Flow Cytometry and Automated Microscopy Integration for Phagocytic Analysis of Hypervirulent Klebsiella pneumoniae in Dictyostelium discoideum.

This chapter introduces protocols for culturing and maintaining Dictyostelium discoideum and methods for conducting virulence assays in this organism to study bacterial pathogenicity. It outlines advanced techniques, such as automated microscopy and flow cytometry, for detailed cellular analysis and traditional microbiological approaches. These comprehensive protocols will enable researchers to probe the virulence factors of pathogens like Klebsiella pneumoniae and to elucidate the details of host-pathogen interactions within a cost-effective and adaptable laboratory framework.

Epinephrine and norepinephrine regulate the expression of virulence factors in Gallibacterium anatis.

Gallibacterium anatis is a member of the Pasteurellaceae family and is an opportunistic pathogen that causes gallibacteriosis in chickens. Stress plays a relevant role in promoting the development of pathogenicity in G. anatis. Epinephrine (E) and norepinephrine (NE) are relevant to stress; however, their effects on G. anatis have not been elucidated. In this work, we evaluated the effects of E and NE on the growth, biofilm formation, expression of adhesins, and proteases of two G. anatis strains, namely, the hemolytic 12656-12 and the nonhemolytic F149T biovars. E (10 µM/mL) and NE (30 and 50 µM/mL) increased the growth of G. anatis 12656-12 by 20 % and 25 %, respectively. E did not affect the growth of F149T, whereas 40 µM/mL NE decreased bacterial growth by 25 %. E and NE at a dose of 30-50 µM/mL upregulated five fibrinogen adhesins in the 12565-12 strain, whereas no effect was observed in the F149T strain. NE increased proteolytic activity in both strains, whereas E diminished proteolytic activity in the 12656-12 strain. E and NE reduced biofilm formation (30 %) and increased Congo red binding (15 %) in both strains. QseBC is the E and NE two-component detection system most common in bacteria. The qseC gene, which is the E and NE receptor in bacteria, was identified in the genomic DNA of the 12565-12 and F149TG. anatis strains via PCR amplification. Our results suggest that QseC can detect host changes in E and NE concentrations and that catecholamines can modulate the expression of several virulence factors in G. anatis.

Auranofin is active against Histoplasma capsulatum and reduces the expression of virulence-related genes.

BACKGROUND: Auranofin is an approved anti-rheumatic drug that has a broad-range inhibitory action against several microorganisms, including human pathogenic fungi. The auranofin activity against Histoplasma capsulatum, the dimorphic fungus that causes histoplasmosis, has not been properly addressed. Since there are few therapeutic options for this life-threatening systemic mycosis, this study evaluated the effects of auranofin on H. capsulatum growth and expression of virulence factors. METHODOLOGY/PRINCIPAL FINDINGS: Minimal inhibitory and fungicidal concentrations (MIC and MFC, respectively) of auranofin against 15 H. capsulatum strains with distinct genetic backgrounds were determined using the yeast form of the fungus and a microdilution protocol. Auranofin activity was also assessed on a macrophage model of infection and on a Tenebrio molitor invertebrate animal model. Expression of virulence-related genes was compared between auranofin treated and untreated H. capsulatum yeast cells using a quantitative PCR assay. Auranofin affected the growth of different strains of H. capsulatum, with MIC and MFC values ranging from 1.25 to 5.0 µM and from 2.5 to >10 µM, respectively. Auranofin was able to kill intracellular H. capsulatum yeast cells and conferred protection against the fungus in the experimental animal model of infection. Moreover, the expression of catalase A, HSP70, superoxide dismutase, thioredoxin reductase, serine proteinase, cytochrome C peroxidase, histone 2B, formamidase, metallopeptidase, Y20 and YPS3 proteins were reduced after six hours of auranofin treatment. CONCLUSIONS/SIGNIFICANCE: Auranofin is fungicidal against H. capsulatum and reduces the expression of several virulence-related genes, which makes this anti-rheumatic drug a good candidate for new medicines against histoplasmosis.

EhVps35, a retromer component, is a key factor in secretion, motility, and tissue invasion by Entamoeba histolytica.

In humans and Drosophila melanogaster, the functional convergence of the endosomal sorting complex required for transport (ESCRT) machinery that is in charge of selecting ubiquitinated proteins for sorting into multivesicular bodies, and the retromer, that is the complex responsible for protein recycling to the plasma membrane and Golgi apparatus. ESCRT and retromer complexes are codependent for protein sorting recycling, degradation, and secretion. In this article, we studied the EhVps35 C isoform (referred to as EhVps35), that is the central member of the Entamoeba histolytica retromer, and its relation with the ESCRT machinery during sorting and protein recycling events and their involvement virulence. Our findings revealed that EhVps35 interacts with at least 300 proteins that participate in multiple cellular processes. Laser confocal and transmission electronic microscopy images, as well as secretion assays, revealed that EhVps35 is secreted in vesicles together with EhVps23 and EhADH (both ESCRT machinery proteins). In addition, immunoprecipitation, immunofluorescence, and molecular docking assays revealed the relationship among EhVps35 and other ESCRT machinery proteins. Red blood cell stimulus increased EhVps35 secretion, and the knockdown of the Ehvps35 gene in trophozoites reduced their capacity to migrate and invade tissues. This also impacts the cellular localization of ubiquitin, EhVps23 (ESCRT-I), and EhVps32 (ESCRT-III) proteins, strongly suggesting their functional relationship. Our results, taken together, give evidence that EhVps35 is a key factor in E. histolytica virulence mechanisms and that it, together with the ESCRT machinery components and other regulatory proteins, is involved in vesicle trafficking, secretion, migration, and cell proliferation.

[Meningitis and myelitis by hypervirulent Klebsiella pneumoniae]. / Meningitis y mielitis por Klebsiella pneumoniae hipervirulenta.

Invasive liver abscess syndrome caused by hypervirulent Klebsiella. pneumoniae is a rare disease. This type of K. pneumoniae is aggressive and invasive, despite its sensitivity profile. We report the case of a 62-year-old man with diabetes mellitus, who was admitted to our hospital with meningeal syndrome. Within 24 hours of admission, Gram negative bacilli were isolated blood and cerebrospinal fluid cultures, which were later identified as K. pneumoniae. Liver abscess was detected by computed tomography. Despite early antibiotic treatment, the patient developed back pain that prevented him from moving and right hemiparesis. Increased signal from the central region of the spinal medulla compatible with myelitis was identified by magnetic resonance, for which he received methylprednisolone 1 g/day for 5 days. The patient evolved favorably. Infections caused by hypermucoviscous K. pneumoniae are aggressive and invasive, and more common in men with a history of diabetes mellitus, as in this case. These infections require early antibiotic treatment and the search of metastatic infections.

El síndrome de absceso hepático invasivo causado por cepas hipermucoviscosas de Klebsiella pneumoniae es una enfermedad poco frecuente. Esta serovariedad de Klebsiella se caracteriza por ser agresiva e invasiva pese a su perfil de sensibilidad. Se presenta el caso de un varón de 62 años con antecedentes de diabetes mellitus, que ingresó a nuestro centro con síndrome meníngeo. A las 24 horas del ingreso se aislaron en hemocultivos y en líquido cefalorraquídeo (LCR) bacilos Gram negativos que luego fueron tipificados como Klebsiella pneumoniae. Se identificó la presencia de un absceso hepático mediante tomografía computarizada. Pese al tratamiento antibiótico instaurado de manera temprana, el paciente evolucionó con dolor dorsal que le impedía movilizarse y hemiparesia derecha. En la resonancia magnética nuclear de columna se identificó aumento de la señal de la región central de la médula espinal compatible con mielitis por lo cual recibió tratamiento con metilprednisolona 1g/día por 5 días consecutivos. El paciente evolucionó de manera favorable. Las infecciones por K. pneumoniae hipermucoviscosas son agresivas e invasoras y más frecuentes en varones con antecedentes de diabetes mellitus, como en este caso. Su control requiere de un tratamiento antibiótico temprano y búsqueda de focos a distancia.

Metabolic Reprogramming of Klebsiella pneumoniae Exposed to Serum and Its Potential Implications in Host Immune System Evasion and Resistance.

The aim of this study was to identify, using proteomics, the molecular alterations caused by human serum exposure to Klebsiella pneumoniae ACH2. The analysis was performed under two different conditions, native serum from healthy donors and heat-inactivated serum (to inactivate the complement system), and at two different times, after 1 and 4 h of serum exposure. More than 1,000 bacterial proteins were identified at each time point. Enterobactin, a siderophore involved in iron uptake, and proteins involved in translation were upregulated at 1 h, while the chaperone ProQ and the glyoxylate cycle were identified after 4 h. Enzymes involved in the stress response were downregulated, and the SOD activity was validated using an enzymatic assay. In addition, an intricate metabolic adaptation was observed, with pyruvate and thiamine possibly involved in survival and virulence in the first hour of serum exposure. The addition of exogenous thiamine contributes to bacterial growth in human serum, corroborating this result. During 4 h of serum exposure, the glyoxylate cycle (GC) probably plays a central role, and the addition of exogenous succinate suppresses the GC, inducing a decrease in serum resistance. Therefore, serum exposure causes important changes in iron acquisition, the expression of virulence factors, and metabolic reprogramming, which could contribute to bacterial serum resistance.

Entomological aspects of leishmaniasis transmission in the urban area of Teófilo Otoni, a municipality endemic for the disease in the Brazilian state of Minas Gerais.

Brazil is endemic for both visceral (VL) and cutaneous (CL) clinical forms of leishmaniasis, poverty-associated diseases with worldwide distribution. Leishmania parasites are the etiological agents of leishmaniases, which are transmitted to humans through the bites of infected phlebotomine sand flies. From 2018 to 2023, 15 cases of VL and 129 cases of CL were reported in Téofilo Otoni, an important economic center in the Brazilian state of Minas Gerais. Owing to the lack of data on the entomological fauna, the present study aimed to clarify this main aspect of leishmaniasis. From May, 2021 to April, 2023, entomological captures were performed monthly in ten neighborhoods in Teófilo Otoni. The influence of bioclimatic variables on insect populations was evaluated, and natural infection by Leishmania spp. was investigated using molecular methods. A total of 306 specimens of 12 species of phlebotomine sand fly were collected. The majority (91.6%) were proven or putative vectors of leishmaniasis agents. The population of insects tended to increase during the cooler and drier months. Although Leishmania infection was not detected in any of the samples, the presence of vectors provides conditions for the maintenance and expansion of the transmission cycle of leishmaniasis in Teófilo Otoni.

Resistance and virulence genes characteristic of a South Asia Clade (I) Candida auris strain isolated from blood in Beijing.

INTRODUCTION: Candida auris is a globally disseminated invasive ascomycetous yeast, that imposes a substantial burden on healthcare systems. It has been documented to have spread to over 40 countries across six continents, necessitating in-depth comprehension through advanced techniques like Whole-Genome Sequencing. METHOD: This study entailed the isolation and Whole-Genome Sequencing of a fluconazole-resistant C. auris strain (CA01) obtained from a patient's blood in Beijing. Genome analysis was conducted to classify the strain, and molecular docking was performed to understand the impact of mutations on drug resistance. RESULTS: Genome analysis revealed that CA01 belongs to the South Asia Clade (I) and shares the closest genetic relationship with previously reported strains BJCA001 and BJCA002. Notably, unlike BJCA001, CA01 exhibits significant resistance to fluconazole primarily due to the A395T mutation in the ERG11 gene. Molecular docking studies demonstrated that this mutation leads to geometric changes in the active site where fluconazole binds, resulting in decreased binding affinity. Additionally, the present findings have identified several core virulence genes in C. auris, such as RBF1. DISCUSSION: The findings from this study expand the understanding of the genetic diversity and adaptive mechanisms of C. auris within the South Asia Clade (I). The observed fluconazole resistance driven by the ERG11 mutation A395T highlights the need for heightened awareness and adaptation in clinical treatment strategies in China. This study provides critical insights into drug resistance and virulence profiles at a genetic level, which could guide future therapeutic and management strategies for C. auris infections.

Genomic insights into the antimicrobial resistance and virulence of Helicobacter pylori isolates from gastritis patients in Pereira, Colombia.

BACKGROUND: Helicobacter pylori infects the stomach and/or small intestines in more than half of the human population. Infection with H. pylori is the most common cause of chronic gastritis, which can lead to more severe gastroduodenal pathologies such as peptic ulcer, mucosa-associated lymphoid tissue lymphoma, and gastric cancer. H. pylori infection is particularly concerning in Colombia in South America, where > 80% of the population is estimated to be infected with H. pylori and the rate of stomach cancer is one of the highest in the continent. RESULTS: We compared the antimicrobial susceptibility profiles and short-read genome sequences of five H. pylori isolates obtained from patients diagnosed with gastritis of varying severity (chronic gastritis, antral erosive gastritis, superficial gastritis) in Pereira, Colombia sampled in 2015. Antimicrobial susceptibility tests revealed the isolates to be resistant to at least one of the five antimicrobials tested: four isolates were resistant to metronidazole, two to clarithromycin, two to levofloxacin, and one to rifampin. All isolates were susceptible to tetracycline and amoxicillin. Comparative genome analyses revealed the presence of genes associated with efflux pump, restriction modification systems, phages and insertion sequences, and virulence genes including the cytotoxin genes cagA and vacA. The five genomes represent three novel sequence types. In the context of the Colombian and global populations, the five H. pylori isolates from Pereira were phylogenetically distant to each other but were closely related to other lineages circulating in the country. CONCLUSIONS: H. pylori from gastritis of different severity varied in their antimicrobial susceptibility profiles and genome content. This knowledge will be useful in implementing appropriate eradication treatment regimens for specific types of gastritis. Understanding the genetic and phenotypic heterogeneity in H. pylori across the geographical landscape is critical in informing health policies for effective disease prevention and management that is most effective at local and country-wide scales. This is especially important in Colombia and other South American countries that are poorly represented in global genomic surveillance studies of bacterial pathogens.

Central and Peripheral Nervous System Manifestations Associated with Dengue Illness.

Dengue illness, caused by the dengue viruses, continues to be a major global health concern, with increasing incidence and the emergence of severe manifestations such as neurological complications. An overview of the current understanding of dengue epidemiology, clinical manifestations, and research priorities is presented here. Dengue transmission has escalated in recent years, exacerbated by factors such as vector expansion, climate change, and socioeconomic challenges. The clinical spectrum of dengue ranges from mild febrile illness to severe manifestations, including hemorrhagic fever and neurological complications. Neurological manifestations of dengue, once considered rare, are now increasingly reported, encompassing encephalitis, myelitis, and Guillain-Barré Syndrome, among others. Diagnosis primarily relies on laboratory methods such as RT/PCR, NS1 antigen detection, and serological assays. Despite advancements in understanding the dengue pathogenesis, there remains a critical need for effective vaccines, antiviral drugs, improved surveillance methods, predictive models for disease severity, and long-term studies on post-Dengue sequelae. Integrated programs and holistic approaches to dengue control are essential for mitigating its impact. Addressing these research priorities will be pivotal in combating dengue and reducing its global burden.

UNAM-HIMFG Bacterial Lysate Activates the Immune Response and Inhibits Colonization of Bladder of Balb/c Mice Infected with the Uropathogenic CFT073 Escherichia coli Strain.

Urinary tract infections (UTIs) represent a clinical and epidemiological problem of worldwide impact that affects the economy and the emotional state of the patient. Control of the condition is complicated due to multidrug resistance of pathogens associated with the disease. Considering the difficulty in carrying out effective treatment with antimicrobials, it is necessary to propose alternatives that improve the clinical status of the patients. With this purpose, in a previous study, the safety and immunostimulant capacity of a polyvalent lysate designated UNAM-HIMFG prepared with different bacteria isolated during a prospective study of chronic urinary tract infection (CUTI) was evaluated. In this work, using an animal model, results are presented on the immunostimulant and protective activity of the polyvalent UNAM-HIMFG lysate to define its potential use in the control and treatment of CUTI. Female Balb/c mice were infected through the urethra with Escherichia coli CFT073 (UPEC O6:K2:H1) strain; urine samples were collected before the infection and every week for up to 60 days. Once the animals were colonized, sublingual doses of UNAM-HIMFG lysate were administrated. The colonization of the bladder and kidneys was evaluated by culture, and their alterations were assessed using histopathological analysis. On the other hand, the immunostimulant activity of the compound was analyzed by qPCR of spleen mRNA. Uninfected animals receiving UNAM-HIMFG lysate and infected animals administered with the physiological saline solution were used as controls. During this study, the clinical status and evolution of the animals were evaluated. At ninety-six hours after infection, the presence of CFT073 was identified in the urine of infected animals, and then, sublingual administration of UNAM-HIMFG lysate was started every week for 60 days. The urine culture of mice treated with UNAM-HIMFG lysate showed the presence of bacteria for three weeks post-treatment; in contrast, in the untreated animals, positive cultures were observed until the 60th day of this study. The histological analysis of bladder samples from untreated animals showed the presence of chronic inflammation and bacteria in the submucosa, while tissues from mice treated with UNAM-HIMFG lysate did not show alterations. The same analysis of kidney samples of the two groups (treated and untreated) did not present alterations. Immunostimulant activity assays of UNAM-HIMFG lysate showed overexpression of TNF-α and IL-10. Results suggest that the lysate activates the expression of cytokines that inhibit the growth of inoculated bacteria and control the inflammation responsible for tissue damage. In conclusion, UNAM-HIMFG lysate is effective for the treatment and control of CUTIs without the use of antimicrobials.

Paracoccidioides spp.: Escape mechanisms and their implications for the development of this mycosis.

Paracoccidioidomycosis (PCM) is a systemic granulomatous mycosis prevalent in individuals who carry out rural activities. Its etiological agent is a thermodimorphic fungus belonging to the genus; Paracoccidioides spp. Seven species of this fungus are known: Paracoccidioides brasiliensis, Paracoccidioides lutzii, Paracoccidioides americana, Paracoccidioides restrepiensis, Paracoccidioides venezuelensis, Paracoccidioides loboi and Paracoccidioides ceti. For a long time, Paracoccidioides brasiliensis was attributed as the only causal agent of this mycosis. What is known about adhesins, virulence, escape mechanisms and fungal involvement with the host's immune system is correlated with the species Paracoccidioides brasiliensis. Interactions between Paracoccidioides spp. and the host are complex and dynamic. The fungus needs nutrients for its needs and must adapt to a hostile environment, evading the host's immune system, thus enabling the development of the infectious process. On the other hand, the host's immune system recognizes Paracoccidioides spp. and employs all protective mechanisms to prevent fungal growth and consequently tissue invasion. Knowing this, understanding how Paracoccidioides spp. escapes the host's immune system, can help to understand the pathogenic mechanisms related to the development of the disease and, therefore, in the design of new specific treatment strategies. In this review we discuss these mechanisms and what are the adhesion molecules of Paracoccidioides spp. uses to escape the hostile environment imposed by the host's defense mechanisms; finally, we suggest how to neutralize them with new antifungal therapies.

Discosia brasiliensis causing Discosia leaf blight on tea plant (Camellia sinensis) in China.

Tea (Camellia sinensis), a perennial evergreen shrub, is one of the most important cash crops in China. Tea leaves with symptoms of wilt disease was observed in Fengqing County, Lincang City, Yunnan Province, China. Large irregular jujube-red necrotic spots appeared on the leaves of tea plants, and the lesions with grayish white edge were accompanied by a certain degree of shrinkage. In the tea garden planting base, the natural disease incidence reached 40%-50 %, which significantly affects the yield of tea. One putative pathogen was isolated from three symptomatic tea plant leaves and was identified as Discosia brasiliensis using morphology and molecular phylogeny of multi-loci (ITS, LSU, tub, rpb2) sequence data. Using D. brasiliensis strains for artificial inoculation assay on the tea plant leaves, leaf atrophy symptom in leaves which is similar to those observed in the tea planting base, and the putative pathogen was re-isolated to fulfill Koch's postulates. This is the first report of wilt disease caused by Discosia brasiliensis in China.

Virulence-Related Genes Expression in Planktonic Mixed Cultures of Candida albicans and Non-Albicans Candida Species.

INTRODUCTION: Candida albicans is the most common opportunistic pathogen causing fungal infections worldwide, especially in high-risk patients. Its pathogenicity is related to virulence factors gene expression, such as hyphal growth (HWP1), cell adhesion (ALS3), and protease secretion (SAP1) during infection spreading mechanisms. In recent years, an increase in non-albicans Candida infections has been reported, which may present coinfection or competitive interactions with C. albicans, potentially aggravating the patient's condition. This study aims to evaluate the expression of genes related to virulence factors of C. albicans and non-albicans Candida during planktonic stage. METHODS: C. albicans (ATCC MYA-3573) as well as with three clinical strains (C. albicans DCA53, C. tropicalis DCT6, and C. parapsilosis DCP1) isolated from blood samples, were grown in 24-well plates at 37°C for 20 h, either in monocultures or mixed cultures. Quantitative real-time polymerase chain reaction was used to evaluate the expression levels of the genes HWP1, ALS3, and SAP1 in cells collected during the planktonic stage. In addition, hyphal filamentation was observed using a Scanning Electron Microscope. RESULTS: The overexpression of HWP1 and ASL3 genes in mixed growth conditions between C. albicans and non-albicans Candida species suggests a synergistic relationship as well as an increased capacity for hyphal growth and adhesion. In contrast, C. parapsilosis versus C. tropicalis interaction shows an antagonistic relationship during mixed culture, suggesting a decreased virulence profile of C. parapsilosis during initial coinfection with C. tropicalis. CONCLUSION: The expression of HWP1, ALS3, and SAP1 genes associated with virulence factors varies under competitive conditions among species of the genus Candida during planktonic stage.

Diagnosis of bacteria from the CMNR group in farm animals.

The CMNR group comprises bacteria of the genera Corynebacterium, Mycobacterium, Nocardia, and Rhodococcus and share cell wall and DNA content characteristics. Many pathogenic CMNR bacteria cause diseases such as mastitis, lymphadenitis, and pneumonia in farmed animals, which cause economic losses for breeders and represent a threat to public health. Traditional diagnosis in CMNR involves isolating target bacteria on general or selective media and conducting metabolic analyses with the assistance of laboratory biochemical identification systems. Advanced mass spectrometry may also support diagnosing these bacteria in the clinic's daily routine despite some challenges, such as the need for isolated bacteria. In difficult identification among some CMNR members, molecular methods using polymerase chain reaction (PCR) emerge as reliable options for correct specification that is sometimes achieved directly from clinical samples such as tracheobronchial aspirates and feces. On the other hand, immunological diagnostics such as the skin test or Enzyme-Linked Immunosorbent Assay (ELISA) for Mycobacterium tuberculosis yield promising results in subclinical infections with no bacterial growth involved. In this review, we present the methods most commonly used to diagnose pathogenic CMNR bacteria and discuss their advantages and limitations, as well as challenges and perspectives on adopting new technologies in diagnostics.

Leishmania major telomerase RNA knockout: From altered cell proliferation to decreased parasite infectivity.

This study focuses on the biological impacts of deleting the telomerase RNA from Leishmania major (LeishTER), a parasite responsible for causing leishmaniases, for which no effective treatment or prevention is available. TER is a critical player in the telomerase ribonucleoprotein complex, containing the template sequence copied by the reverse transcriptase component during telomere elongation. The success of knocking out both LeishTER alleles was confirmed, and no off-targets were detected. LmTER-/- cells share similar characteristics with other TER-depleted eukaryotes, such as altered growth patterns and partial G0/G1 cell cycle arrest in early passages, telomere shortening, and elevated TERRA expression. They also exhibit increased γH2A phosphorylation, suggesting that the loss of LeishTER induces DNA damage signaling. Moreover, pro-survival autophagic signals and mitochondrion alterations were shown without any detectable plasma membrane modifications. LmTER-/- retained the ability to transform into metacyclics, but their infectivity capacity was compromised. Furthermore, the overexpression of LeishTER was also deleterious, inducing a dominant negative effect that led to telomere shortening and growth impairments. These findings highlight TER's vital role in parasite homeostasis, opening discussions about its potential as a drug target candidate against Leishmania.

Paracoccidioides lutzii Infects Galleria mellonella Employing Formamidase as a Virulence Factor.

The formamidase (FMD) enzyme plays an important role in fungal thriving by releasing a secondary nitrogen source as a product of its activity. In Paracoccidioides species, previous studies have demonstrated the upregulation of this enzyme in a wide range of starvation and infective-like conditions. However, Paracoccidioides lutzii formamidase has not yet been defined as a virulence factor. Here, by employing in vivo infections using an fmd-silenced strain in Galleria mellonella larvae model, we demonstrate the influence of formamidase in P. lutzii's immune stimulation and pathogenicity. The formamidase silencing resulted in improper arrangement of the nodules, poor melanogenesis and decreased fungal burden. Thus, we suggest that formamidase may be a piece composing the process of molecular recognition by Galleria immune cells. Furthermore, formamidase silencing doubled the observed survival rate of the larvae, demonstrating its importance in fungal virulence in vivo. Therefore, our findings indicate that formamidase contributes to Galleria's immune incitement and establishes the role of this enzyme as a P. lutzii virulence factor.

Genomic Dissection of an Enteroaggregative Escherichia coli Strain Isolated from Bacteremia Reveals Insights into Its Hybrid Pathogenic Potential.

Escherichia coli is a frequent pathogen isolated from bloodstream infections. This study aimed to characterize the genetic features of EC092, an E. coli strain isolated from bacteremia that harbors enteroaggregative E. coli (EAEC) genetic markers, indicating its hybrid pathogenic potential. Whole-genome sequencing showed that EC092 belongs to phylogroup B1, ST278, and serotype O165:H4. Genes encoding virulence factors such as fimbriae, toxins, iron-uptake systems, autotransporter proteins (Pet, Pic, Sat, and SepA), and secretion systems were detected, as well as EAEC virulence genes (aggR, aatA, aaiC, and aap). EC092 was found to be closely related to the other EAEC prototype strains and highly similar in terms of virulence to three EAEC strains isolated from diarrhea. The genomic neighborhood of pet, pic, sat, sepA, and the EAEC virulence genes of EC092 and its three genetically related fecal EAEC strains showed an identical genomic organization and nucleotide sequences. Also, EC092 produced and secreted Pet, Pic, Sat, and SepA in the culture supernatant and resisted the bactericidal activity of normal human serum. Our results demonstrate that the strain EC092, isolated from bacteremia, is a hybrid pathogenic extraintestinal E. coli (ExPEC)/EAEC with virulence features that could mediate both extraintestinal and intestinal infections.

Infection of South American coatis (Nasua nasua) with highly pathogenic avian influenza H5N1 virus displaying mammalian adaptive mutations.

Deadly outbreaks among poultry, wild birds, and carnivorous mammals by the highly pathogenic H5N1 virus of the clade 2.3.4.4b have been reported in South America. The increasing virus incidence in various mammal species poses a severe zoonotic and pandemic threat. In Uruguay, the clade 2.3.4.4b viruses were first detected in February 2023, affecting wild birds and backyard poultry. Three months after the first reported case in Uruguay, the disease affected a population of 23 coatis (Nasua) in an ecological park. Most animals became infected, likely directly or indirectly from wild birds in the park, and experienced sudden death. Five animals from the colony survived, and four of them developed antibodies. The genomes of the H5N1 strains infecting coatis belonged to the B3.2 genotype of the clade 2.3.4.4b. Genomes from coatis were closely associated with those infecting backyard poultry, but transmission likely occurred through wild birds. Notable, two genomes have a 627K substitution in the RNA polymerase PB2 subunit, a hallmark amino acid linked to mammalian adaptation. Our findings support the ability of the avian influenza virus of the 2.3.4.4b clade to infect and transmit among terrestrial mammals with high pathogenicity and undergo rapid adaptive changes. It also highlights the coatis' ability to develop immunity and naturally clear the infection.