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Little is known about shifts in the fecal microbiome of dairy calves preceding and following the incidence of gastrointestinal disease. The objective of this cohort study was to describe the fecal microbiome of preweaned dairy calves before, during, and after gastrointestinal disease. A total of 111 Holstein dairy calves were enrolled on 2 dairies (D1 and D2) and followed until 5 weeks old. Health assessments were performed weekly and fecal samples were collected every other week. Of the 111 calves, 12 calves from D1 and 12 calves from D2 were retrospectively defined as healthy, and 7 calves from D1 and 11 calves from D2 were defined as diarrheic. Samples from these calves were sequenced targeting the 16S rRNA gene and compared based on health status within age groups and farms: healthy (0-1 week old) vs. pre-diarrheic (0-1 week old), healthy (2-3 weeks old) vs. diarrheic (2-3 weeks old), and healthy (4-5 weeks old) vs. post-diarrheic (4-5 weeks old) calves. Healthy and diarrheic samples clustered together based on age rather than health status on both farms. Based on linear discriminant analysis, a few species were identified to be differently enriched when comparing health status within age groups and farm. Among them, Bifidobacterium sp. was differently enriched in pre-diarrheic calves at D1 (0-1 week old) whereas healthy calves of the same age group and farm showed a higher abundance of Escherichia coli. Bifidobacterium sp. was identified as a biomarker of fecal samples from healthy calves (2-3 weeks old) on D1 when compared with diarrheic calves of the same age group and farm. Feces from diarrheic calves on D2 (2-3 weeks old) were characterized by taxa from Peptostreptococcus and Anaerovibrio genera whereas fecal samples of age-matched healthy calves were characterized by Collinsella aerofaciens and Bifidobacterium longum. After resolution of uncomplicated diarrhea (4-5 weeks old), Collinsella aerofaciens was more abundant in D2 calves whereas Bacteriodes uniformis was more abundant in D1 calves. Taken together, these findings suggest that the age of the preweaned calf is the major driver of changes to fecal microbiome composition and diversity even in the face of uncomplicated gastrointestinal disease.

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Background: Clostridioides difficile Infection (CDI) is a healthcare-associated diarrheal disease prevalent worldwide. A common diagnostic algorithm relies on a two-step protocol that employs stool enzyme immunoassays (EIAs) to detect the pathogen, and its toxins, respectively. Active CDI is deemed less likely when the Toxin EIA result is negative, even if the pathogen-specific EIA is positive for C. difficile. We recently reported, however, that low-toxin-producing C. difficile strains recovered from Toxin-negative ('discrepant') clinical stool specimens can be fully pathogenic, and cause lethality in a rodent CDI model. To document frequency of discrepant CDI specimens, and evaluate C. difficile strain diversity, we performed longitudinal surveillance at a Southern Arizona tertiary-care hospital. Methods: Diarrheic stool specimens from patients with clinical suspicion of CDI were obtained over an eight-year period (2015-2022) from all inpatient and outpatient Units of a > 600-bed Medical Center in Southern Arizona. Clinical laboratory EIA testing identified C. difficile-containing specimens, and classified them as Toxin-positive or Toxin-negative. C. difficile isolates recovered from the stool specimens were DNA fingerprinted using an international phylogenetic lineage assignment system ("ribotyping"). For select isolates, toxin abundance in stationary phase supernatants of pure cultures was quantified via EIA. Results: Of 8,910 diarrheic specimens that underwent diagnostic testing, 1733 (19.4%) harbored C. difficile. Our major findings were that: (1) C. difficile prevalence and phylogenetic diversity was stable over the 8-year period; (2) toxigenic C. difficile was recovered from 69% of clinically Tox-neg ('discrepant') specimens; (3) the six most prevalent USA ribotypes were recovered in significant proportions (>60%) from Tox-neg specimens; and (4) toxin-producing C. difficile recovered from discrepant specimens produced less toxin than strains of the same ribotype isolated from non-discrepant specimens. Conclusion: Our study highlights the dominance of Toxin EIA-negative CDI specimens in a clinical setting and the high frequency of known virulent ribotypes in these specimens. Therefore, a careful reevaluation of the clinical relevance of diagnostically-discrepant specimens particularly in the context of missed CDI diagnoses and C. difficile persistence, is warranted.

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Bovine respiratory disease (BRD) is a leading cause of calf morbidity and mortality, and prevalence remains high despite current management practices. Differential gene expression (DGE) provides detailed insight into individual immune responses and can illuminate enriched pathways and biomarkers that contribute to disease susceptibility and outcomes. The aims of this study were to investigate differences in peripheral leukocyte gene expression in Holstein preweaned heifer calves 1) with and without BRD, and 2) across weeks of age. Calves were enrolled for this short-term longitudinal study on two commercial dairies in Washington State. Calves were assessed every two weeks throughout the pre-weaning period using clinical respiratory scoring (CRS) and thoracic ultrasonography (TUS), and blood samples were collected. Calves were selected that were either healthy (n = 10) or had BRD diagnosed by CRS (n = 7), TUS (n = 6), or both (n = 6) in weeks 5 or 7 of life). Three consecutive time point samples were analyzed for each BRD calf consisting of PRE, ONSET, and POST samples. Nineteen genes of interest were selected based on previous gene expression studies in cattle: ALOX15, BPI, CATHL6, CXCL8, DHX58, GZMB, HPGD, IFNG, IL17D, IL1R2, ISG15, LCN2, LIF, MX1, OAS2, PGLYRP1, S100A8, SELP, and TNF. Comparisons were made between age and disease time point matched BRD and healthy calves as well as between calf weeks of age. No DGE was observed between diseased and healthy calves; however, DGE was observed between calf weeks of age regardless of disease state. Developmental differences in leukocyte gene expression, phenotype, and functionality make pre-weaned calves immunologically distinct from mature cattle, and early life shifts in calf leukocyte populations likely contribute to the age-related gene expression differences we observed. Age overshadows disease impacts to influence gene expression in young calves, and immune development progresses upon a common trajectory regardless of disease during the preweaning period.

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Clostridioides difficile infection (CDI) is a major healthcare-associated diarrheal disease. Consistent with trends across the United States, C. difficile RT106 was the second-most prevalent molecular type in our surveillance in Arizona from 2015 to 2018. A representative RT106 strain displayed robust virulence and 100% lethality in the hamster model of acute CDI. We identified a unique 46 KB genomic island (GI1) in all RT106 strains sequenced to date, including those in public databases. GI1 was not found in its entirety in any other C. difficile clade, or indeed, in any other microbial genome; however, smaller segments were detected in Enterococcus faecium strains. Molecular clock analyses suggested that GI1 was horizontally acquired and sequentially assembled over time. GI1 encodes homologs of VanZ and a SrtB-anchored collagen-binding adhesin, and correspondingly, all tested RT106 strains had increased teicoplanin resistance, and a majority displayed collagen-dependent biofilm formation. Two additional genomic islands (GI2 and GI3) were also present in a subset of RT106 strains. All three islands are predicted to encode mobile genetic elements as well as virulence factors. Emergent phenotypes associated with these genetic islands may have contributed to the relatively rapid expansion of RT106 in US healthcare and community settings.

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Morbidity and mortality attributed to Clostridium difficile infection (CDI) have increased over the past 20 years. Currently, antibiotics are the only US FDA-approved treatment for primary C. difficile infection, and these are, ironically, associated with disease relapse and the threat of burgeoning drug resistance. We previously showed that non-toxin virulence factors play key roles in CDI, and that colonization factors are critical for disease. Specifically, a C. difficile adhesin, Surface Layer Protein A (SlpA) is a major contributor to host cell attachment. In this work, we engineered Syn-LAB 2.0 and Syn-LAB 2.1, two synthetic biologic agents derived from lactic acid bacteria, to stably and constitutively express a host-cell binding fragment of the C. difficile adhesin SlpA on their cell-surface. Both agents harbor conditional suicide plasmids expressing a codon-optimized chimera of the lactic acid bacterium's cell-wall anchoring surface-protein domain, fused to the conserved, highly adherent, host-cell-binding domain of C. difficile SlpA. Both agents also incorporate engineered biocontrol, obviating the need for any antibiotic selection. Syn-LAB 2.0 and Syn-LAB 2.1 possess positive biophysical and in vivo properties compared with their parental antecedents in that they robustly and constitutively display the SlpA chimera on their cell surface, potentiate human intestinal epithelial barrier function in vitro, are safe, tolerable and palatable to Golden Syrian hamsters and neonatal piglets at high daily doses, and are detectable in animal feces within 24 h of dosing, confirming robust colonization. In combination, the engineered strains also delay (in fixed doses) or prevent (when continuously administered) death of infected hamsters upon challenge with high doses of virulent C. difficile. Finally, fixed-dose Syn-LAB ameliorates diarrhea in a non-lethal model of neonatal piglet enteritis. Taken together, our findings suggest that the two synthetic biologics may be effectively employed as non-antibiotic interventions for CDI.