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To evaluate the association between respiratory tract microorganisms at birth and the subsequent development and severity of bronchopulmonary dysplasia (BPD) in preterm infants. This prospective cohort study enrolled 98 preterm infants (gestational age < 32 weeks, birth weight < 2,000 g). Tracheal aspirate samples were collected through endotracheal intubation within 2 h after birth. Using 16S rRNA sequencing, we characterized the airway microbiome and performed co-occurrence network analysis with compositionally robust methods. Among 98 preterm infants analyzed, the incidence of BPD was 68.4%, comprising 31 grade I, 20 grade II, and 16 grade III cases. Airway microbiota in infants with BPD exhibited distinct severity-stage patterns: Escherichia-Shigella and Streptococcus were significantly enriched in grade I, while Chryseobacterium increased markedly in grade III, accompanied by a significant reduction in Streptococcus. Microbial co-occurrence network analysis yielded three key insights. (i) Network complexity declined sharply with BPD severity, being sparsest in grade III. (ii) Distinct keystone taxa were identified across different groups: Acinetobacter and Fusobacterium in the non-BPD group; Brevundimonas and Fusobacterium in grade I; Fusobacterium and Acinetobacter at grade II and grade III. (iii) In a multivariable model adjusted for key clinical confounders, a higher microbial network density at birth was independently associated with a substantially reduced risk (OR = 0.12, P < 0.05). The ecological architecture of the neonatal airway microbiome at birth, defined by network complexity and keystone taxa, is associated with BPD severity. This highlights microbial network stability as a novel factor and ecological interactions as a target for future research.

Importance: Bronchopulmonary dysplasia (BPD) remains the most common chronic lung disease in preterm infants. While its pathogenesis is incompletely understood, the role of the early respiratory microbe is increasingly recognized. Previous studies have largely focused on individual pathogenic taxa, overlooking the complex ecological interactions within microbial communities. Our analysis reveals that the architecture of microbial co-occurrence networks in the neonatal airway varies significantly with BPD severity. Notably, network complexity decreased markedly as BPD severity increased. We identified specific keystone taxa uniquely associated with disease outcomes, suggesting that microbial ecosystem stability rather than individual species may be a critical factor in BPD pathogenesis. These findings shift the focus from single microbes to the stability of the microbial ecosystem as a novel risk factor for severe BPD, offering new avenues for risk stratification and early intervention.

Cryptococcus neoformans, the etiological agent of cryptococcal meningitis (CM) is a globally distributed environmental yeast that mainly causes infections in immunocompromised individuals. Particularly in low-resource countries, the mortality rate of CM can reach 81% and accounts for ~19% of HIV/AIDS-related deaths each year. In immunocompromised individuals, once inhaled, C. neoformans escapes from the lungs and disseminates with special predilection for the central nervous system (CNS). Once in the brain, C. neoformans interacts with microglia, the tissue-resident macrophages of the CNS. Previous studies indirectly showed that microglia are ineffective at controlling this fungal infection. The mechanisms underlying this fungal survival and proliferation within the CNS, however, remain unclear. In this study, we use and validate the C20 immortalized human microglia cell line to study cryptococcal-microglia interactions. We show that microglia have limited phagocytic activity that is specific to C. neoformans and partly dependent on cryptococcal antiphagocytic proteins that alter cell size and cell wall structure. We also show that human microglia respond to cryptococcal strains differently than peripheral macrophages. Further, we show that human microglia are ineffective at killing phagocytosed C. neoformans, and that this could be due to the ability of this yeast to disrupt phagosome maturation and induce phagosome membrane damage in these cells. These findings provide us with fundamental knowledge regarding cryptococcal pathogenesis in the CNS, specifically the insight into how C. neoformans is recognized by microglia under different conditions and demonstrate the usefulness of C20 cells to further study how this yeast survives and replicates within the CNS environment.

Importance: While Cryptococcus neoformans is acquired through inhalation, the fatal pathology of cryptococcal infection occurs when the yeast disseminates to the central nervous system (CNS) and causes cryptococcal meningitis. Microglia are the first immune cells that C. neoformans will encounter once it reaches the CNS, and they are the largest population of macrophages in the brain. While microglia are professional phagocytes, they are unable to control C. neoformans infection. The mechanisms behind uncontrolled growth of C. neoformans within the CNS remain understudied, partly due to incomplete knowledge regarding microglia-cryptococcal interactions. This study provides fundamental knowledge into these interactions and establishes a powerful model to specifically study how C. neoformans is recognized by microglia and how cryptococcal phagosomes mature in these phagocytes. This work opens new avenues of research to further our understanding of cryptococcal-host interactions, which can be leveraged to develop more effective therapeutics for cryptococcal meningitis.

Studies on human gut microbiota have recently highlighted a significant decline in bacterial diversity associated with urbanization, driven by shifts toward processed diets, increased antibiotic usage, and improved sanitation practices. This phenomenon has been largely overlooked in the Colombian Amazon, despite rapid urbanization in the region. In this study, we investigate the composition of gut bacterial microbiota and intestinal protozoa and soil-transmitted helminths (STHs) in both urban and rural areas of Leticia, located in the southern Colombian Amazon. Despite their geographic proximity, the urban population is predominantly non-indigenous, while indigenous communities mostly inhabit the rural area, resulting in notable lifestyle differences between the two settings. Our analyses reveal a reduction in bacterial families linked to non-processed diets, such as Lachnospiraceae, Spirochaetaceae, and Succinivibrionaceae, in the urban environment compared to their rural counterparts. Prevotellaceae, typically associated with non-processed food consumption, shows a significantly higher abundance in urban Leticia. STH infections were primarily detected in rural Leticia, while intestinal protozoa were ubiquitous in both rural and urban areas. Both types of parasites were associated with higher gut bacterial richness and diversity. Additionally, microbial metabolic prediction analysis indicated differences in pathways related to unsaturated fatty acid production and aerobic respiration between rural and urban bacterial microbiomes. This suggests a tendency toward changes in the urban microbiota that may lead to increased susceptibility to non-communicable chronic diseases. These findings provide new insights into the impact of urbanization on gut microbiota dynamics in the Amazonian context and underscore the need for further research into any associated health outcomes.IMPORTANCEChanges in the diversity and composition of gut microbiota in urban populations have been linked to the rise of non-communicable chronic diseases, such as autoimmune conditions, diabetes, and cancer. As developing countries undergo a demographic shift toward increased urbanization, accompanied by changes in diet, housing, and medication use, there is a concerning loss of microbial diversity. Therefore, it is essential to investigate microbiota changes in overlooked populations, such as indigenous communities in the Colombian Amazon basin. A better understanding of local and generalizable changes in gut microbial composition through urbanization may facilitate the development of targeted programs aimed at promoting lifestyle and diet changes to prevent diseases that healthcare systems may be ill-equipped to effectively address.

Glycosaminoglycans (GAGs), comprising uronic acids and amino sugars, are widely distributed in human tissues such as the intestine and oral cavity. Various bacteria colonize these tissues by assimilating GAGs. During GAG degradation, 4-deoxy-l-threo-5-hexosulose uronate (DHU) is produced. Pectin, an abundant plant component, is also degraded into DHU. DHU is metabolized in a stepwise manner by the isomerase KduI or its nonhomologous isofunctional enzyme DhuI, followed by the reductase KduD or DhuD, belonging to the same reductase-dehydrogenase family. Previous studies have found that the genes encoding isomerase and reductase (kduI-kduD and dhuD-dhuI, respectively) are usually organized in clusters. Therefore, it was believed that the kduI-kduD and dhuD-dhuI clusters evolved independently. However, the discovery of a hybrid kduI-dhuD cluster raised questions regarding the evolution of these clusters. This study investigated the diversity of clusters through a pan-genomic phylogenetic analysis across 3,550 bacterial strains. Among 16 possible cluster structures, 10 types were involved in DHU metabolism. Bacteroidota possessed a hybrid-type kduI-dhuD cluster, while Bacillota, but not Pseudomonadota or Bacteroidota, possessed the cluster dhuD-dhuI. Using public data sets from the human fecal microbiome and environmental habitats, we detected the prevalence of kduI-dhuD and dhuD-dhuI clusters in gut microbes. Although DHU is generated from oligomerized GAG degradation by unsaturated glucuronyl hydrolase (UGL), the UGL gene was frequently found in pathogenic strains containing kduD-kduI, dhuD-dhuI, kduI-dhuD, or dhuD-kduI, indicating that the acquisition of these clusters is advantageous for human colonization.IMPORTANCEGlycosaminoglycans (GAGs), crucial components of the extracellular matrix, play vital roles in host infection by pathogenic bacteria and host colonization by commensal bacteria. The dhuD-dhuI cluster is well conserved within certain phyla, and it appears to have a strong association with GAG metabolism. In contrast, kduI-containing clusters are more widely distributed across bacterial species. Based on the possession ratios of genes encoding the enzymes involved in the production of 4-deoxy-l-threo-5-hexosulose uronate, this study indicates that the substrates differ depending on the specific cluster type.

The apicoplast is an essential organelle found in Apicomplexa, a large phylum of intracellular eukaryotic pathogens. The apicoplast produces metabolites that are utilized for membrane biogenesis and energy production. A majority of apicoplast-resident proteins are encoded by the nuclear genome and are trafficked to the apicoplast and are referred to as nuclear-encoded and apicoplast-trafficked (NEAT) proteins. In this study, we characterized a NEAT protein named TgBipA, which is a homolog of the highly conserved prokaryotic translational GTPase BipA. BipA is essential for bacterial survival in stress conditions and functions through interactions with the prokaryotic ribosome, although its role is not fully understood. Through genetic knockouts of TgBipA and immunofluorescence imaging, we show that the loss of TgBipA results in apicoplast genome replication defects, disruption of NEAT trafficking, loss of the apicoplast, and ultimately parasite death. Furthermore, we show through comparative studies that this phenotype closely resembles the delayed death phenomenon observed when inhibiting apicoplast translation. Finally, we show that TgBipA is an active GTPase in vitro, and its GTP hydrolysis activity is critical for its cellular function. Our findings demonstrate that TgBipA is a GTPase that has an essential role in apicoplast maintenance, providing new insights into the cellular processes of the organelle.IMPORTANCEToxoplasma gondii, and many other parasites in the phylum Apicomplexa, are pathogens with significant medical and veterinary importance. Most Apicomplexa contain a non-photosynthetic plastid organelle named the apicoplast. This organelle produces essential metabolites, and perturbation of apicoplast function results in parasite death. The apicoplast contains bacterial-like pathways for apicoplast genome replication and expression. Thus, the discovery of the apicoplast leads to optimism that this organelle would provide a wealth of anti-parasitic drug targets. Therefore, the identification and characterization of new apicoplast proteins could provide new opportunities for therapeutic development. In this study, we characterized the function of a protein called TgBipA, a homolog of a highly conserved bacterial GTPase BipA, which has been implicated in the maturation of the 50S ribosomal subunit and adaptation to cellular stress. We show that TgBipA is essential for apicoplast maintenance and parasite survival.

A dysfunctional gut microbiome has become increasingly common in infants born in high-income countries as Bifidobacterium strains no longer dominate the gut microbiome. Probiotics containing Bifidobacterium infantis have been used in breastfed newborns to successfully restore the gut microbiome; however, no studies to date have demonstrated this effect in older breastfed infants whose gut microbiomes are transitioning toward stability and maturity. This is a 9-week randomized controlled trial wherein 2-4 months old exclusively breastfed infants (n = 40) received 0 CFU/day B. infantis EVC001 (placebo), 4.0 × 10⁹ CFU/day B. infantis EVC001 (low), 8.0 × 10⁹ CFU/day B. infantis EVC001 (medium), or 1.8 × 10¹⁰ CFU/day B. infantis EVC001 (high) in equal allocation for 28 consecutive days beginning on day 8. Stool samples were collected on study days 7, 10, 14, 21, 28, 35, 42, and 63. Fecal B. infantis levels were significantly higher in all supplement groups compared with placebo on day 28 and day 63. On day 28, fecal B. infantis levels were significantly higher in infants who received any (low, medium, and high) dose compared with baseline. The abundance of fecal Bifidobacteriaceae significantly increased nearly 2-fold in response to B. infantis EVC001 supplementation. No matter the dose, probiotic supplementation with B. infantis in 2- to 4-month-old exclusively breastfed infants resulted in colonization until at least 1 month post-supplementation.

Importance: This study found that supplementing exclusively breastfed infants with a probiotic, Bifidobacterium infantis EVC001, between 2 and 4 months of age can successfully restore beneficial bacteria in their gut, even after the newborn period. Although previous research showed this effect in newborns, this is the first study to demonstrate that older infants, whose gut microbiomes are typically more stable, can still benefit. The probiotic was effective at all tested doses, with higher levels of B. infantis and overall Bifidobacteriaceae in infants' stool during and even 1 month after supplementation. This study demonstrates that B. infantis can take hold in the gut and potentially improve gut health in older breastfed babies, offering a promising approach to support infant health in settings where beneficial gut bacteria are often missing.

Clinical trials: This study was registered at clinicaltrials.gov as NCT03476447.

The situation regarding drug resistance among gram-negative bacteria is becoming increasingly severe. While antimicrobial peptides are an ideal alternative to traditional antibiotics, single-target natural antimicrobial peptides exhibit limitations, including high toxicity and poor permeability. Given the numerous advantages of dual-target peptides for disease treatment, we designed and synthesized the first membrane/ribosome dual-target antimicrobial peptide, FPON, through a functional peptide splicing strategy utilizing FP-CATH and Oncocin as templates. FPON specifically targets gram-negative bacteria and possesses dual functionalities: the ability to disrupt bacterial membrane integrity and the ability to inhibit protein translation. Additionally, FPON exhibited low toxicity and demonstrated significant activity against drug-resistant bacteria in vitro and in vivo. In conclusion, the results presented in this study provide further evidence that dual-targeted antimicrobial peptides constitute an effective treatment strategy against gram-negative drug-resistant bacteria.IMPORTANCEThe issue of antibiotic drug resistance in gram-negative bacteria is one of grave urgency. While single-target antimicrobial peptides offer a potential solution to antibiotic resistance, therapeutic applications are constrained by their high toxicity and poor penetration. In this study, FP-CATH and Oncocin were used as templates for functional peptide splicing to develop FPON, a novel antimicrobial peptide. FPON was shown to disrupt bacterial membranes and inhibit protein synthesis, effectively eliminating gram-negative bacteria. Moreover, FPON exhibits low toxicity and has a significant effect against drug-resistant bacteria. Our research demonstrates that a dual-target design offers a promising avenue for addressing drug-resistant infections.

Mosquito-borne viruses represent a major global public health threat, with transmission dynamics governed by climatic, ecological, and anthropogenic factors. Yantai City, Shandong Province, situated in a warm-temperate monsoon climate zone, shares geographical and ecological characteristics with regions where mosquito-borne viruses are endemic, creating potential for virus introduction. We used metagenomics to systematically analyze viral communities in mosquitoes from the Yantai region. We collected 8,111 mosquitoes representing four genera and six species, with Culex being predominant (89.8%). High-throughput sequencing revealed 11 viral species spanning 9 families, including Peribunyaviridae and Picornaviridae. Notably, Serbia mononega-like virus 1 and Biggievirus Mos11 represent the first reports from China, with quantitative reverse transcription PCR revealing minimum infection rates of 0.34% and 0.68%, respectively. Phylogenetic analysis revealed close relationships to known viral strains, with several isolates potentially representing novel genera or species. Analysis revealed that Culex quinquefasciatus harbored the greatest viral diversity (five species), with significantly higher viral diversity in agricultural versus urban areas (P < 0.001). Several viruses demonstrated cross-species transmission potential, including Zhee mosquito virus, Zhejiang mosquito virus 3, and Culex tritaeniorhynchus rhabdovirus, all detected across multiple mosquito species. While most viruses appear mosquito-specific, several show close phylogenetic relationships to known pathogens, potentially posing public health risks warranting surveillance. This study addresses knowledge gaps regarding mosquito-borne viruses in the Bohai Rim region and provides a scientific foundation for regional viral surveillance and early warning systems.IMPORTANCEMosquito-borne viruses are a significant global health threat, with the potential to cause widespread disease outbreaks. This study investigated the viral diversity within mosquito populations in Yantai, China, and characterized the molecular features of two emerging RNA viruses. These findings highlight the remarkable viral diversity harbored by Culex mosquitoes and reveal higher viral diversity in agricultural areas compared to urban settings. Several identified viruses exhibit cross-species transmission potential and close phylogenetic relationships to known pathogens, suggesting that they may pose public health risks. Understanding these interactions is essential for predicting how environmental changes may affect virus transmission and the resilience of surveillance and control strategies.

Signal transduction allows bacterial pathogens to sense the host environment and regulate gene expression accordingly for adaptation and survival. While the success of infection largely depends on the timely induction of virulence genes, the activity of the regulatory pathways controlling their expression must be tightly regulated for pathogens to cause disease. Here, we establish that a small RNA (sRNA) promotes the negative feedback control of a master virulence regulator in Salmonella enterica serovar Typhimurium (S. Typhimurium) by repressing a signaling protein essential for its induction in response to an intracellular cue. We show that the virulence regulatory PhoP/PhoQ pathway is inhibited by the PhoP-activated sRNA PinT in mildly acidic pH, an infection-relevant condition encountered by S. Typhimurium inside macrophages. PinT directly represses the translation of ugtL mRNA, which encodes the PhoP activator UgtL. This negative feedback regulation reduces PhoP activity, thereby decreasing the expression of PhoP-activated virulence genes like pagC. PinT-mediated repression of ugtL is predicted to be conserved in Salmonella enterica, but not in the nonpathogenic species Salmonella bongori, thus suggesting that the regulation is relevant for virulence. Our findings uncover how pathogens achieve proper levels of induction of their virulence programs through the post-transcriptional negative feedback regulation of factors enhancing the signaling activity of virulence pathways.

Importance: To cause disease, pathogens must express their virulence genes at the right time and in proper levels. Here, we establish that a small RNA (sRNA) restricts the activation of a regulator critical for the virulence of Salmonella enterica serovar Typhimurium (S. Typhimurium). We show that the sRNA PinT inhibits the activity of the master virulence regulator PhoP by repressing its activator UgtL through a direct interaction with ugtL mRNA. This regulation reduces the expression of PhoP-activated genes. Because PhoP activates PinT and UgtL, the three regulators form a negative feedback loop. That the PinT-mediated repression of ugtL is predicted to occur in Salmonella enterica but not in the nonpathogenic species S. bongori suggests it may be a key virulence determinant. Our results unveil a novel layer of fine-tuning of PhoP activity ensuring that S. Typhimurium induces the proper level of its virulence program in response to an infection-relevant stress condition.