Microorganisms, Volume 12, Issue 4 (April 2024) – 209 articles

Microbial degradation of feathers offers potential for bioremediation, yet the microbial response mechanisms warrant additional investigation. In prior work, Pseudomonas aeruginosa Gxun-7, which demonstrated robust degradation of feathers at elevated concentrations, was isolated. However, the molecular mechanism of this degradation remains only partially understood. To investigate this, we used RNA sequencing (RNA-seq) to examine the genes that were expressed differentially in P. aeruginosa Gxun-7 when exposed to 25 g/L of feather substrate. The RNA-seq analysis identified 5571 differentially expressed genes; of these, 795 were upregulated and 603 were downregulated. Upregulated genes primarily participated in proteolysis, amino acid, and pyruvate metabolism. Genes encoding proteases, as well as those involved in sulfur metabolism, phenazine synthesis, and type VI secretion systems, were notably elevated, highlighting their crucial function in feather decomposition. Integration of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) taxonomies, combined with a review of the literature, led us to propose that metabolic feather degradation involves environmental activation, reducing agent secretion, protease release, peptide/amino acid uptake, and metabolic processes. Sulfite has emerged as a critical activator of keratinase catalysis, while cysteine serves as a regulatory mediator. qRT–PCR assay results for 11 selected gene subset corroborated the RNA-seq findings. This study enhances our understanding of the transcriptomic responses of P. aeruginosa Gxun-7 to feather degradation and offers insights into potential degradation mechanisms, thereby aiding in the formulation of effective feather waste management strategies in poultry farming. Full article

Representatives of the genus Bifidobacterium are widely used as probiotics to modulate the gut microbiome and alleviate various health conditions. The action mechanisms of probiotics rely on their direct effect on the gut microbiota and the local and systemic effect of its metabolites. The main purpose of this animal experiment was to assess the biosafety of the Bifidobacterium longum strain BIOCC1719. Additional aims were to characterise the influence of the strain on the intestinal microbiota and the effect on several health parameters of the host during 15- and 30-day oral administration of the strain to mice. The strain altered the gut microbial community, thereby altering luminal short-chain fatty acid metabolism, resulting in a shift in the proportions of acetic, butyric, and propionic acids in the faeces and serum of the test group mice. Targeted metabolic profiling of serum revealed the possible ability of the strain to positively affect the hosts’ amino acids and bile acids metabolism, as the cholic acid, deoxycholic acid, aspartate, and glutamate concentration were significantly higher in the test group. The tendency to increase anti-inflammatory polyamines (spermidine, putrescine) and neuroprotective 3-indolepropionic acid metabolism and to lower uremic toxins (P-cresol-SO₄, indoxyl-SO₄) was registered. Thus, B. longum BIOCC1719 may exert health-promoting effects on the host through modulation of the gut microbiome and the host metabolome via inducing the production of health-promoting bioactive compounds. The health effects of the strain need to be confirmed in clinical trials with human volunteers. Full article

The worldwide reduction in the use of antibiotics in animal feed is fueling the need for alternatives for the prevention and control of poultry intestinal diseases such as necrotic enteritis (NE), which is caused by Clostridium perfringens. This is the first report on the use of an intestinal epithelial chicken cell line (CHIC-8E11) to study the pathogenic traits of C. perfringens and to investigate the mode of action of cell-free supernatants (CFS) from probiotic Lactobacillus acidophilus AG01 and Bifidobacterium animalis subsp. lactis AG02 in reducing the pathogenicity of C. perfringens. The cell adhesion, permeability and cytotoxicity were assessed under challenge with four C. perfringens strains isolated from broiler NE episodes of differing geographical origin (CP1–UK; CP10–Sweden; 25037–CP01 and CP22–USA). All the C. perfringens strains could adhere to the CHIC-8E11 cells, with varying affinity (0.05–0.48% adhesion across the strains). The CFS from one out of two strains (CP22) increased the cell permeability (+4.5-fold vs. the control, p < 0.01), as measured by the fluorescein isothiocyanate-dextran (FD4) content, with NetB toxin implicated in this effect. The CFS from all the strains was cytotoxic against the CHIC-8E11 cells in a dose- and strain-dependent manner (cytotoxicity 23–62% across the strains when dosed at 50 µL/mL, as assessed by the MTT cell viability assay). Pre-treatment of the cells with CFS from B. animalis subsp. lactis AG02 but not L. acidophilus AG01 reduced the cell adhesion of three out of four C. perfringens strains (by 77–85% vs. the control, p < 0.001) and reduced the negative effect of two NetB-positive strains on the cell permeability. The CFS of both probiotics alleviated the cytotoxicity of all the C. perfringens strains, which was dependent on the dose. The results confirm the suitability of the CHIC-8E11 cell line for the study of host–pathogen cell interactions in the context of NE caused by C. perfringens and reveal a beneficial mode of action of B. animalis subsp. lactis AG02 in reducing C. perfringens cell adhesion and, together with L. acidophilus AG01, in reducing C. perfringens cytotoxicity. Full article

This research examined the positivity ratio of Crimean–Congo haemorrhagic fever (CCHF) antibodies in cattle and sheep within Namibia’s Omaheke region after a human disease outbreak in the same geographical area. A total of 200 samples (100 cattle and 100 sheep) were randomly collected from animals brought to two regional auction sites, and then tested using the ID Screen^® CCHF Double Antigen Multi-Species Enzyme-Linked Immunosorbent Assay kit. Of the cattle samples, 36% tested positive, while 22% of the sheep samples were seropositive. The cattle had a significantly higher positivity ratio than sheep at the individual animal level (p = 0.0291). At the herd level, 62.5% of cattle herds and 45.5% of sheep flocks had at least one positive animal, but this difference was statistically insignificant (p = 0.2475). The fourteen cattle farms with at least one seropositive animal were dispersed across the Omaheke region. In contrast, the ten sheep farms with seropositive cases were predominantly situated in the southern half of the region. The study concluded that the CCHF is endemic in the Omaheke region and likely in most of Namibia, underscoring the importance of continued surveillance and preventive measures to mitigate the impact of CCHFV on animal health and potential spillover into human populations. Full article

In the world’s first pig-to-human cardiac cytomegalovirus (PCMV), xenotransplant and elevated levels of porcine key factors contributing to patient mortality were considered. This has renewed attention on PCMV, a virus widely prevalent in pigs. Currently, there are no effective drugs or vaccines targeting PCMV, and its high detection difficulty poses challenges for prevention and control research. In this study, antiviral small hairpin RNA (shRNA) was selected and inserted into the Rosa26 and miR-17-92 loci of pigs via a CRISPR/Cas9-mediated knock-in strategy. Further in vitro viral challenge experiments demonstrated that these genetically edited pig cells could effectively limit PCMV replication. Through this process, we constructed a PCMV-infected cell model, validated partial viral interference sites, enhanced gene knock-in efficiency, performed gene editing at two different gene loci, and ultimately demonstrated that RNA interference (RNAi) technology combined with CRISPR/Cas9 has the potential to generate pig cells with enhanced antiviral infection capabilities. This opens up possibilities for the future production of pig populations with antiviral functionalities. Full article

The benefits of probiotics on dysbiotic microbiomes and inflammation are dependent on the tested strain, host factors, and the resident microbiome. There is limited knowledge on the effects of probiotics in A. actinomycetemcomitans-associated periodontitis. Thus, Lactobacillus acidophilus LA5 (LA5) was orally inoculated for 30 days in C57Bl/6 mice infected with A. actinomycetemcomitans JP2 (Aa) and S. gordonii (Sg). Alveolar bone loss, gingival gene expression, and oral and gut microbiomes were determined. LA5 controlled bone loss in Aa+Sg-infected mice, downregulated the expression of Il-1β and upregulated Il-10 in gingival tissues, and altered the oral and gut microbiomes. LA5 increased the diversity of the oral microbiome of Aa+Sg infected mice, and Aa+Sg and Aa+Sg+LA5 oral or gut microbiomes clustered apart. LA5 induced shifts in Aa+Sg infected mice by increasing the abundance of Muribaculaceae and decreasing Bifidobacteriaceae in the oral cavity and increasing the abundance of Verrucomicrobiae and Eggerthellales in the gut. In conclusion, LA5 oral administration controls experimental Aa-associated periodontitis by altering inflammatory gene expression and the oral and gut microbiomes. Full article

Certain microalgal species can grow with different trophic strategies depending on the availability of nutrient resources. They can use the energy from light or an organic substrate, or both, and can therefore be called autotrophs, heterotrophs, or mixotrophs. We recently isolated a microalgal strain from the microplastic biofilm, which was identified as Auxenochlorella protothecoides, AS-1. Strain AS-1 grew rapidly in bacterial culture media and exhibited different growth rates and cell sizes under different trophic conditions. We compared the growth performance of AS-1 under the three different trophic modes. AS-1 reached a high biomass (>4 g/L) in 6 days under mixotrophic growth conditions with a few organic carbons as a substrate. In contrast, poor autotrophic growth was observed for AS-1. Different cell sizes, including daughter and mother cells, were observed under the different growth modes. We applied a Coulter Counter to measure the size distribution patterns of AS-1 under different trophic modes. We showed that the cell size distribution of AS-1 was affected by different growth modes. Compared to the auto-, hetero- and mixotrophic modes, AS-1 achieved higher biomass productivity by increasing cell number and cell size in the presence of organic substrate. The mechanisms and advantages of having more mother cells with organic substrates are still unclear and warrant further investigations. The work here provides the growth information of a newly isolated A. protothecoides AS-1 which will be beneficial to future downstream applications. Full article

The increasing worldwide demand for antimicrobial agents has significantly contributed to the alarming rise of antimicrobial resistance, posing a grave threat to human life. Consequently, there is a pressing need to explore uncharted environments, seeking out novel antimicrobial compounds that display exceptionally efficient capabilities. Hot springs harbor microorganisms possessing remarkable properties, rendering them an invaluable resource for uncovering groundbreaking antimicrobial compounds. In this study, thermophilic bacteria were isolated from Mahallat Hot Spring, Iran. Out of the 30 isolates examined, 3 strains exhibited the most significant antibacterial activities against Escherichia coli and Staphylococcus aureus. Furthermore, the supernatants of the isolated strains exhibited remarkable antibacterial activity, displaying notable resistance to temperatures as high as 75 °C for 30 min. It was determined that the two strains showed high similarity to the Bacillus genus, while strain Kh3 was classified as Saccharomonospora azurea. All three strains exhibited tolerance to NaCl. Bacillus strains demonstrated optimal growth at pH 5 and 40 °C, whereas S. azurea exhibited optimal growth at pH 9 and 45 °C. Accordingly, hot springs present promising natural reservoirs for the isolation of resilient strains possessing antibacterial properties, which can be utilized in disease treatment or within the food industry. Full article

Currently, there is a wide application in the literature of the use of the Fourier Transform Infrared Spectroscopy (FTIR) technique. This basic tool has also proven to be efficient for detecting molecules associated with hosts and pathogens in infections, as well as other molecules present in humans and animals’ biological samples. However, there is a crisis in science data reproducibility. This crisis can also be observed in data from experimental animal models (EAMs). When it comes to rodents, a major challenge is to carry out sanitary monitoring, which is currently expensive and requires a large volume of biological samples, generating ethical, legal, and psychological conflicts for professionals and researchers. We carried out a survey of data from the relevant literature on the use of this technique in different diagnostic protocols and combined the data with the aim of presenting the technique as a promising tool for use in EAM. Since FTIR can detect molecules associated with different diseases and has advantages such as the low volume of samples required, low cost, sustainability, and provides diagnostic tests with high specificity and sensitivity, we believe that the technique is highly promising for the sanitary and stress and the detection of molecules of interest of infectious or non-infectious origin. Full article

Pickled cabbage, a traditional fermented food rich in functional microorganisms, can effectively control hyperuricemia and gout. In this study, a Priestia megaterium ASC-1 strain with strong uric acid (UA) degradation ability was isolated from pickled cabbage. After oral administration for 15 days, ASC-1 was stably colonized in the rats in this study. ASC-1 significantly reduced UA levels (67.24%) in hyperuricemic rats. Additionally, ASC-1 alleviated hyperuricemia-related inflammatory response, oxidative stress, and blood urea nitrogen. Intestinal microbial diversity results showed that ASC-1 restored intestinal injury and gut flora dysbiosis caused by hyperuricemia. These findings suggest that P. megaterium ASC-1 may be used as a therapeutic adjuvant for the treatment of hyperuricemia. Full article

The probiotic potential of Lactobacillus helveticus LH10, derived from vinegar Pei, a brewing mixture, was assessed through genotype and phenotype analyses. The assembled genome was comprised of 1,810,276 bp and predicted a total of 2044 coding sequences (CDSs). Based on the whole genome sequence analysis, two bacteriocin gene clusters were identified, while no pathogenic genes were detected. In in vitro experiments, L. helveticus LH10 exhibited excellent tolerance to simulated gastrointestinal fluid, a positive hydrophobic interaction with xylene, and good auto-aggregation properties. Additionally, this strain demonstrated varying degrees of resistance to five antibiotics, strong antagonistic activity against four tested pathogens, and no hemolytic activity. Therefore, L. helveticus LH10 holds great promise as a potential probiotic candidate deserving further investigation for its beneficial effects on human health. Full article

Healthcare-associated infections (HAIa) and antimicrobial resistance are expected to be the next threat to human health and are most frequent in people with severe acquired brain injury (SABI), who can be more easily colonized by multidrug-resistant organisms (MDROs). The study’s aim is to investigate the impact of MDRO colonizations and infections on SABI rehabilitation outcomes. This retrospective observational study was performed in a tertiary referral specialized rehabilitation hospital. The main outcomes were the presence of carbapenemase-producing Enterobacteriaceae (CPE) colonization, type and timing of HAI and MDRO HAI, and the number of CPE transmissions. We included 48 patients, 31% carrying CPE on admission and 33% colonized during the hospitalization. A total of 101 HAI were identified in 40 patients, with an overall incidence of 10.5/1000 patient days. Some 37% of patients had at least one MDRO infection, with a MDRO infection incidence of 2.8/1000 patient days. The number of HAIs was significantly correlated with the length of stay (LOS) (r = 0.453, p = 0.001). A significant correlation was found between colonization and type of hospital room (p = 0.013). Complications and HAI significantly affected LOS. We suggest that CPE carriers might be at risk of HAI and worse outcomes compared with non-CPE carriers. Full article

This report describes acidic microbial mats containing cyanobacteria that are strongly associated to precipitated minerals in the source area of Río Tinto. Río Tinto (Huelva, Southwestern Spain) is an extreme acidic environment where iron and sulfur cycles play a fundamental role in sustaining the extremely low pH and the high concentration of heavy metals, while maintaining a high level of microbial diversity. These multi-layered mineral deposits are stable all year round and are characterized by a succession of thick greenish-blue and brownish layers mainly composed of natrojarosite. The temperature and absorbance above and below the mineral precipitates were followed and stable conditions were detected inside the mineral precipitates. Different methodologies, scanning and transmission electron microscopy, immunological detection, fluorescence in situ hybridization, and metagenomic analysis were used to describe the biodiversity existing in these microbial mats, demonstrating, for the first time, the existence of acid-tolerant cyanobacteria in a hyperacidic environment of below pH 1. Up to 0.46% of the classified sequences belong to cyanobacterial microorganisms, and 1.47% of the aligned DNA reads belong to the Cyanobacteria clade. Full article

Short chain fatty acids (SCFAs) are primarily produced in the caecum and proximal colon via the bacterial fermentation of undigested carbohydrates that have avoided digestion in the small intestine. Increasing evidence supports the critical role that SCFAs play in health and homeostasis. Microbial SCFAs, namely butyric acid, serve as a principal energy source for colonocytes, and their production is essential for gut integrity. A direct link between SCFAs and some human pathological conditions, such as inflammatory bowel disease, irritable bowel syndrome, diarrhea, and cancer, has been proposed. The direct measurement of SCFAs in feces provides a non-invasive approach to demonstrating connections between SCFAs, microbiota, and metabolic diseases to estimate their potential applicability as meaningful biomarkers of intestinal health. This study aimed to adapt a robust analytical method (liquid–liquid extraction, followed by isobutyl chloroformate derivatization and GC–MS analysis), with comparable performances to methods from the literature, and to use this tool to tackle the question of pre-analytical conditions, namely stool processing. We focused on the methodology of managing stool samples before the analysis (fresh stool or dilution in either ethanol/methanol, lyophilized stool, or RNAlater^®), as this is a significant issue to consider for standardizing results between clinical laboratories. The objective was to standardize methods for future applications as diagnostic tools. In this paper, we propose a validated GC–MS method for SCFA quantification in stool samples, including pre- and post-analytical comparison studies that could be easily used for clinical laboratory purposes. Our results show that using lyophilization as a stool-processing method would be the best method to achieve this goal. Full article

Neutrophils are the most abundant polymorphonuclear granular leukocytes in human blood and are an essential part of the innate immune system. Neutrophils are efficient cells that eliminate pathogenic bacteria and fungi, but their role in dealing with protozoan parasitic infections remains controversial. At sites of protozoan parasite infections, a large number of infiltrating neutrophils is observed, suggesting that neutrophils are important cells for controlling the infection. Yet, in most cases, there is also a strong inflammatory response that can provoke tissue damage. Diseases like malaria, trichomoniasis, leishmaniasis, Chagas disease, and amoebiasis affect millions of people globally. In this review, we summarize these protozoan diseases and describe the novel view on how neutrophils are involved in protection from these parasites. Also, we present recent evidence that neutrophils play a double role in these infections participating both in control of the parasite and in the pathogenesis of the disease. Full article

This study described 17 cases of children admitted to the Bambino Gesù Children’s Hospital with acute hepatitis of unknown origin between mid-April and November 2022. Following the World Health Organization’s working case definition of probable cases, 17 children, with a median age of 2.1 years (interquartile range: 1.0–7.1), presenting with acute hepatitis non-AE, with serum transaminase >500 IU/L, were included in the study. A pre-specified set of microbiological tests was performed on different biological specimens for all pediatric patients. All patients resulted negative for the common hepatotropic viruses. The most common pathogen detected in blood specimens was human-herpes-virus-7 (52.9%). Adenovirus was detected more frequently in stool specimens (62.5%) than in respiratory (20.0%) or blood samples (17.6%). Regarding Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection, one child tested positive two days after admission, while antibodies against spike and nucleoprotein were present in 82.3% of patients. A co-pathogen detection was observed in 94.1% of children. Overall, 16 children recovered without clinical complications, while one patient required liver transplantation. In these cases of acute hepatitis of unknown origin, adenovirus was mainly detected in stool samples. A co-pathogen detection was also frequently observed, suggesting that the etiology of this acute hepatitis is most probably multifactorial. Full article

Training food handlers is essential to ensure food safety. However, the efficacy of training programs relying solely on theoretical information remains uncertain and often fails to induce significant changes in inadequate food practices. Training programs in good hygiene and food safety practices that integrate theoretical and practical approaches have emerged as a vital tool, enabling food handlers to apply their knowledge during work hours and clarify doubts. This study aimed to assess the impact of food safety training based on theoretical and on-the-job training on the microbiological counts of equipment, surfaces, utensils, and food handler (FH) hands. The hygiene and food safety conditions of four restaurants were analyzed through facility checklists, employee questionnaires, and microbiological analyses conducted before and after training. Eight sample collection moments were conducted at each restaurant before and after training. The pre-training results indicate that 15% and 26% of analyses for Enterobacteriaceae and total mesophilic aerobic bacteria (TMB), respectively, did not comply with hygiene safety limits. Additionally, 31% and 64% of Enterobacteriaceae and TMB values, respectively, exceeded safety limits on food handler hands. Positive cases of coagulase-positive Staphylococcus (CoPS) resulted from unprotected wounds on some FH hands. The presence of Listeria monocytogenes in drains was also identified as a concern. Following training, significant differences in results were observed. In many cases, there was a reduction of over 80% in microbial load for Enterobacteriaceae and TMB collected from equipment, surfaces, utensils, and food handler hands. The presence of L. monocytogenes in drains was also eliminated after food safety training. In conclusion, this study underscores the importance of effective training in improving food safety practices. Full article

Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) is one of the important zoonotic and opportunistic pathogens. In recent years, there has been growing evidence that supports the potential role of S. zooepidemicus in severe diseases in horses and other animals, including humans. Furthermore, the clinical isolation and drug resistance rates of S. zooepidemicus have been increasing yearly, leading to interest in its in-depth genomic analysis. In order to deepen the understanding of the S. zooepidemicus characteristics and genomic features, we investigated the genomic islands, mobile genetic elements, virulence and resistance genes, and phenotype of S. zooepidemicus strain ZHZ 211 (ST147), isolated from an equine farm in China. We obtained a 2.18 Mb, high-quality chromosome and found eight genomic islands. According to a comparative genomic investigation with other reference strains, ZHZ 211 has more virulence factors, like an iron uptake system, adherence, exoenzymes, and antiphagocytosis. More interestingly, ZHZ 211 has acquired a mobile genetic element (MGE), prophage Ph01, which was found to be in the chromosome of this strain and included two hyaluronidase (hyl) genes, important virulence factors of the strain. Moreover, two transposons and two virulence (virD4) genes were found to be located in the same genome island of ZHZ 211. In vitro phenotypic results showed that ZHZ 211 grows faster and is resistant to clarithromycin, enrofloxacin, and sulfonamides. The higher biofilm-forming capabilities of ZHZ 211 may provide a competitive advantage for survival in its niche. The results expand our understanding of the genomic, pathogenicity, and resistance characterization of Streptococcus zooepidemicus and facilitate further exploration of its molecular pathogenic mechanism. Full article

Haemaphysalis longicornis, a three-host tick with a wide host range, is widely distributed in different countries and regions. It stands out among ticks due to its unique feature of having both parthenogenetic and bisexual populations. Despite their morphological resemblance, the characteristics of the parthenogenetic population have been overlooked. In this comprehensive study, we systematically compared the similarities and differences between these two populations. Our investigation revealed that the parthenogenetic H. longicornis, widely distributed in China, was found in ten provinces, surpassing the previously reported distribution. Notably, individuals from the parthenogenetic population exhibited a prolonged blood-feeding duration during the larval and nymph stages compared to their bisexual counterparts. Additionally, the life cycle of the parthenogenetic population was observed to be longer. A flow cytometry analysis indicated a DNA content ratio of approximately 2:3 between the bisexual and parthenogenetic populations. A phylogenetic analysis using whole mitochondrial genome sequences resulted in the separation of the phylogenetic tree into two distinct branches. A molecular analysis unveiled a consistent single T-base deletion at nucleotide 8497 in the parthenogenetic population compared to the bisexual population. Both populations displayed high viral infection capability and significant resistance to ivermectin. Intriguingly, despite these differences, the parthenogenetic population exhibited a similar life cycle to the bisexual population, retaining the ability to transmit pathogens such as Severe fever with thrombocytopenia syndrome virus (SFTSV) and Heartland Virus (HRTV). These findings contribute to a deeper understanding of the distinct characteristics and similarities between different populations of H. longicornis, laying the foundation for future research in this field. Full article

Leizhou goats are famous for their delicious meat but have inferior growth performance. There is little information on rumen-protected fat (RPF) from the Leizhou goat. Hence, we observed the effects of RPF on growth, fecal short-chain fatty acids, and bacteria community with respect to Leizhou goats. Twelve goats (13.34 ± 0.024 kg) were selected and assigned randomly to one of two treatments: (1) a control diet (CON) and (2) 2.4% RPF with a control diet (RPF). The final body weight and average daily gain (ADG) were greater (p < 0.05), and the dry matter intake (DMI): ADG was lower (p < 0.05) in the RPF group than in the CON group. There were no differences in DMI between the CON and RPF groups. The concentrations of total short-chain fatty acids, acetate, propionate, and butyrate were lower (p < 0.05) in the RPF group than in the CON group. The relative abundances of Ruminococcus, Rikenellaceae_RC9_gut_group, Treponema, norank_f__norank_o__RF39, Eubacterium_siraeum_group, and Ruminococcus_torques_group were lower (p < 0.05) in the RPF group than in the CON group. The relative abundances of Bacteroides, norank_f__norank_o__Clostridia_UCG-014, norank_f__Eubacterium_coprostanoligenes_group, Eubacterium_ruminantium_group, norank_f__Oscillospirale-UCG-010, Oscillospiraceae_UCG-002, and Family_XIII_AD3011_group were greater (p < 0.05) in the RPF group than in the CON group. It was concluded that RPF could improve the goats’ growth performance by regulating their fecal bacteria communities. Full article

Sex and gender are fundamental health determinants and their role as modifiers of treatment response is increasingly recognized. Norepinephrine is a cornerstone of septic shock management and its use is based on the highest level of evidence compared to dopamine. The related 2021 Surviving Sepsis Campaign (SCC) recommendation is presumably applicable to both females and males; however, a sex- and gender-based analysis is lacking, thus not allowing generalizable conclusions. This paper was aimed at exploring whether sex- and gender-disaggregated data are available in the evidence supporting this recommendation. For all the studies underpinning it, four pairs of authors, including a woman and a man, extracted data concerning sex and gender, according to the Sex and Gender Equity in Research guidelines. Nine manuscripts were included with an overall population of 2126 patients, of which 43.2% were females. No sex analysis was performed and gender was never reported. In conclusion, the present manuscript highlighted that the clinical studies underlying the SCC recommendation of NE administration in septic shock have neglected the likely role of sex and gender as modifiers of treatment response, thus missing the opportunity of sex- and gender-specific guidelines. Full article

The spread of antibiotic-resistant bacteria and the rise of emerging and re-emerging viruses in recent years constitute significant public health problems. Therefore, it is necessary to develop new antimicrobial strategies to overcome these challenges. Herein, we describe an innovative method to synthesize ligand-free silver nanoparticles by Pulsed Laser Ablation in Liquid (PLAL-AgNPs). Thus produced, nanoparticles were characterized by total X-ray fluorescence, zeta potential analysis, transmission electron microscopy (TEM), and nanoparticle tracking analysis (NTA). A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to evaluate the nanoparticles’ cytotoxicity. Their potential was evaluated against the enveloped herpes simplex virus type 1 (HSV-1) and the naked poliovirus type 1 (PV-1) by plaque reduction assays and confirmed by real-time PCR and fluorescence microscopy, showing that nanoparticles interfered with the early stage of infection. Their action was also examined against different bacteria. We observed that the PLAL-AgNPs exerted a strong effect against both methicillin-resistant Staphylococcus aureus (S. aureus MRSA) and Escherichia coli (E. coli) producing extended-spectrum β-lactamase (ESBL). In detail, the PLAL-AgNPs exhibited a bacteriostatic action against S. aureus and a bactericidal activity against E. coli. Finally, we proved that the PLAL-AgNPs were able to inhibit/degrade the biofilm of S. aureus and E. coli. Full article

Leishmania infantum, a zoonotic vector-born parasite, is endemic in the Mediterranean region, presenting mostly as visceral (VL), but also as cutaneous (CL) and mucosal leishmaniasis (ML). This study aimed to describe the epidemiological and clinical aspects of the CL and ML cases diagnosed in mainland Portugal between 2010 and 2020. Collaboration was requested from every hospital of the Portuguese National Health System. Cases were screened through a search of diagnostic discharge codes or positive laboratory results for Leishmania infection. Simultaneously, a comprehensive literature search was performed. Descriptive statistics and hypothesis testing were performed using IBM^® SPSS^® Statistics. A total of 43 CL and 7 ML cases were identified, with a predominance of autochthonous cases (86%). In CL, immunosuppressed individuals constituted a significant proportion of patients (48%), and in this group, disseminated CL (22%) and simultaneous VL (54%) were common. In autochthonous cases, lesions, mostly papules/nodules (62%), were frequently observed on the head (48%). The approach to treatment was very heterogeneous. ML cases were all autochthonous, were diagnosed primarily in older immunosuppressed individuals, and were generally treated with liposomal amphotericin B. The findings suggest a need for enhanced surveillance and reporting, clinical awareness, and diagnostic capacity of these forms of leishmaniasis to mitigate underdiagnosis and improve patient outcomes. A holistic One Health approach is advocated to address the multifaceted challenges posed by leishmaniases in Portugal and beyond. Full article

In recent years, the number of foodborne infections with non-O1 and non-O139 Vibrio cholerae (NOVC) has increased worldwide. These have ranged from sporadic infection cases to localized outbreaks. The majority of case reports describe self-limiting gastroenteritis. However, severe gastroenteritis and even cholera-like symptoms have also been described. All reported diarrheal cases can be traced back to the consumption of contaminated seafood. As climate change alters the habitats and distribution patterns of aquatic bacteria, there is a possibility that the number of infections and outbreaks caused by Vibrio spp. will further increase, especially in countries where raw or undercooked seafood is consumed or clean drinking water is lacking. Against this background, this review article focuses on a possible infection pathway and how NOVC can survive in the human host after oral ingestion, colonize intestinal epithelial cells, express virulence factors causing diarrhea, and is excreted by the human host to return to the environment. Full article

Listeria monocytogenes is a ubiquitous pathogen found both in the environment and food. It can cause listeriosis in a wide range of animals as well as in humans. Investigations on presence, spread and virulence are still limited to terrestrial and human environments. Embracing the One Health Approach, investigating the presence and spread of L. monocytogenes in marine ecosystems and among wildlife, would provide us with useful information for human health. This study investigated the presence of L. monocytogenes and Listeria spp. in two species of sea turtles common in the Mediterranean Sea (Caretta caretta and Chelonia mydas). A total of one hundred and sixty-four carcasses of sea turtles (C. caretta n = 161 and C. mydas n = 3) stranded along the Abruzzo, Molise, Campania, and Calabria coasts, were collected. Brain and fecal samples were taken, enriched, and cultured for the detection of Listeria spp. From the specimens collected, strains of L. monocytogenes (brain n = 1, brain and feces n = 1, multiorgan n = 1 and feces n = 1), L. innocua (feces n = 1 and brain n = 1), and L. ivanovii (brain n = 1) were isolated. Typical colonies were isolated for Whole Genome Sequencing (WGS). Virulence genes, disinfectants/metal resistance, and antimicrobial resistance were also investigated. L. monocytogenes, L. innocua, and L. ivanovii were detected in C. caretta, whilst only L. monocytogenes and L. innocua in C. mydas. Notable among the results is the lack of significant differences in gene distribution between human and sea turtle strains. Furthermore, potentially pathogenic strains of L. monocytogenes were found in sea turtles. Full article

Equine piroplasmosis (EP) is caused by Theileria (T.) equi and/or Babesia (B.) caballi. The aim was to assess the percentage of positive test results for EP in horses in Europe and to identify risk factors for pathogen contact/infection. This study included results from PCR and competitive enzyme-linked immunosorbent assay testing requested by European veterinarians between 2008 and 2021. Binary bivariate logistic regression was used to analyze risk factors. A total of 4060 horses were included. PCR testing was positive in 9.7% (154/1589), serology for T. equi in 15.2% (393/2591) and for B. caballi in 6.8% (175/2578). The odds of positive serology increased by 6.8% (B. caballi, p = 0.008) and 9.5% (T. equi, p < 0.001) each year. Regionality had a statistically significant impact on PCR (Eastern p = 0.047/OR = 1.605; Southern p = 0.029/OR = 1.451; Central p = 0.007/OR = 0.617) and serological testing for T. equi (Southern p < 0.001/OR = 2.521; Central p < 0.001/OR = 0.537; Northern p = 0.003/OR = 0.462), as well as breeds on seroprevalence of B. caballi (heavy horses: p = 0.016/OR = 2.239) and T. equi (ponies: p = 0.007/OR = 0.340; warmbloods: p = 0.025/OR = 1.602). In conclusion, there was a significant geographical impact on the results of PCR and serology, consistent with known vector habitats. The rising numbers of horses tested serologically positive highlights the importance of surveillance. Full article

Extended reality (XR) devices, including virtual and augmented reality head-mounted displays (HMDs), are increasingly utilised within healthcare to provide clinical interventions and education. Currently, XR devices are utilised to assist in reducing pain and improving psychological outcomes for immunocompromised patients in intensive care units, palliative care environments and surgical theatres. However, there is a paucity of research on the risks of infection from such devices in healthcare settings. Identify existing literature providing insights into the infection control risk XR HMDs pose within healthcare facilities and the efficacy of current infection control and cleaning procedures. Three databases (PubMed, Embase and CINAHL) in addition to Google Scholar were systematically searched. A total of seven studies were identified for this review. Microorganisms, including pathogenic bacteria (e.g., Staphylococcus aureus and Pseudomonas aeruginosa), were found to be present on XR HMDs. Published cleaning and infection control protocols designed to disinfect XR HMDs and protect users were heterogeneous in nature. Current cleaning protocols displayed varying levels of efficacy with microbial load affected by multiple factors, including time in use, number of users and XR HMD design features. In healthcare settings, fitting XR HMDs harbouring microorganisms near biological and mucosal entry points presents an infection control risk. An urgent revision of the Spaulding classification is required to ensure flexibility that allows for these devices to be reclassified from ‘Non-critical’ to ‘Semi-Critical’ depending on the healthcare setting and patient population (surgery, immunocompromised, burns, etc.). This review identified evidence supporting the presence of microorganisms on XR HMDs. Due to the potential for HMDs to contact mucosal entry points, devices must be re-considered within the Spaulding classification as ‘Semi-critical’. The existence of microbial contaminated XR HMDs in high-risk medical settings such as operating wards, intensive care units, emergency departments, labour and delivery wards and clinical areas with immunosuppressed patients requires urgent attention. Public health authorities have a duty of care to develop revised guidelines or new recommendations to ensure efficient sanitation of such devices. Full article

The past decades have witnessed intensive research on the biological effects of graphene-based nanomaterials (GBNs) and the application of GBNs in different fields. The published literature shows that GBNs exhibit inhibitory effects on almost all microorganisms under pure culture conditions, and that this inhibitory effect is influenced by the microbial species, the GBN’s physicochemical properties, the GBN’s concentration, treatment time, and experimental surroundings. In addition, microorganisms exist in the soil in the form of microbial communities. Considering the complex interactions between different soil components, different microbial communities, and GBNs in the soil environment, the effects of GBNs on soil microbial communities are undoubtedly intertwined. Since bacteria and fungi are major players in terrestrial biogeochemistry, this review focuses on the antibacterial and antifungal performance of GBNs, their antimicrobial mechanisms and influencing factors, as well as the impact of this effect on soil microbial communities. This review will provide a better understanding of the effects of GBNs on microorganisms at both the individual and population scales, thus providing an ecologically safe reference for the release of GBNs to different soil environments. Full article

Vibrio parahaemolyticus is an important human pathogen that is currently the leading cause of shellfish-borne gastroenteritis in the world. Particularly, the pandemic strain has the capacity to induce cytotoxicity and enterotoxicity through its Type 3 Secretion System (T3SS2) that leads to massive cell death. However, the specific mechanism by which the T3SS2 induces cell death remains unclear and its contribution to mitochondrial stress is not fully understood. In this work, we evaluated the contribution of the T3SS2 of V. parahaemolyticus in generating mitochondrial stress during infection in human intestinal HT-29 cells. To evaluate the contribution of the T3SS2 of V. parahaemolyticus in mitochondrial stress, infection assays were carried out to evaluate mitochondrial transition pore opening, mitochondrial fragmentation, ATP quantification, and cell viability during infection. Our results showed that the Δvscn1 (T3SS2+) mutant strain contributes to generating the sustained opening of the mitochondrial transition pore. Furthermore, it generates perturbations in the ATP production in infected cells, leading to a significant decrease in cell viability and loss of membrane integrity. Our results suggest that the T3SS2 from V. parahaemolyticus plays a role in generating mitochondrial stress that leads to cell death in human intestinal HT-29 cells. It is important to highlight that this study represents the first report indicating the possible role of the V. parahaemolyticus T3SS2 and its effector proteins involvement in generating mitochondrial stress, its impact on the mitochondrial pore, and its effect on ATP production in human cells. Full article

Objective: The gut microbiome has been associated with visceral fat (VAT) in European and Asian populations; however, associations with VAT and with ectopic fats among African-ancestry individuals are not known. Our objective was to investigate cross-sectional associations of fecal microbiota diversity and composition with VAT and ectopic fat, as well as body mass index (BMI), among middle-aged and older African Caribbean men. Methods: We included in our analysis n = 193 men (mean age = 62.2 ± 7.6 years; mean BMI = 28.3 ± 4.9 kg/m²) from the Tobago Health Study. We assessed fecal microbiota using V4 16s rRNA gene sequencing. We evaluated multivariable-adjusted associations of microbiota features (alpha diversity, beta diversity, microbiota differential abundance) with BMI and with computed tomography-measured VAT and ectopic fats (pericardial and intermuscular fat; muscle and liver attenuation). Results: Lower alpha diversity was associated with higher VAT and BMI, and somewhat with higher pericardial and liver fat. VAT, BMI, and pericardial fat each explained similar levels of variance in beta diversity. Gram-negative Prevotellaceae and Negativicutes microbiota showed positive associations, while gram-positive Ruminococcaceae microbiota showed inverse associations, with ectopic fats. Conclusions: Fecal microbiota features associated with measures of general adiposity also extend to metabolically pernicious VAT and ectopic fat accumulation in older African-ancestry men. Full article