Pathogenic Species Research Articles

High, but variable prevalence of Sarcocystis cruzi infections in farm-raised American bison (Bison bison) beef destined for human consumption.

Bison (Bison bison) and cattle (Bos taurus) are closely related (can interbreed) and they also share many parasites. Cattle are commonly infected with one or more of the eight named Sarcocystis species: Sarcocystis hirsuta, S. cruzi, S. hominis, S. bovifelis, S. heydorni, S. bovini, S. sigmoideus and S. rommeli. Among these, the full life-cycle is known only for S. cruzi. Sarcocystis cruzi (transmitted via canids) is recognized as the most pathogenic Sarcocystis species, causing abortion, low milk yield and poor body growth. It has been experimentally cross-transmitted from cattle to bison and vice versa. We tested 200 bison tongues from three commercial sources (farms) (Nebraska #141; South Dakota #36; New Jersey and Pennsylvania #23). Frozen tongues were purchased and examined for Sarcocystis infection using light microscopy, histology and quantitative PCR (qPCR) targeting 18S ribosomal DNA (18S rRNA) of S. cruzi. Lesions associated with degenerating sarcocysts were studied. The intensity of Sarcocystis infection in histological sections was quantitated. Sarcocystis cruzi-like infections were detected in 129 of 141 (91.5%) tongues from Nebraska, 36 of 36 (100%) tongues from South Dakota and two of 23 (8.6%) tongues from New Jersey and Pennsylvania. Sarcocysts were detected in histological sections stained with hematoxylin and eosin in 167 of 200 samples. Light microscopy examination revealed that the sarcocysts had thin walls (< 1 µm thick) and appeared to be S. cruzi. However, in two samples, sarcocysts had thicker walls measuring up to 2.3 µm wide and 154 µm long and the sarcocyst wall was not striated; these two samples could not be characterized further. In three tongues, degenerating sarcocysts were recognized; two of these were associated with thick-walled sarcocysts. Molecularly, S. cruzi from bison was identical to that in cattle. In the present study of bison tongues, S. cruzi was the only species identified in bison using both molecular and morphological methods. An unidentified species of Sarcocystis found in two bison samples needs further study.

Assessment of pathogenic yeast species in the oral cavity of dogs: Antifungal susceptibility and implications for human health.

Companions' animals can present a risk for the transmission of opportunistic diseases to their owners, including those caused by yeasts of the Candida genus residing in their oral microbiota. This study aimed to isolate and identify yeasts from the oral cavity of dogs and assess their susceptibility to antifungals. Yeast species were identified using automated methods MALDI-TOF-MS and VITEK 2 from 50 dogs (aged 2-4years, various breeds). Among the evaluated animals Candida albicans (37.5%) and Candida parapsilosis (25%) were predominant, followed by other species including C. haemuloni, C. tropicalis, C. krusei, and Trichosporon asahii (12.5% each). In this study, antifungal susceptibility testing revealed a general susceptibility with limited resistance. Although most antifungals exhibited good efficacy, some Candida strains demonstrated resistance to fluconazole (FLC), caspofungin (CAS), and amphotericin B (AMPB). These findings highlight the importance of conducting fungal cultures and antifungigram testing for the effective management of fungal infections in humans and animals. Therefore, vigilant monitoring and control measures are essential to reduce the transmission of opportunistic diseases from pets to humans.

Survey of foodborne pathogenic Vibrio species in commercial Tasmanian bivalve shellfish and predictors of risk at harvest.

There has been an increase in foodborne vibriosis outbreaks globally, with Vibrio parahaemolyticus emerging as a foodborne issue in temperate commercial shellfish growing regions, including southern Australia. The food safety concerns associated with these microorganisms have led to the need for specific guidance on potential risk management strategies for their control. This is the first Australian multi-seasonal survey of V. parahaemolyticus and Vibrio vulnificus in commercial shellfish growing areas aimed at understanding their occurrence and regional environmental drivers of risk at harvest. Eleven commercial Tasmanian shellfish (oyster and mussel) growing areas were surveyed for the prevalence and levels of V. parahaemolyticus, including presence of pathogenicity associated tdh and trh genes, and V. vulnificus at harvest between 2020 and 2022. Vibrio parahaemolyticus was detected in all surveyed growing areas during the three summer/autumn sampling periods, with a prevalence of 8-100%. Prevalence was generally higher in the north (north-west, Moulting Bay and upper east coast) as compared with the mid-east coast and south-east and Bruny regions. Vibrio parahaemolyticus was only detected in five of the eleven growing areas during the two surveyed winter/spring seasons: Duck Bay, Moulting Bay, Great Swanport, Little Swanport and Boomer Bay East. Where it was detected, the prevalence was much lower in the winter/spring seasons (17-33%). Levels of V. parahaemolyticus detected during the survey were generally low (<10 MPN/g) for most growing areas. Some higher levels (100-1100 MPN/g) were observed in Duck Bay, Moulting Bay, Great Swanport and Little Swanport. Only one sample from Great Swanport had a level of over 1000 MPN/g (1,100 MPN/g). The higher levels were only observed in the summer/autumn sampling periods. Vibrio parahaemolyticus tdh, trh or tdh/trh gene detections only occurred in the summer/autumn months with a prevalence of 0-21%, 0-18% or 0-7%, respectively, depending on the growing area surveyed. Despite low levels of V. parahaemolyticus being detected in southern commercial growing areas, five sporadic cases of vibriosis associated with oysters from southern Tasmania were reported during the survey period, predominantly from oysters harvested recreationally. Levels of V. vulnificus detected were generally very low in most Tasmanian growing areas (<1 MPN/g). However, levels of 35-460 MPN/g were detected in shellfish at harvest in one area (Great Swanport). Predictive models for V. parahaemolyticus at harvest were developed from survey data which were area specific. Water temperature was the sole or primary driver in most areas. Predictive models for V. vulnificus at harvest were developed for Great Swanport and were driven by river flow and rainfall.

Huang-Lian-Jie-Du decoction ameliorates sepsis through dynamic regulation of immune response and gut microbiota-metabolite axis.

Sepsis remains a life-threatening condition with high mortality rates despite current therapeutic approaches. While Huang-Lian-Jie-Du Decoction (HLJDD), a traditional Chinese medicine formula, has been historically used to treat inflammatory conditions, its therapeutic potential in sepsis and underlying mechanisms remain unexplored. This study investigated HLJDD's comprehensive effects on sepsis pathophysiology using a rat cecal ligation and puncture (CLP) model. HLJDD significantly improved survival rates and demonstrated sophisticated immunomodulatory effects through temporal regulation of the biphasic immune response characteristic of sepsis. In early sepsis, HLJDD suppressed pro-inflammatory cytokines (IL-1β, IL-6) while maintaining defensive inflammation. During late sepsis, it counteracted immunosuppression by reducing IL-10 levels and CD4+CD25+ T cell populations while protecting CD4+ and CD8+ T cells from apoptosis. Notably, HLJDD demonstrated dynamic regulation of the gut microbiota-metabolite axis. It enhanced beneficial bacterial populations (Firmicutes, Lactobacillus) while suppressing potentially pathogenic species (Bacteroides, Parabacteroides). Metabolomic analysis revealed time-dependent modulation of short-chain fatty acids, with elevated levels at 12h followed by strategic reduction at 18-30h, coordinating with changes in SCFA-producing bacteria. This temporal metabolic regulation corresponded with improved intestinal barrier function and balanced immune responses. The study unveils HLJDD's novel mechanism of action through synchronized modulation of immune responses, gut microbiota, and metabolite profiles, presenting a multi-target therapeutic approach that addresses the complex pathophysiology of sepsis. These findings provide a strong foundation for further clinical investigation of HLJDD as an innovative treatment strategy for sepsis.

Role of respiratory secretion culture in the surgical outcome prediction of bacterial empyema.

Thoracic empyema is a serious infection. Video-assisted thoracoscopic surgery is a recommended treatment, and pleural fluid and tissue cultures are collected intraoperatively. The combination of a pleural peels tissue culture and a pleural fluid culture improves the positive culture rate. We aimed to investigate the role of respiratory secretion cultures to determine the optimal management for improving surgical outcome. The study analyzed 225 adult patients with phase II/III thoracic empyema who underwent thoracoscopic decortication. Respiratory secretion cultures were obtained and compared with pleural cultures. Key outcomes were culture positivity and pathogen consistency, with secondary outcomes including intensive care unit stay, hospital stay, and mortality. There were 225 empyema patients with either a positive pleural fluid culture or a positive pleural peel tissue culture. Of these, 76 patients had positive respiratory secretion culture findings during hospitalization. The most common pathogen species were Pseudomonas aeruginosa (44%) and Klebsiella pneumoniae (16%) in the respiratory secretion cultures and Streptococcus spp. (38%) and Klebsiella pneumoniae (12%) in the pleural cultures. There were 30 patients having a common pathogen in the respiratory secretion culture and in the pleural fluid/tissue culture. Poor outcome measures were found in these patients, including the longer use of antibiotics preoperatively [2.50 (1.00-6.00) days versus 5.00 (2.75-11.00) days, p = 0.006] and a higher mortality rate during hospitalization (40.0% versus 17.4%, p = 0.002). Respiratory secretion cultures are vital for predicting surgical outcomes in bacterial empyema, and prompt specimen collection can improve patient survival.

First report of Colletotrichum truncatum causing anthracnose of ashwagandha (Withania somnifera) in India.

Ashwagandha (Withania somnifera), enriched in alkaloids, steroidal lactones and saponins, is a valuable herb in Indian Ayurvedic medicine. During December 2023, Va-1 (Vallabh Ashwagandha-1) plants at ICAR -Central Tobacco Research Institute, Vedasandur, Tamil Nadu (10.53717ºN, 77.9507°E) were noticed with anthracnose-like disease symptoms, characterized by irregular necrotic lesions on stem, leaves and pods. Infected plants dried entirely in the advanced stage and had pin-head-shaped fruiting bodies. In the assessment of 185 plants, 42 were symptomatic, while five were dried, and the disease severity varied between 17 to 22%. Pathogen was isolated from infected leaf and stem tissues (3 to 5 mm2) using a potato dextrose agar (PDA) medium amended with streptomycin sulphate (20 mg/l). Infected tissues were surface sterilized using 1% sodium hypochlorite, and the PDA plates were incubated at 27°C for three days. The pathogen was purified, and two cultures, AVCT01 and AVCT02, were used for the downstream characterization. Colonies on the PDA medium were initially pale white and later turned to dark grey, with an average growth of 42.6 mm (diameter) on the 7th day after inoculation (DAI). Conidia were smooth, hyaline, aseptate and curved, with a tapering end and truncate base. The conidial dimensions (n=30) were 22.8 ± 1.32 × 3.4 ± 0.29 µm (20.6 to 25.8 × 2.9 to 4.2). Acervuli were scattered, containing dark brown conidiomata with septate, pointed setae and measured 119 ± 16.6 × 4.2 ± 0.5 µm (81 to 148 × 3.1 to 5.3) in dimension. Morphological and cultural characteristics were identical to Colletotrichum truncatum described by Damm et al. (2009). Further, the molecular characterization was executed with primers targeting ITS (ITS1/ITS4) and three housekeeping genes; act (ACT-512F/ACT-783R), tub2 (Bt2a/Bt2b) and gapdh (GDF1/GDFR1) (Kim et al. 2020). The amplified products were sequenced bidirectionally and in BLASTn analysis, sequences revealed 100% identity with C. truncatum [(NCBI GenBank OM327672 (ITS), OR147336 (act), MG214130 (tub2) and MK675260 (gapdh)]. In the maximum likelihood phylogeny of concatenated alignments, these cultures clustered with CBS119189. All sequences were deposited in NCBI GenBank (ITS: PP919605 and PP919606; act: PP921525 and PP921526; tub2: PP921527 and PP921528; gapdh: PP921529 and PP921530). Pathogenicity was done by spraying conidial suspension of AVCT01 (1 × 106/ml) on leaves of three plants (two months old). Non-injury technique of Gupta et al. (2020) was conducted for stem inoculation. The inoculated and control plants (sprayed with sterile distilled water) were maintained separately at 25°C with 90% relative humidity. Necrotic lesions were observed on leaves and stems of inoculated plants on 8th DAI confirming Koch's postulates and the pathogen re-isolation was also done. Disease severity was assessed using the scale of Yang and Hartman, 2015. Previously, leaf spots caused by C. gloeosporioides and C. dematium were reported in ashwagandha from West Bengal (Sarkar and Dasgupta, 2017) and Uttar Pradesh (Wijeskara et al. 2005) states of India. However, our study marks the first report of C. truncatum in ashwagandha, alerting the adaptation of multiple Colletotrichum species to this highly valued medicinal herb. Elucidating the etiology of newly adapted pathogen species paves the way for rapid disease diagnostics to implement effective disease management strategies.

P-772. Whole Genome Analysis of Mycobacterium intracellulare isolates from Colonic Biopsy In Inflammatory Bowel Disease Patients

Abstract Background A long debate has prevailed over cause of Crohn's disease with published evidence for Mycobacterium paratuberculosis (MAP).Our study was designed to look for mycobacterial pathogens by culture and DNA PCR in patients with inflammatory bowel disease(IBD). Phylogenetic Analysis of M. intracellulare isolate genomes compared with reference M. intracellular strain ATCC 13950T Circular representation of four M. intracellulare isolate genomes compared with reference M. intracellular strain ATCC 13950T. Inner to outer four concentric rings show isolates from this study. The information is read from outer circle to inner as follows: genome size, genes on the forward strand, genes on the reverse strand, t-RNA, and r-RNA of reference strain.The percentage similarity is represented by different color codes. Zoomed portion represent absent genetic region in four isolates. Methods Colonic biopsy and Blood Buffy coat samples were processed for cultures and DNA detection for mycobacterial pathogens (including MAP) in 889 patients with inflammatory bowel disease; Crohn’s disease (CD)( clinical manifestations, endoscopic,radiological and histological features as per ECCO consensus guidelines), Intestinal tuberculosis,Controls (Adult patients with hemorrhoidal bleeding and no other colonic disease undergoing sigmoidoscopy). Results Four cultures from CD patients grew Mycobacterium intracellulare. Whole Genome Sequencing identified deletion regions in the studied isolates, with 156 protein-coding genes absent in all four isolates compared to the reference genome (M. intracellulare Strain ATCC 13950T). Antibiotic resistance and virulence factor analyses indicated resistance to rifampicin and isoniazid and an abundance of virulence factor genes in M. Intracellulare species. Pan and core gene analyses suggested a closed pan-genome in M.intracellulare, with limited distribution of antibiotic resistance genes. Conclusion M. Intracellulare is reported in immune-compromised patientsin several studies. The results of the core genome phylogenetic analysis suggest that factors beyond the isolation sources of M.intracellulare strains may contribute to shape their evolutionary trajectory. Functional analysis indicated a gradual decrease in metabolic capacity across clusters, suggesting an ongoing genome streamlining process in M. Intracellulare species. Essential gene prediction identified strain specific essential genes, emphasizing the importance of energy-related processes. Homologous recombination rate analysis suggested recombination in M. intracellulare strains, with specific parameters showing variation compared to other pathogenic species. This comprehensive study contributes valuable information about the genomic characteristics, evolution, and functional profiles of M. intracellulare strains. Disclosures All Authors: No reported disclosures

P-2237. Mortality and Causative Pathogens of Sepsis Differ by Molecular Phenotype in a Secondary Analysis of MARS

Abstract Background Heterogeneity of host response has been a barrier to identifying novel therapies in sepsis. Two molecular phenotypes of critical illness, called the Hyperinflammatory and Hypoinflammatory, have been identified in multiple cohorts of sepsis and ARDS ( &amp;gt;12,000 patients), with divergent biology, clinical course, and responses to therapy1. We evaluated whether these phenotypes were identifiable in MARS-- a large observational cohort of critically ill patients-- and whether the pathogens present in each phenotype differed. Methods MARS recruited critically ill patients from two Dutch ICUs. Only patients meeting sepsis or ARDS diagnostic criteria were included for this analysis. We applied latent class analysis (LCA) to vital signs, clinical laboratory values, and protein biomarkers from Day 1 of ICU admission in 1,485 patients. LCA models with 1-5 classes were fitted. We assigned class membership using the best fitting model and compared characteristics between classes. The present classes’ overlap with prior phenotypes was evaluated by comparison to an extensively validated parsimonious model,2 trained previously to classify molecular phenotypes. Results A two-class model best fit the cohort (VLMR p&amp;lt; .001), and the parsimonious model and multivariate profiles suggested high overlap (AUC=.929) with previously described ‘Hypoinflammatory’ (Class 1; n=895) and ‘Hyperinflammatory’ (Class 2; n=590) phenotypes (Figure 1). Bacteremia was more prevalent in the Hyperinflammatory phenotype (27.2% vs 7.8%; p&amp;lt; .001), with Enterobacteriaceae as the most prevalent species. For primary infection sites, lower respiratory tract was more common in the Hypoinflammatory (56% vs 36%) and abdomen more common in the Hyperinflammatory phenotype (26% vs 9%). Mortality was significantly higher in the Hyperinflammatory phenotype at both 30 days (OR=2.54, CI95%= 2.003—3.235 ) and 90 days (OR=2.41, CI95% = 1.925—3.016; both p&amp;lt; .001). Conclusion In MARS, we identified the previously described Hypo- and Hyperinflammatory phenotypes with divergent host response, pathogen profiles, and outcomes. These findings suggest that host phenotype signatures in part may be driven by sites of infection and pathogen species. Disclosures Lieuwe D.J. Bos, MD-PhD, Astra Zeneca: Advisor/Consultant|CSL Behring: Advisor/Consultant|Impentri: Advisor/Consultant|Novartis: Advisor/Consultant|Scailyte: Advisor/Consultant|Sobi: Advisor/Consultant Pratik Sinha, MBChB/PhD, AstraZeneca: Advisor/Consultant|Prenosis Inc: Board Member Carolyn S. Calfee, MD, Cellenkos: Advisor/Consultant|Gen1e Life Sciences: Advisor/Consultant|Janssen: Advisor/Consultant|NGMBio: Advisor/Consultant|Santhera: Advisor/Consultant|Vasomune: Advisor/Consultant

510. Bacteriophages as Drug Delivery Vehicles for Polymyxin B

Abstract Background The goal of this project is to develop a safer, more effective formulation of polymyxin B for E. coli bacteremia. Polymyxins are a family of cyclic lipopeptides that display exceptional antibiotic activities toward gram-negative pathogens such as E. coli, Pseudomonas aeruginosa, and Acinetobacter baumannii. The PMB label includes an FDA black box warning for serious nephrotoxicity and neurotoxicity. However, today, with the growing emergence of “superbugs” and a dry antibiotic discovery pipeline, PMB is increasingly used as a last-line therapy to treat systemic infections. Thus, although PMB is a potent antibacterial agent, its toxicity currently prevents its use as an earlier or more widespread treatment. Transmission electron microscopy (TEM; negative stain) images of anti-LPS phage bound to gram-negative organisms. Phages were labeled with gold nanoparticles (dark spheres) coated with antibodies against pVIII, allowing phages to be easily identified (red darts). Gold-labeled anti-LPS phage are shown (A) without bacteria; or incubated with (B) E. coli DH5α; (C) E. coli ATCC BAA 1161; (D) A. baumannii ATCC 19606. Phage width is increased by the labeling reagents. Scale bars = 500 nm. Methods We engineered M13 to bind the core antigen of lipopolysaccharide (LPS), enabling binding to a variety of top-priority pathogenic gram-negative bacterial species, including E. coli, P. aeruginosa, A. baumannii, Klebsiella pneumoniae, and Burkholderia cepacia. Since the anti-LPS phages are able to bind but do not infect (nonviable), they are not cytotoxic by themselves. However, delivery of PMB by the anti-LPS phage confers antibiotic activity. Thus, in our strategy, PMB molecules are conjugated to the anti-LPS phage capsid, and delivered specifically to gram-negative bacterial cells through the specificity and high affinity of the anti-LPS binding domain. We have shown that the PMB – anti-LPS phage conjugate (termed “PMB–Phage” hereafter) effectively lowers the minimal inhibitory concentration of PMB by multiple orders of magnitude in vitro compared to PMB sulfate. MIC and MBC of PMB and PMB-Phage, determined in vitro for several gram-negative organisms. The heat map corresponds to the MIC or MBC values. Equivalent PMB concentrations for PMB-Phage MIC and MBC were calculated from the measured phage concentration and the average amount of PMB loaded per virion. * = resistant. ND = not determined due to technical limitation on phage concentration. Results We have already demonstrated following results.A broad-spectrum anti-LPS phage was engineered and verified to bind to several gram-negative bacterial species including E. coli.Polymyxin B was conjugated to the anti-LPS phage, creating PMB-Phage, which was highly antibacterial in vitro.PMB-Phage is non-toxic to mammalian cells in vitro and non-toxic in mice.PMB-Phage is distributed to various organs within 1 hour and has an elimination half-life of approximately 1 day.PMB-Phage appears to be effective in treating E. coli bacteremia in a mouse model. PMB-Phage Biodistribution Figure 9: Time course biodistribution of PMB-Phage in various organs, over time in male mice with (red) or without (blue) 1×10^7cfu (non-lethal dose) of E. coli ATCC BAA 1161 injected (n=3). Signals are measured as a percentage of the injected dose (ID) per volume (per cc). Representative biodistribution images (j) of 1 animal. Conclusion PMB-Phage will greatly reduce the nephrotoxicity of PMB, enabling safe treatment for a broader patient population. Liver and kidney blood biomarkers at end point of 7-day toxicity studies. Animals in experimental groups were injected 100µL of test materials through IV tail vein injection daily for 7 consecutive days. Experimental group animals for each condition (N=3), receiving either PMB or PMB-Phage at different doses show no significant difference comparing to control groups of animals group receiving no treatment (N=5) and 1xPBS buffer (N=3), in light and dark grey columns respectively. The only exception being all animals receiving 72 mg/kg body weight PMB (N=3) did not survive a single dose and died on day 1 (indicated by red cross in figure). Disclosures All Authors: No reported disclosures

Examining the Biophysical Properties of the Inner Membrane of Gram-Negative ESKAPE Pathogens.

The World Health Organization has identified multidrug-resistant bacteria as a serious global health threat. Gram-negative bacteria are particularly prone to antibiotic resistance, and their high rate of antibiotic resistance has been suggested to be related to the complex structure of their cell membrane. The outer membrane of Gram-negative bacteria contains lipopolysaccharides that protect the bacteria against threats such as antibiotics, while the inner membrane houses 20-30% of the bacterial cellular proteins. Given the cell membrane's critical role in bacterial survival, antibiotics targeting the cell membrane have been proposed to combat bacterial infections. However, a deeper understanding of the biophysical properties of the bacterial cell membrane is crucial to developing effective and specific antibiotics. In this study, Martini coarse-grain molecular dynamics simulations were used to investigate the interplay between membrane composition and biophysical properties of the inner membrane across four pathogenic bacterial species: Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterobacter cloacae, and Escherichia coli. The simulations indicate the impact of species-specific membrane composition on the overall membrane properties. Specifically, the cardiolipin concentration in the inner membrane is a key factor influencing the membrane features. Model membranes with varying concentrations of bacterial lipids (phosphatidylglycerol, phosphatidylethanolamine, and cardiolipin) further support the significant role of cardiolipin in determining the membrane biophysical properties. The bacterial inner membrane models developed in this work pave the way for future simulations of bacterial membrane proteins and for simulations investigating novel strategies aimed at disrupting the bacterial membrane to treat antibiotic-resistant infections.

Salivary Microbiome Profiling of HPV+ and HPV− Oropharyngeal Head and Neck Cancer Patients Undergoing Durvalumab Immunotherapy Suggests Prevotella melaninogenica and Veillonella atypica as Key Players: A Pilot Study

Objective: Head and neck cancer (HNC) is a common cancer represented by nearly 80% oral cavity (OC) and oropharyngeal cancers (OPCs). Seventy percent of OPCs are associated with the Human Papilloma Virus (HPV). Immunotherapy holds the promise of future improvements in treating HNC patients. The study objective was to determine whether durvalumab immunotherapy alone, prior to curative surgery, would significantly impact the oral salivary microbiome in a pilot cohort of HPV negative and positive OC and OPC patients. Methods: Early stage OPC patients with squamous cell carcinoma were recruited: 5 HPV+ and 12 HPV−, and treated with two or three administrations of durvalumab given every two weeks, prior to surgery. Unstimulated saliva was collected and processed for bacterial DNA Isolation and V1–V3 16S rRNA gene next generation sequencing, taxa identification, and determination of relative abundance at four time points: baseline prior to surgery (A) and weekly durvalumab treatment timepoints (B, C, and D). Alpha- and beta-diversity differences for the time series were determined in Primerv7. MaAsLin2 in R was used to identify potential associations with the time series and/or HPV status. Linear decomposition model (LDM) R-package was used to investigate the relationship of salivary microbiome with HPV status. ROC curves were plotted for significant species in common between MaAsLin2 analysis and FDR-corrected Mann-Whitney U-test using XLSTAT. Results: Longitudinal microbiome data across four timepoints (A, B, C, D) were obtained (HPV+: n = 18 samples; HPV−: n = 46 samples). A total of 416 taxa were detected across all time points, ranging from 336 to 373 per group. There were no differences in α- and β-diversities for all longitudinal comparisons (C vs. BCD, AB vs. CD, or A vs. B, C, or D). However, comparison A vs. D showed a significant increase in Prevotella melaninogenica relative abundance, a potentially pathogenic species able to evade the immune system, after three weeks treatment. Moreover, differences in beta-diversity based on HPV status were found. LDM analysis identified Veillonella atypica, overrepresented in HPV+ group, as the top species accounting for HPV status. Conclusions: The results are consistent with findings from previous studies investigating HNC patients treated with chemoradiotherapy. More research is needed to understand possible impact of immunotherapy on opportunistic bacterial species, although negligible impact from durvalumab treatment on salivary microbiome was observed.

Crop rotation effects on the population density of soybean soilborne pathogens under no-till cropping system.

Soilborne diseases are persistent problems in soybean production. Long-term crop rotation can contribute to soilborne disease management. However, the response of soilborne pathogens to crop rotation is inconsistent, and rotation efficacy is highly variable. Selection of proper crop plants and crop sequences for disease management is needed. In this research, the effects of crop rotation on soybean soilborne pathogens were evaluated in a long-term no-till crop rotation field trial in South Dakota. Five rotation treatments were evaluated in this field trial, including corn-soybean (CS; Zea mays L., Glycine max (L.) Merr.), corn-soybean-spring wheat-pea (CSSwP; wheat: Triticum aestivum L., pea: Pisum sativum subsp. arvense (L.) Asch.), corn-soybean-spring wheat-sunflower (CSSwSf; sunflower: Helianthus annus L.), corn-oat-winter wheat-soybean (COWwS; oat: Avena sativa L.), and corn-pea-winter wheat-soybean (CPWwS). Six soilborne pathogens that consistently threaten the United States soybean production were quantified in the field soils using quantitative PCR or egg extraction for soybean cyst nematode. Three soilborne pathogens, Macrophomina phaseolina, Fusarium oxysporum, and Pythium irregulare, were detected, whereas the population density of F. graminearum and F. virguliforme was not measurable in the soil samples. The number of soybean cyst nematode eggs or juveniles was zero or very low in all soil samples. Overall, crop rotation treatments affected the population density of three detected pathogens but varied by crop phase, year, and pathogen species. The population density of three detected pathogens was positively correlated with soil temperature but negatively correlated with soil volumetric water content. Notably, the CPWwS rotation treatment had a consistently lower M. phaseolina population compared with the other rotation treatments, regardless of crop phase and year. This study provided potential crop sequences that limit soilborne pathogen populations in the soil and may reduce disease incidence on the host crops.

Herbicide glyphosate efficiently inhibits growth of pathogenic Prototheca algae species, suggesting the presence of novel pathways for the development of anti-algal drugs.

Prototheca are ubiquitous algae and occasional pathogens of humans and animals. While rare, the infection is often fatal and treatment options are limited to antifungals with low efficiency. Here, using growth curve assays, we demonstrate that five pathogenic species of Prototheca (P. blaschkeae, P. wickerhamii, P. cutis, P. ciferrii, P. bovis) were fully inhibited by 50-100 μg/mL of herbicide glyphosate, suggesting novel pathways that can be considered for anti-algal drug development.IMPORTANCEPrototheca are algae frequently found in the environment that occasionally cause infections in humans and animals. Although these infections are rare, they are often deadly for immunocompromised patients. Considering the rising ambient temperatures that promote algal bloom and a growing number of immunocompromised patients globally, such cases are likely to increase and will require efficient medications. Currently, the treatment is limited to antifungals that affect algal and animal membranes alike at concentrations close to toxic. Here, we hypothesized that targeting a pathway that is present in plants but not animals may be a new approach to the development of novel anti-algal compounds with high efficiency and lower toxicity. In this proof-of-principle study, we found that herbicide glyphosate, which targets the shikimate pathway found in plants but not in animals, efficiently inhibits all five tested pathogenic Prototheca, suggesting that the shikimate pathway may be a promising target for anti-algal drug development.

Trichosporon asahii: emerging challenges in pathogenesis and drug resistance.

Trichosporon asahii (T. asahii) is an opportunistic pathogenic fungus that often causes severe infections in immunosuppressed patients. Among Trichosporon species, T. asahii is the most pathogenic and lethal species. Current research faces challenges related to unknown pathogenic mechanisms, complex resistance mechanisms, insufficiently rapid and accurate diagnostic methods, and insufficient research on susceptibility to infection. These issues need to be explored in depth. This review summarizes research progress on the origin and classification of T. asahii, its virulence factors and pathogenic mechanisms, epidemiological characteristics, infection modes, diagnostic methods, drug treatment options, and drug resistance mechanisms. Traditional culture combined with molecular biology techniques, such as polymerase chain reaction and gene sequencing, has improved the accuracy and speed of detection. Treatment relies mainly on azole antifungal drugs and amphotericin B; however, patients are facing the problem of drug resistance. New techniques, such as gene knockout and gene sequencing, have identified resistance mechanisms, thus supporting the development of novel antifungal drugs. In summary, an in-depth study of T. asahii will aid in developing more effective diagnostic and therapeutic methods and improve patient prognosis.

Codon usage analysis in selected virulence genes of Staphylococcal species.

The Staphylococcus genus, composed of Gram-positive bacteria, includes several pathogenic species such as Staphylococcus aureus, S. epidermidis, S. haemolyticus, and S. saprophyticus, each implicated in a range of infections. This study investigates the codon usage patterns in key virulence genes, including Autolysin (alt), Elastin Binding protein (EbpS), Lipase, Thermonuclease, Intercellular Adhesion Protein (IcaR), and V8 Protease, across four Staphylococcus species. Using metrics such as the Effective Number of Codons (ENc), Relative Synonymous Codon Usage (RSCU), Codon Adaptation Index (CAI), alongside neutrality and parity plots, we explored the codon preferences and nucleotide composition biases. Our findings revealed a pronounced AT-rich codon preference, with AT-rich genomes likely aiding in energy-efficient translation and bacterial survival in host environments. These insights provide a deeper understanding of the evolutionary adaptations and translational efficiency mechanisms that contribute to the pathogenicity of Staphylococcus species. This knowledge could pave the way for novel therapeutic interventions targeting codon usage to disrupt virulence gene expression.

Global prevalence of Eimeria species in goats: a systematic review and meta-analysis

BackgroundCoccidiosis is a protozoal disease caused by Eimeria species, the main symptom of which is diarrhea. Eimeria spp. infection can cause weight loss and ill-thrift in goats, and in severe cases, it can lead to mortality in kids, resulting in economic losses for the goat industry. This study aimed to determine the global prevalence of Eimeria spp. in goats and to identify the possible predictors of heterogeneity among selected studies.MethodsData were retrieved from five databases of major global importance (PubMed, Web of Science, CAB Direct, Scopus, and Google Scholar), with 255 studies published between 1963 and 2022 being included. A random-effects model was used to calculate pooled prevalence estimates with 95% confidence intervals (CI), followed by subgroup meta-analysis and meta-regression analysis to identify factors contributing to high prevalence and explore sources of heterogeneity among studies.ResultsThe estimated global prevalence of Eimeria spp. in goats was 62.9% (95% CI: 58.6–67.2). Our results indicated high inter-study variability (inconsistency index (I2) = 99.7%, p &amp;lt; 0.01). Among the variables analyzed, regions and quality of studies were the most significant predictors of heterogeneity. According to the region-based subgroup meta-analysis, North America had the highest estimated prevalence of Eimeria spp. (92.2, 95% CI: 82.7–98.2), followed by Europe (86.6, 95% CI: 79.8–92.3), while Asia had the lowest prevalence (52.0, 95% CI: 45.9–58.1). Most countries (n = 42/56) had an estimated prevalence above the overall pooled estimate (&amp;gt;62.9%). The subgroup of studies conducted in 2000 or later presented a lower prevalence of 59.6% (95% CI: 54.7–64.3). Studies with a score of 5–7 had a significantly higher prevalence (72.4, 95% CI: 66.2–78.2) than studies with low or medium scores (p &amp;lt; 0.01). The prevalence of Eimeria spp. in goats detected with conventional and molecular methods was 67.3% (95% CI: 47.0–84.7). Only 47% (119/255) of the studies provided details on identifying Eimeria at the species level. Overall, more than 26 Eimeria spp. have been identified in goats globally. Among these, the most frequently reported and pathogenic species were E. arloingi (115/119), E. ninakohlyakimovae (108/119), E. christenseni (94/119), and E. caprina (71/119). Other valid species that were reported less frequently include E. alijevi, E. hirci, E. caprovina, E. aspheronica and E. jolchijevi.ConclusionThese findings suggest that the pathogenic Eimeria spp. are widespread in goats globally. Given the high prevalence and the extensive distribution of pathogenic Eimeria spp. in goats, it is recommended that integrated parasite management approaches be implemented for the effective control of coccidiosis in goats.

Molecular detection and typing of pathogenic Leptospira species from livestock and small mammals in Uganda.

Molecular detection and typing of pathogenic Leptospira species from livestock and small mammals in Uganda.

Distribution of plant pathogenic Aphanomyces species in Sweden, Denmark and Lithuania and its relationship with soil factors

Abstract Three species of Aphanomyces, A. euteiches, A. cochlioides and A. cladogamus are devastating agricultural plant pathogens causing root rot disease on pea, sugar beet and spinach, respectively. Here we investigated the distribution of the three Aphanomyces species in parts of Sweden, Denmark and Lithuania using climate chamber soil bioassays, and tested for correlations with macronutrient levels in the soil. Aphanomyces euteiches was the most widespread pathogen found in 28% of the tested samples in Sweden and Denmark, while A. cochlioides had a more limited distribution, and was found in 22% of the samples. Aphanomyces cladogamus was only detected in Sweden and was present in 16% of the tested samples. Aphanomyces euteiches only infected pea plants, while A. cochlioides and A. cladogamus infected both sugar beet and spinach. There were significant (P &lt; 0.001) differences in disease severity index (DSI) between regions, pathogens and for the interaction between regions and crops. Soil calcium (Ca) content ranged from 82 to 3600 mg/100 g dry soil. No species of Aphanomyces was detected in soils with Ca levels above 250 mg/100 g soil, indicating a pathogen-suppressive effect from calcium. Similar results were observed from 1350 soil samples collected from commercial pea growing fields, as DSI values on pea remained very low (below 20 on a 0–100 scale) in soil samples with confirmed presence of A. euteiches and a Ca value above 210 mg/100 g soil. High Ca levels was a significant (P &lt; 0.05) predictor for low probability of presence of Aphanomyces spp. in soil.

Controlling Oral Polymicrobial Biofilm Using Usnic Acid on the Surface of Titanium in the Artificial Saliva Media

Background/Objectives: Titanium dental implants, while highly successful, face challenges due to polymicrobial infections leading to peri-implantitis and implant failure. Biofilm formation on implant surfaces is the primary cause of these infections, with factors such as matrix production and cross-kingdom interactions contributing to the microbial accumulation of bacterial and fungal pathogens species. To combat this issue, naturally derived molecules have been reported to overcome the hurdle of antimicrobial resistance against the application of conventional antibiotics and antifungals. Methods: The present study aimed to employ the lichen-derived molecules, usnic acid (UA), to retard the development of biofilms of bacterial and fungal pathogens on the surface of titanium kept in the human artificial saliva (HAS) working as a growth-supporting, host-mimicking media. Results: The minimum inhibitory concentration of UA in HAS towards Candida albicans was &gt;512 µg/mL, whereas against Staphylococcus aureus and Streptococcus mutans, it was determined to be 512 µg/mL. Whereas, in the standard growth media, the MIC value of UA towards S. mutans and S. aureus were 8 and 16 µg/mL; however, against C. albicans, it was 512 µg/mL. UA synergistically enhanced the efficacy of the antibiotics toward bacterial pathogens and the efficacy of antifungals against C. albicans. The antibiofilm results depict the fact that in the HAS, UA significantly reduced both mono-species of S. mutans, S. aureus, and C. albicans and mixed-species biofilm of C. albicans with S. mutans and S. aureus on the surface of the titanium. Conclusions: The present study showed that UA is a promising natural drug that can control oral polymicrobial disease as a result of the application of dental implants.

Cdr1 in focus: a personal reflection on multidrug transporter research.

Drug resistance mechanisms in human pathogenic Candida species are constantly evolving. Over time, these species have developed diverse strategies to counter the effects of various drug classes, making them a significant threat to human health. In addition to well-known mechanisms such as drug target modification, overexpression, and chromosome duplication, Candida species have also developed permeability barriers to antifungal drugs through reduced drug import or increased efflux. The genomes of Candida species contain a multitude of drug resistance genes, many of which encode membrane efflux transporters that actively expel drugs, preventing their toxic accumulation inside the cells and contributing to multidrug resistance. This brief personal retrospective piece for the "Thematic Issue on Celebrating 30 Years of Cdr1 Research: new trends in antifungal therapy and drug resistance" looks back as to how antifungal research has shifted focus since the identification of the first multidrug transporter gene, CDR1 (Candida Drug Resistance 1), leading to new insights into how reduced azole permeability across Candida cell membranes influences antifungal susceptibility.