Microbial Pathogenesis

Enteric infections caused by Salmonella enterica remain a major global health concern and are increasingly associated with antimicrobial resistance. Therefore, new strategies to combat this important pathogen are needed. The interactions between S. enterica and the human host have been the subject of intense investigation over the last several decades, yet new findings continue to emerge. We previously showed that 15-deoxy-{Delta}12,14-prostaglandin J2 (15d-PGJ2) reduces Salmonella colonization of macrophages, but the mechanisms underlying this protective effect were still unknown. Here, we demonstrate that 15d-PGJ2 limits Salmonella infection by suppressing TLR4 signaling and inflammasome activation. Treatment with 15d-PGJ2 reduced TLR4 expression, NF-{kappa}B activation, iNOS, COX-2, nitric oxide production, IL-1{beta} release, and inflammasome-related targets, including NLRP3 and caspase-1 activity, while only partially reversing macrophage polarization. Combined treatment with the TLR4 antagonist TAK-242 further reduced bacterial colonization of and IL-1{beta} release by macrophages, supporting the involvement of TLR4 signaling in the effects of 15d-PGJ2. During mouse infections, 15d-PGJ2 reduced bacterial burdens in a tissue-dependent manner. Together, these findings demonstrate that 15d-PGJ2 limits Salmonella infection through selective modulation of TLR4 signaling and inflammasome activation.

BackgroundEscherichia coli contamination of chicken eggs is an important food-safety concern, while antimicrobial-resistant E. coli may contribute to the dissemination of antimicrobial resistance through the food chain. However, information on egg-associated E. coli and its antimicrobial susceptibility in Natore District, Bangladesh, is limited. ObjectivesThis study aimed to determine the prevalence of E. coli in chicken eggs collected from commercial farms, markets and indigenous/backyard flocks in Natore District, identify the isolates based on cultural, morphological and biochemical characteristics, and assess their antimicrobial susceptibility. Materials and MethodsA total of 84 egg-shell swab samples, comprising 28 samples each from commercial farms, markets and indigenous chicken flocks, were collected from seven upazillas of Natore District between January and June 2023. Samples were cultured on selective and differential media, and presumptive isolates were confirmed by Gram staining, motility and biochemical tests. Antimicrobial susceptibility was determined using the Kirby-Bauer disc-diffusion method against seven antimicrobial agents. ResultsE. coli was detected in 56/84 (66.67%) egg samples. Prevalence was highest in indigenous eggs (22/28, 78.57%), followed by farm eggs (18/28, 64.28%) and market eggs (16/28, 57.14%). Among 22 confirmed isolates tested for antimicrobial susceptibility, resistance was highest to neomycin (90.91%) and erythromycin (86.36%), followed by oxytetracycline (77.27%), amoxicillin (68.18%), ciprofloxacin (63.63%), levofloxacin (59.09%) and doxycycline (36.36%). No isolate was sensitive to neomycin or erythromycin. ConclusionThe high prevalence of E. coli and substantial antimicrobial resistance among egg-associated isolates indicate an important food-safety and public-health concern. Improved hygienic egg handling, prudent antimicrobial use and continued antimicrobial-resistance surveillance are warranted throughout the poultry production and marketing chain.

BackgroundLive bird markets (LBMs) may facilitate the persistence and dissemination of Escherichia coli and antimicrobial-resistant bacteria because of intensive bird handling, environmental contamination and inadequate sanitation. However, information on E. coli contamination and antimicrobial susceptibility in LBM environments in Rajshahi District, Bangladesh, remains limited. ObjectiveThis study aimed to determine the prevalence, identify the cultural and biochemical characteristics, and assess the antimicrobial susceptibility pattern of E. coli isolated from water, soil and bird-dropping samples collected from LBMs in Rajshahi District. MethodsA total of 60 environmental samples, comprising 20 water, 20 soil and 20 bird-dropping samples, were collected from LBMs across all ten upazillas of Rajshahi District between January and June 2023. E. coli was isolated and identified using cultural characteristics, Gram staining and biochemical tests. Antimicrobial susceptibility was determined by the Kirby- Bauer disc diffusion method against seven antimicrobial agents using CLSI interpretive criteria. ResultsE. coli was detected in 33 of 60 samples, giving an overall prevalence of 55.00%. Prevalence was highest in bird-dropping samples (75.00%), followed by water (55.00%) and soil (35.00%). Among the 33 isolates, resistance was highest to oxytetracycline (78.79%) and amoxicillin (63.64%), followed by ciprofloxacin (48.48%), doxycycline (33.33%), levofloxacin (9.09%), erythromycin (9.09%) and neomycin (6.06%). Sensitivity was highest to neomycin (60.61%), followed by levofloxacin and erythromycin (51.51% each). ConclusionThe high prevalence of E. coli and substantial resistance to several commonly used antimicrobials indicate considerable microbiological and antimicrobial-resistance concerns in LBM environments. Improved sanitation, biosecurity, hygienic poultry handling and prudent antimicrobial use are warranted to reduce environmental contamination and potential transmission of resistant bacteria.

Scrub typhus, caused by Orientia tsutsugamushi, is an obligate intracellular gram-negative pathogen that remains a cause of acute febrile illness in India. Culture isolation of Orientia tsutsugamushi clinical isolates is infrequent because it is technically more challenging than PCR-based molecular identification. In this report, we describe the culture isolation of Orientia from the whole blood of a 64-year-old farmer with acute febrile illness. Whole blood was inoculated onto an 80% confluent L929 cell line. Real-time PCR targeting the 47-kDa and 56-kDa genes, combined with Sanger sequencing, confirmed the isolate. Transmission electron microscopy of infected L929 cells revealed multiple oval-shaped bacteria within the host cytoplasm. Confocal microscopy demonstrated progressive accumulation of CFSE-labelled bacteria within infected cells over time. These findings support the successful isolation and visualization of a blood-derived O. tsutsugamushi isolate and provide a platform for downstream assays of host-pathogen interactions, antimicrobial susceptibility testing, and vaccine development.

Candida auris (currently Candidozyma auris) is an emerging fungal pathogen responsible for dramatic global increase in invasive candidiasis with high mortality. Most concerning, C. auris has a high propensity to colonize patients and persist and develop multidrug resistance to main classes of antifungals. In this study, we investigated the genetic and phenotypic diversity and resistance mechanisms of C. auris clinical isolates recovered from hospitalized infected patients. A total of 53 isolates from 38 unique patients were recovered from various clinical sources and evaluated for susceptibility to routine antifungal drugs. Whole genome sequencing (WGS) and single nucleotide polymorphism (SNP) analysis were performed to generate a phylogenetic network to infer population structure and identify mutations associated with drug resistance development. Isolates were also phenotypically evaluated for ability to form biofilms and aggregate, and cell wall adhesins gene expression studies were performed to provide mechanistic insights into C. auris phenotypic plasticity. Except for one clade III isolate, all isolates belonged to clade I and all were resistant to fluconazole with incidence of resistance to amphotericin B, echinocandins or both. Non-synonymous SNPs were found in genes associated with antifungal resistance including ERG11, TAC1B, CDR1 and FKS1. Phenotypically, isolates varied in their ability to form biofilm and aggregate which correlated with expression of the Scf1 and Als4112 cell wall adhesins genes highlighting C. auris phenotypic plasticity in circulating clinical strains. These findings underscore the growing clinical threat posed by C. auris and reinforce the need for optimized surveillance and treatment strategies for controlling its spread.

PurposeElucidate the influence of glucose metabolic pathways on A. fumigatus lung and corneal infection. MethodsThe A. fumigatus acuF and creA genes were deleted in an mcherry-expressing strain. The mutants were tested for alterations in radial growth, cell wall composition by fluorescence staining assays, and antifungal sensitivity through broth microdilution assays. Hyphal penetration of the strains through explanted porcine corneas was tracked by confocal microscopy using the mCherry signal. Virulence was evaluated in established models of invasive pulmonary aspergillosis (IPA) and fungal keratitis (FK) using C57BL/6J mice. ResultsDeletion of the A. fumigatus phosphoenolpyruvate carboxykinase (acuF) resulted in a dependency on exogenous glucose to support growth in vitro, but did not impact virulence in either the IPA or FK models. Loss of the carbon catabolite repressor CreA resulted in a broad dysregulation of carbon metabolic pathways and altered cell wall homeostasis. Surprisingly, whereas the{Delta} creA remained fully virulent in the lung, the mutant was unable to establish infection in the FK model. This in vivo phenotype corresponded to an inability of{Delta} creA to physically invade porcine corneal explants, which we attributed to a marked reduction in cell wall chitin content. ConclusionsGluconeogenesis is dispensable for A. fumigatus lung and corneal infection, suggesting tissue-derived glucose supports fungal growth in both environments. Loss of CreA disrupts glucose assimilation, its synthesis into chitin and, consequently, cell rigidity and hyphal invasion into the dense corneal stroma. Thus, CreA and other cell wall regulatory proteins may serve as targets for novel FK antifungals.

The marine bacterium Vibrio alginolyticus and the food poisoning bacterium V. parahaemolyticus possess two types of flagella in one cell: proton-driven lateral flagella (Laf) extending from the periphery of the cell body, and sodium ion-driven polar flagella (Pof) extending from a cell pole. For swimming in seawater they use polar flagella, suppressing the expression of lateral flagella. When they attach to the body surface of fish or intestinal tract, lateral flagella are induced, allowing it to crawl along the surface or through mucus. The dynamometer hypothesis, which proposes that polar flagella sense rotation and control the expression of lateral flagellar genes, has been widely accepted. However, how rotation is sensed and how expression is controlled remains unclear. Although swarming has recently been analyzed by physical, biological, or biochemical perspectives, it remains unclear how this motility is controlled, or which substances and conditions are necessary for swarming ability. In this study, we discovered that adding gelatin to agar medium promotes swarming on the agar surface by the lateral flagella of Vibrio. Our data suggested that surfactants or viscous polysaccharides secreted extracellularly are important for promoting swarming on the agar surface and we identified that swarming is likely to be driven by S (social)-motility, in which bacteria move by interacting with each other, and A (adventure)-motility, in which bacteria move by interacting with the agar surface. Our study provides clues that help clarify the mechanism of bacterial swarming IMPORTANCEWe discovered that adding gelatin to hard agar medium promoted swarming on agar surfaces by the lateral flagella of Vibrio cells. The surfactants or viscous polysaccharides secreted extracellularly seem to be important for swarming ability on agar surfaces. We proposed that the swarming is thought to occur through S(social)-motility, where cells move by interacting with cell bodies each other, and A(adventure)-motility, where cells move by interacting with the agar surface and cell body. The present study should provide the clues to clarify the mechanism of bacterial swarming and how to move in a viscous environment.

Background: Pulmonary arterial hypertension (PAH) is a serious disease with poor prognosis, especially in infants or preterm babies and there is still no optimal treatment for this disease. Noradrenalin (NE) is a vasoactive mediator which is released by sympathetic ganglion. According to previous studies, NE/1-adrenoreceptors is not only in regulating normal physiologic responses, but also in the pathogenesis of PAH. However, the mechanisms of NE in PAH are not fully understood. Methods: Human PASMC (PASMC) was used in this study. Cell viability assay and Wound healing assay were used to evaluate the proliferation and migration of PASMC. Immunoprecipitation and western blots analysis were used to investigate the mechanisms which involved in NE-induced PASMC proliferation. Results: We investigated that NE could induce human PASMC proliferation and migration. Furthermore, we first find that endothelin 1 (ET-1) signaling pathway plays an important role in NE-induced PASMC proliferation. ET1 is a critical molecular which is known for regulating cell growth and migration. We investigated that NE could increase NE-1 secretion, further enhancing ET-1 bind to its receptors. For further clarifying the downstream signals in NE/ET-1 induced PASMC proliferation, we detected the phosphorylation and expression levels of ERK and JNK. Conclusions: By combining the results from ours and previous studies, we believed that JNK/c-jun pathway may play an important role in NE-induced PASMC proliferation. Key Words: Noradrenaline; Pulmonary Arterial Hypertension; Pulmonary Artery Smooth Muscle Cells; Endothelin-1; JNK/c-Jun Signaling.

Coxiella burnetii alternates morphologies to survive in two niches: the external environment and a degradative intracellular compartment. The small cell variant (SCV) is adapted for environmental persistence and transmission of Q fever to ruminants and humans. The large cell variant (LCV) is intracellular, and despite not being a major source of transmission, is infectious in vitro. When modeling infection, researchers typically apply a mixed population of these cell types as inocula. As this practice does not mimic natural infection, it may confound our understanding of early Q fever infection events. We separated SCV and LCV by density gradient centrifugation and compared their replication in primary murine macrophages and a fibroblast cell line. SCV inocula replicated more efficiently than LCVs in both host cell types. LCV replication was delayed for four days in macrophages compared with SCV inocula, which had completed logarithmic growth by that time point. We found no difference in pathogenic vacuole size, but there was a modest difference in their respective bacterial burdens. Interestingly, IL-6 and CXCL2 secretion was significantly elevated in LCV-infected macrophages as compared to SCV at 24 hours, suggesting a difference in the host response to each. This is the first study to demonstrate that C. burnetii developmental status influences the progression of infection.

With the rise of antimicrobial resistance, anti-virulence therapeutics are a viable alternative to circumvent resistance pressures. Hypothetical genes and proteins are an under-studied source of potential virulence factor targets. We performed bioinformatic analyses to identify conserved hypothetical genes enriched in pathogenic Pseudomonas aeruginosa but not in non-pathogenic strains. This analysis identified an atypical BlaIR system, which we named pvmSR, that regulated P. aeruginosa virulence in a Caenorhabditis elegans infection model. This is in contrast with the typical BlaIR system from Staphylococcus aureus, which regulates resistance to {beta}-lac-tam antibiotics. The{Delta} pvmSR mutant showed reduced virulence in a C. elegans slow-killing assay. To understand how PvmSR regulated virulence in vivo, we performed dual RNA-seq to analyze transcriptomic changes in both C. elegans and P. aeruginosa. We found that C. elegans responded to P. aeruginosa {Delta}pvmSR infection by decreasing expression of lysosome and phagocytosis pathways. In P. aeruginosa {Delta}pvmSR, we observed decreased gene expression of several known virulence factors including the hydrogen cyanide synthase, hcnC, and heparinase, hepP. Additionally, we observed dysregulation in genes important for quorum sensing and biofilm formation. Collectively, our findings indicated that PvmSR contributed to virulence regulation and may serve as a potential anti-virulence target.

BackgroundHair tools such as hairbrushes and combs allow for various styling options to produce desired hairstyles among various people. However, there is the risk of they serving as fomites for infection and contamination especially among people in close habitation. ObjectivesThis study therefore decided to investigate the trend of microbial populations on these hair grooming tools in universities such as KNUST to inform student hygiene practices and disease prevention strategies. Methods30 students were randomly selected for the study, and swab samples from different hairbrushes and combs were taken for microbial investigation. Microbial isolates were identified based on their morphological and biochemical characteristics. Determination of efficacy of different cleaning methods for hair tools was also done. ResultsThe study found an average bacterial and fungal count of 7.4x102 CFU/ml and 4.6x103 CFU/ml, respectively. The bacterial isolates suspected included Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus sp., Bacillus subtilis, and Corynebacterium sp. The fungal isolates included Aspergillus species, Penicillium sp., Rhizopus sp., Neurospora sp., Colletotrichum gloeosporioides and Curlvularia sp. Correlation analysis showed higher bacterial numbers significantly associated with the presence of hair diseases such as dandruff (p=0.046). Water and detergent were found to be the most effective method of eliminating microbial content from hairbrushes and combs. ConclusionThis study uncovered a variety of microbes on KNUST students combs and hairbrushes, which is evident of microbial contamination. While these numbers are relatively low, this study highlights the need for students to still follow good hygiene procedures and implement efficient cleaning techniques of hair tools, as they may still serve as an ideal environment to harbor and transfer microbes

Hemochromatosis and hemosiderosis are iron overload disorders that cause immune dysfunction and increase susceptibility to bacterial infections. There have been numerous case studies reporting septic-like outcomes for hemochromatosis patients infected with enteric Yersiniae; however, research regarding hemosiderosis and Yersinia infection is limited. Here, we have established a mouse model of hemosiderosis by feeding C57BL/6 mice a high-iron diet. These mice exhibit several indicators of iron overload that are seen clinically, including elevated serum iron levels and iron deposition in various tissues. Characterization of the iron overload mouse model shows that a high-iron diet induces local inflammation in the small intestine and systemic inflammation in a time-dependent manner. Oral infection with Yersinia enterocolitica causes complete mortality in the iron-overloaded mice, while wild-type mice all survive and effectively clear the infection. Lastly, we have observed that iron chelation therapies such as Deferoxamine and Deferisarox are detrimental to iron-overloaded mice during Yersinia infection. This work provides a model to further study iron overload disorders and Yersinia infection.

Spodoptera frugiperda is one of the most important agronomical pests due to its migratory capacity and broad host range. Since it is resistant to several insecticides, novel control strategies are being explored to control it. In this way, Spodoptera frugiperda Multiple Nucleopolyhedrovirus, a natural pathogen, has been proposed for its biocontrol. In this work, we performed a small RNA-seq on uninfected larvae and larvae infected with SfMNPV to identify expressed miRNA, characterize them, and identify differentially expressed (DE) miRNA in the infected condition. We identified several known and putative novel miRNAs, some of which are encoded in multiple copies and may be expressed within miRNA clusters. We also found 13 DE miRNA, most of them previously reported, two of them are putative novel miRNAs identified in this work. We predicted miRNA targets and found that their putative biological role could be related with processes relevant to the infection such as proliferative and apoptotic pathways, cell cycle regulation, autophagy, DNA damage response (DDR), vesicle transport, cytoskeleton remodelling, JAK/STAT and Toll signaling pathway, and immune response activation, among others. Moreover, we observed that several of the putative targets were hub genes in a predicted protein - protein interaction network. Finally, we found DE miRNA putatively associated with the regulation of viral gene expression, suggesting they might have a role in modulating the infection. Our results contribute to better understanding the miRNA landscape in S. frugiperda, and their putative role upon SfMNPV infection.

Certain strains of the facultative intracellular bacterial pathogen Listeria monocytogenes are thought to invade cranial nerves in the gut and disseminate directly to the brainstem to cause rhombencephalitis in both humans and ruminants. Bacteria with actin tails were previously observed within neurons of naturally infected sheep, but the mechanism for how these neurotropic strains access the nervous system has not been well characterized. Using a foodborne mouse model of listeriosis, we show here that bypassing the gut phase of infection prevents colonization of the brain, confirming that invasion of the nervous system happens in the intestinal tract. L. monocytogenes did not efficiently invade neuronal cell lines, although they could replicate exponentially in the cytosol and form actin tails. Instead, the neurotropic strains displayed a preferential ability to invade enteric glial cells, a specialized subset of glia that support neurons and are critical for intestinal homeostasis. Using an in vitro co-culture system, we demonstrated that neurotropic L. monocytogenes could readily invade enteric glial cells and use ActA-mediated actin-based motility to spread to adjacent neurons. These results suggest that invasion of enteric glial cells is a novel virulence strategy that can promote brainstem infection following foodborne transmission of L. monocytogenes. IMPORTANCEThis study provides further evidence for dissemination of neurotropic strains of L. monocytogenes from the gut directly to the brain via axonal migration using foodborne mouse model of listeriosis. It is the first report showing that enteric glial cells, a specialized subset of cells in the gut that support intestinal neurons, are susceptible to pathogenic bacterial infection.

Bartonella henselae is a zoonotic pathogen associated with cat-scratch disease. Although multilocus sequence typing (MLST) has been used for strain classification, its resolution for distinguishing between B. henselae isolates remains limited. We herein developed a B. henselae-specific core genome MLST (cgMLST) scheme based on whole-genome sequencing data and examined the genetic and phenotypic diversities of 80 strains derived from cats, humans, mongooses, and masked palm civets. Using the conventional MLST scheme, the 80 strains were classified into nine sequence types (STs), while cgMLST subdivided them into 72 cgSTs, demonstrating a marked improvement in discriminatory power. The cgMLST scheme comprised 1,183 core genes and showed high applicability across the 80 strains. A phylogenetic analysis revealed that ST1, which has been associated with cat-scratch disease, was further subdivided into three major clusters and two singletons, indicating high genetic heterogeneity within this ST. We also found that the bafA subtypes clustered in a manner that was largely consistent with the cgMLST-based phylogenetic structure, suggesting a close relationship between bafA variations and the genomic background of B. henselae strains. In a human umbilical vein endothelial cell proliferation assay, strains belonging to distinct cgSTs exhibited strain-dependent differences in proliferative capacity, which were associated with the bafA subtype classification. Some strains induced focal cell fragmentation and a reduced cell density at a high multiplicity of infection, indicating strain-dependent differences in endothelial cell injury. Collectively, the present results establish a high-resolution cgMLST framework for B. henselae and demonstrate that genetically distinct strains have diverse endothelial cell phenotypes.

Pseudocercospora fijiensis is the causal agent of Black Leaf Streak Disease (BLSD), also known as black Sigatoka, in banana. The disease affects many banana varieties, including the highly susceptible Cavendish banana that dominates global production and the export trade, and several cooking bananas that are a staple food for hundreds of millions of people worldwide. Currently, the disease is controlled using preventative fungicide treatments with up to 70 applications per year in Cavendish plantations, which accounts for approximately 30% of the production costs. Resistant cultivars are required for more sustainable production, but no resistance gene to BLSD has been identified. This is partly due to the poor genetic amenability of P. fijiensis and the lack of methods for functional gene analysis. To address these limitations, we developed a CRISPR/Cas9-mediated transformation system specifically optimized for P. fijiensis. We established a protocol to produce protoplasts, evaluated their capacity to regenerate into new colonies, and assessed antibiotic sensitivity. Subsequently, we confirmed the integration of foreign DNA, including resistance markers, using PEG-mediated transformation. We demonstrated targeted transformation using CRISPR-Cas9 to knockout the polyketide synthase gene PKS10-1, which is responsible for the production of the pigment melanin, and the mitogen-activated protein kinase (MAPK) gene Fus3. Following the successful generation of knockout mutants for these genes, achieving gene targeting efficiencies of respectively 96% and 58%, we subsequently generated knockout mutants of the renowned effector Avr4 in P. fijiensis. The resulting mutants exhibited no reduction in virulence on the susceptible banana cultivar Cavendish. In addition, we used the wild-type isolate and Avr4 knockout strains to test the resistant banana accession Calcutta 4. Contrary to a previous study, we demonstrate that Avr4 does not explain the resistance of Calcutta 4, suggesting that resistance is instead triggered by the recognition of other hitherto unknown effectors. The established CRISPR/Cas9-mediated disruption system is highly efficient and enables routine functional gene characterization, which will help to elucidate genes involved in banana-P. fijiensis interaction, thereby supporting the discovery of resistance genes against BLSD.

Methicillin-resistant Staphylococcus aureus (MRSA) is an opportunistic pathogen that colonizes a significant proportion of humans, contains numerous virulence factors promoting infection, and continues to threaten human lives and burden healthcare systems globally. Many MRSA virulence factors are known to be either secreted or anchored on the outer leaflet of the cell surface. Although many virulence factors have been studied intensively in MRSA, there remains a significant proportion of secreted and surface proteins that are unstudied for their potential as virulence factors. We began with identifying proteins secreted from MRSA in axenic culture using an unbiased mass-spectrometry based approach. 2 secreted proteins thus identified mapped to an operon of 6 genes, SAUSA300_1739 to SAUSA300_1744. Mutation of each of the individual genes in the operon resulted in attenuation in a mouse model of subcutaneous infection. We demonstrate that two genes in the operon, SAUSA300_1739, and SAUSA300_1740, encode nucleases with DNase activity. Genetic analysis of the SAUSA300_1739 to SAUSA300_1744 operon across several Staphylococcus aureus strains indicate that the operon is highly conserved, highlighting its importance for virulence.

S. mutans and V. parvula cooperate in dental plaque to assemble a healthy biofilm and are associated with increased caries risk. S. mutans produces lactic acid from carbohydrates resulting in a final biofilm pH[~]4, while V. parvula metabolizes lactate to acetic and propionic acids resulting in pH[~]5. This process results in healthier biofilms that still generate a pH capable of demineralizing tooth surfaces (pH<5.5). The purpose of this study was to identify V. parvula genes required for deacidification of S. mutans biofilms and determine whether the ability of V. parvula to deacidify S. mutans biofilms correlates with enhanced biofilm health. Using transposon mutagenesis in V. parvula we identified several genes required for deacidification of S. mutans biofilms. These included numerous V. parvula transposon mutations in the previously unstudied lutABC lactate utilization operon. To assess biofilm health, S. mutans in the presence of various V. parvula mutants were stained with a LIVE/DEAD stain and imaged by fluorescence microscopy. An intact lutABC operon was required to enhance biofilm health, as demonstrated by plasmid-based complementation of a lutB transposon mutant. Transposon insertions in other loci unrelated to deacidification had no impact on biofilm health. Addition of HEPES buffer at the time of S. mutans biofilm assembly prevented full acidification of the biofilm and resulted in improved biofilm health, even without the addition of V. parvula. Finally, we found V. parvula can use either nitrate or fumarate as a final ETC electron acceptor during lactate utilization. In all, we found the lutABC lactate utilization operon of V. parvula is critical for the ability of V. parvula to deacidify S. mutans biofilms and promote biofilm health. Interfering with this pathway would interrupt the mutually beneficial relationship between S. mutans and V. parvula that leads to their co-association in caries, root caries, and early childhood caries.

Ticks are obligate hematophagous arthropods and feed exclusively on blood. As blood is nutrient-poor, ticks rely on bacterial endosymbionts to synthesise nutrients, yet the diversity and functional roles of these symbionts in Australian ticks remain largely uncharacterised. This is critical to address as these ticks are of high medical importance in Australia. In this study, shotgun metagenomic sequencing was performed on Bothriocroton concolor, Bothriocroton hydrosauri, Haemaphysalis longicornis and Ixodes holocyclus, enabling the recovery of six complete or partial metagenome-assembled genomes (MAGs). These comprised Coxiella-like endosymbionts (CLE), a facultative Rickettsia symbiont, and two Midichloria mitochondrii strains (Ixholo1 and Ixholo2). Functional annotation of these taxon-specific symbionts revealed the absence of virulence factors and the presence of B-vitamin and/or heme biosynthesis genes, indicative of nutritional mutualism, which is essential for tick hematophagy. The CLEs additionally harbour genes of the shikimate pathway, which modulate blood feeding in ticks by regulating serotonin biosynthesis. Furthermore, functional annotation and pangenomic analysis of Midichloria spp. found evidence that the genus may encompass multiple species, as well as the retention of genes potentially associated with an intramitochondrial lifestyle in M. mitochondrii Ixholo2. Tick microbiomes are dominated by non-pathogenic microorganisms, which are often overshadowed by pathogens. These include the endosymbionts, which can influence host biology and pathogen transmission, and are fundamental for the development of diagnostic tools and taxon-specific tick biocontrols.

Background: Viral myocarditis presents a significant burden of disease, particularly among children and young adults. However, clinical guidelines and treatment strategies for pediatric patients are derived from those for adult patients due to a lack of pediatric data. Current animal models of viral myocarditis use adult mice, so conclusions from these models cannot necessarily be extrapolated to the pediatric population. We sought to develop a juvenile mouse model of myocarditis to examine differences between these two distinct clinical populations. Methods: Male and female BALB/c 3-4-week-old 'juvenile' and 8-week-old 'adult' mice were infected intraperitoneally with 103 PFU of heart-passaged coxsackievirus B3. Sera was used to evaluate testosterone and estradiol levels. Cardiac histological evaluations included overall inflammation, fibrosis, and specific cell-type infiltration. RNA was extracted from cardiac tissue and evaluated for changes in gene expression of cell-type markers, complement components, and NLRP3 inflammasome components. Results: Juvenile mice exhibited more severe inflammation than adult mice but no sex differences in overall inflammation. Juvenile mice demonstrated increased infiltration of CD11b+ cells, F4/80+ cells, and CD3+ T-cells vs. adults. Inflammasome genes NLRP3 and caspase-1 were significantly increased in juvenile compared with adult myocarditis. Conclusions: This paper is the first to describe a juvenile mouse model of coxsackievirus B3 myocarditis and provides a direct comparison to a translational adult mouse model. Juvenile mice had greater cardiac inflammation than adults. This model replicates clinical populations and provides a valuable tool to study age as a factor in the pathogenesis of myocarditis.