risk factors for small ruminant lentiviruses (SRLVs) seropositivity. Ewes were serologically tested semiannually at pre-mating and pre-lambing, and their age, breed, and body condition score (BCS) were recorded. Εwes were categorized as constantly seronegative, constantly seropositive, seroconverted, seroreverted, or animals with an intermittent presence of antibodies. Mixed binary logistic regression models were used to estimate the adjusted relative risks of the studied risk factors for (i) the individual ewes’ seropositivity, (ii) the manifestation of specific serological patterns, and (iii) the occurrence of seroconversion and seroreversion incidents. Increased age was associated with seropositivity and constantly seropositive status (p < 0.001 in both cases). On the other hand, age was negatively associated with constantly seronegative pattern, seroconversion incident, and the intermittent presence of antibodies (p < 0.05 in all cases). Moreover, breed was recognized as a risk factor: Lacaune ewes demonstrated increased seropositivity, whereas Chios ewes were more likely to demonstrate an intermittent presence of antibodies (p < 0.01 in both cases). Seropositive status (p < 0.001), seropositivity in animals with an intermittent presence of antibodies (p = 0.001), and seroconversion incidents (p < 0.001) were significantly increased at pre-lambing compared to pre-mating. The risk factors recognized in our study contribute to a better understanding of SRLVs epidemiology and the evidence-based designation of SRLVs’ control programs in intensive dairy sheep farms in Greece. Full article

Rotavirus is a major cause of diarrhea globally in animals and young children under 5 years old. Here, molecular detection and genetic characterization of porcine rotavirus in smallholder and commercial pig farms in the Lusaka Province of Zambia were conducted. Screening of 148 stool samples by RT-PCR targeting the VP6 gene revealed a prevalence of 22.9% (34/148). Further testing of VP6-positive samples with VP7-specific primers produced 12 positives, which were then Sanger-sequenced. BLASTn of the VP7 positives showed sequence similarity to porcine and human rotavirus strains with identities ranging from 87.5% to 97.1%. By next-generation sequencing, the full-length genetic constellation of the representative strains RVA/pig-wt/ZMB/LSK0137 and RVA/pig-wt/ZMB/LSK0147 were determined. Genotyping of these strains revealed a known Wa-like genetic backbone, and their genetic constellations were G4-P[6]-I5-R1-C1-M1-A8-N1-T1-E1-H1 and G9-P[13]-I5-R1-C1-M1-A8-N1-T1-E1-H1, respectively. Phylogenetic analysis revealed that these two viruses might have their ancestral origin from pigs, though some of their gene segments were related to human strains. The study shows evidence of reassortment and possible interspecies transmission between pigs and humans in Zambia. Therefore, the “One Health” surveillance approach for rotavirus A in animals and humans is recommended to inform the design of effective control measures. Full article

Spontaneous HBsAg seroclearance has been mainly studied in populations from Asia, Australia, the Pacific Islands, and Polynesia. For the first time, we evaluated the spontaneous HBsAg seroclearance and its possible associated factors and the risk of disease progression in HBeAg-negative patients with inactive infection all coming from the same region in South Italy. In this multicenter retrospective study, 146 patients were selected after 18 months of observation and followed for a median of 82 months (IQR 60–107). For our analyses, they were divided into three groups based on their HBsAg levels: <100 IU/mL, 100–1000 IU/mL, and >1000 IU/mL. Crude and adjusted hazard ratios (HRs) for HBsAg seroclearance were determined. During the follow-up period, three patients (2.0%) showed a disease progression with an increased liver stiffness, whereas 17 (11.6%) cleared the HBsAg. Patients with HBsAg levels <100 IU/mL had the highest probability of HBsAg seroclearance compared to the other two groups (p = 0.009). In the multivariate analysis, the HBsAg level <100 IU/mL was the only parameter independently associated with HBsAg seroclearance (adjusted HR = 3.53; CI 1.29–9.69; p = 0.01). In patients with chronic HBV inactive infection, HBsAg levels <100 IU/mL predicted the highest probability of HBsAg seroclearance. Full article

This study aimed to investigate the histopathological changes associated with SARS-CoV-2 infection in placentas. A case series of anatomopathological analysis was conducted on the placentas of pregnant women with SARS-CoV-2 who delivered between March and December 2020 at Santo Amaro Hospital (HSA) in Salvador, Brazil. Out of the 29 placentas examined, the median weight was 423.0 (IQR: 385.0–521.0) g. Among them, 58.3% (n = 14) had inadequate weight relative to the newborn’s weight. The histopathological findings revealed that 86.2% (n = 25) of the placentas had poorly defined lobes, and the fetal and maternal surface color was normal in 89.7% (n = 26) and 93.1% (n = 27), respectively. Additionally, 51.7% (n = 15) of the umbilical cords displayed hypercoiling. The most frequent microscopic finding was infarction, present in 35.3% (n = 6) of the cases, followed by 11.8% (n = 2) for each of chorioamnionitis, chronic villitis, focal perivillositis, and laminar necrosis. Analysis of the umbilical cords identified 23.5% (n = 4) cases of intervillous thrombosis, while amnion analysis showed 13.8% (n = 4) cases of squamous metaplasia. Extraplacental membrane examination revealed fibrin deposition in 93.1% (n = 27) of the cases, necrosis in 62.0% (n = 18), calcifications in 51.7% (n = 15), cysts in 37.9% (n = 11), neutrophilic exudate in 17.2% (n = 5), thrombosis in 13.7% (n = 4), and delayed placental maturation in 6.9% (n = 2). All analyzed placentas exhibited histopathological changes, primarily vascular and inflammatory, which indicate SARS-CoV-2 infection in term pregnancies. These alterations could be associated with impaired placental function, fetal growth restriction, preeclampsia, and prematurity. However, further prospective studies are required to validate the type, prevalence, and prognosis of each of these changes. Full article

The detection of hepatitis E virus (HEV) RNA is the gold standard for HEV infection diagnosis. In order to address the quality control requirements for HEV RNA detection kits within China, we aimed to establish the first Chinese national standard for HEV RNA detection through a collaborative study. The candidate standard was quantified using digital PCR (dPCR). A total of five laboratories were invited to determine the estimated mean value of this national standard relative to the World Health Organization International Standard (WHO IS). Additionally, four commercial kits were used to assess the applicability of the candidate standard. The stability was determined by freeze–thaw cycles and storage at 37 °C, 25 °C and 4 °C. The estimated mean value of this national standard relative to the WHO IS was 5.67 log₁₀ IU/mL. Two out of the four commercial kits can detect as low as the estimated limit of detection (LOD). The degradation rates of samples in the stability study ranged from 4% to 19%. In conclusion, we have established the first Chinese national standard for HEV nucleic acid detection against WHO IS, which can be employed to evaluate the quality of HEV RNA detection kits. Full article

Oral microbiome disruptions in periodontitis are related to the chronic inflammatory reactions that could in turn lead to the development of multiple oral diseases. The objective of the study was to assess the frequencies of Streptococcus mitis, Prevotella melaninogenica, and Prevotella intermedia in oral benign lesions, oral potentially malignant disorders (OPMDs), and oral squamous cell carcinomas (OSCCs) and investigate the impact of these bacteria on the expression patterns of the selected (potential) target genes (PI3CA/AKT2/mTOR, DUSP16/MAPK14, and COX2). After sample collection (25 benign lesions, 30 OPMDs, and 35 OSCCs) and DNA/RNA extraction, quantitative real-time polymerase chain reaction (qPCR) was performed to detect bacterial presence and assess relative gene expression levels in different lesion groups. Prevotella melaninogenica was the most prevalent of the three analyzed bacteria, with the frequency being 60% in benign lesions, 87% in OPMDs (p = 0.024), and 77% in OSCC. The OPMD tissues in which Prevotella melaninogenica was present exhibited a higher expression level of AKT2 (p = 0.042). Significantly lower expression of DUSP16 was observed in OSCC tissues containing Streptococcus mitis (p = 0.011). The obtained results indicate a substantial contribution of P. melaninogenica and Str. mitis in the pathogenesis of oral mucosal lesions, possibly via AKT2 upregulation and DUSP16 downregulation. Full article

Naturally abundant antimicrobial lipids, such as fatty acids and monoglycerides, that disrupt membrane-enveloped viruses are promising mitigants to inhibit African swine fever virus (ASFV). Among mitigant candidates in this class, glycerol monolaurate (GML) has demonstrated particularly high antiviral activity against laboratory-adapted ASFV strains. However, there is an outstanding need to further determine the effects of GML on wild-type ASFV strains, which can have different virulence levels and sensitivities to membrane-disrupting compounds as compared to laboratory-adapted strains. Herein, we investigated the antiviral effects of GML on a highly virulent strain of a wild-type ASFV isolate (Armenia/07) in an in vitro porcine macrophage model. GML treatment caused a concentration-dependent reduction in viral infectivity, and there was a sharp transition between inactive and active GML concentrations. Low GML concentrations had negligible effect on viral infectivity, whereas sufficiently high GML concentrations caused a >99% decrease in viral infectivity. The concentration onset of antiviral activity matched the critical micelle concentration (CMC) value of GML, reinforcing that GML micelles play a critical role in enabling anti-ASFV activity. These findings validate that GML can potently inhibit wild-type ASFV infection of porcine macrophages and support a biophysical explanation to guide antimicrobial lipid performance optimization for pathogen mitigation applications. Full article

Leptospirosis is a global zoonosis caused by pathogenic bacteria of the genus Leptospira. The application of the CRISPR/Cas9 system has facilitated the generation of mutants and subsequent evaluation of phenotypes. Since DNA breaks induced by RNA-guided Cas9 nuclease are lethal to Leptospira, different methodologies were implemented to overcome this limitation. Initially, CRISPR interference (CRISPRi) was employed to create knockdown mutants, utilizing a catalytically inactive Cas9 (dCas9). Subsequently, the co-expression of CRISPR/Cas9 and a DNA repair system from Mycobacterium smegmatis enabled the generation of scarless knockout mutants. We eliminated plasmids from the lipL32 knockout L. interrogans strain and further achieved multiple gene mutations via gene silencing in this knockout background. Strains lacking both LipL41 and LipL32 and LigA, LigB, and LipL32, were evaluated. The absence of proteins LipL32 and LipL41 had no effect on leptospiral virulence. On the other hand, mutants lacking LigA, LigB, and LipL32 were unable to cause acute disease. The expanded apparatus for genetic manipulation of pathogenic leptospires via the CRISPR/Cas9 system has allowed the evaluation of multiple mutations upon leptospiral virulence. This work shows that LipL32 and LipL41 are not required for acute disease and consolidates LigA and LigB proteins as virulence factors. Full article

Phlebotomine sand flies (Diptera: Psychodidae) are the principal vectors of phleboviruses and Leishmania spp., the causative agents of leishmaniases. The Mediterranean sand fly fauna is diverse, and leishmaniasis, mainly caused by Leishmania infantum, is endemic in the Balkan countries. Despite recent entomological surveys, only some districts of Kosovo have been sampled for sand flies, with no proof/confirmation of L. infantum. This study aimed to gain further insights into the species composition of natural sand fly populations in previously unsampled districts and areas in Kosovo without reports of leishmaniasis and to detect Leishmania DNA in sand flies. A sand fly survey was conducted in 2022 in all seven districts of Kosovo. Collected females were screened for Leishmania DNA by PCR. Positive samples were sequenced and subjected to maximum likelihood analysis with reference sequences for further molecular characterization. The trapping activities at 114 different localities resulted in 3272 caught specimens, comprising seven sand fly species of two genera, namely Phlebotomus neglectus, Ph. perfiliewi, Ph. tobbi, Ph. papatasi, Ph. simici, Ph. balcanicus and Sergentomyia minuta. Leishmania infantum DNA was detected in three individual sand flies of Ph. neglectus and Ph. perfiliewi. This study provides the most extensive sand fly survey in Kosovo and reports the first record of L. infantum DNA in sand flies, indicating autochthonous circulation of L. infantum. Full article

Brucella is an intracellular parasitic bacterium that uses multiple strategies to evade the host’s defense mechanisms. However, how Brucella manipulates the host-induced oxidative stress and relevant biological processes are still poorly understood. In this study, a comparative transcriptome assay of macrophages infected with Brucella abortus S2308 and its rough mutant RB14 was performed to investigate the differentially expressed genes which might be associated with the pathogenic mechanism of Brucella. Our results showed that numerous host pro-oxidative and antioxidative stress genes were differentially expressed in macrophages infected with B. abortus S2308 and mutant RB14 at 4, 8, 24, and 48 h post-infection. Interestingly, we found that several ferroptosis-associated genes were differentially expressed during B. abortus RB14 infection. Moreover, we found that the rough mutant RB14-induced macrophage death was associated with reduced levels of host glutathione and glutathione peroxidase 4, together with increased free iron, lipid peroxidation, and ROS, all of which are important hallmarks of ferroptosis. The ferroptosis occurring during infection with RB14 was reduced by treatment with the inhibitor ferrostatin-1. However, B. abortus S2308 infection did not induce these hallmarks of ferroptosis. Taken together, our results demonstrate that ferroptosis is involved in rough B. abortus infection. Investigating how Brucella manipulates oxidative stress and ferroptosis in its host will be helpful to clarify the pathogenicity of B. abortus. Full article

Staphylococci, including Staphylococcus aureus and Staphylococcus epidermidis, are important human pathogens associated with potentially life-threatening infections. Their great biofilm-producing ability and the development of resistance mechanisms often account for therapeutic failure. Hence, the scientific community has devoted intensive efforts to the development of antimicrobial compounds active against both planktonic and sessile bacterial populations. Contextually, antimicrobial peptides (AMPs) are natural peptides produced by the innate immunity of every organism, representing a potential new therapeutic solution against human microbial pathogens. Our work focused on the in vitro activity of Oreoch-1, an AMP from the gills of Nile tilapia (Oreochromis niloticus), against standard and clinical S. aureus and S. epidermidis strains. Firstly, the cytotoxicity profile of Oreoch-1 was determined in human colon carcinoma cells. Secondly, its antibacterial spectrum was explored against staphylococcal strains to set up the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC). Our results highlighted an antibacterial activity in the range 6.25–25 μM, with a general bacteriostatic effect. Therefore, the biofilm-inhibitory property was assessed against S. aureus ATCC 25923 and S. epidermidis ATCC 35984, indicating a significant reduction in S. aureus biomass at sub-MIC concentrations. Overall, our study indicates Oreoch-1 as a promising new therapeutic weapon against staphylococcal infections. Full article