Clostridioides difficile superoxide reductase mitigates oxygen sensitivity.

Clostridioides difficile causes a serious diarrheal disease and is a common healthcare-associated bacterial pathogen. Although it has a major impact on human health, the mechanistic details of C. difficile intestinal colonization remain undefined. C. difficile is highly sensitive to oxygen and requires anaerobic conditions for in vitro growth. However, the mammalian gut is not devoid of oxygen, and C. difficile tolerates moderate oxidative stress in vivo. The C. difficile genome encodes several antioxidant proteins, including a predicted superoxide reductase (SOR) that is upregulated upon exposure to antimicrobial peptides. The goal of this study was to establish SOR enzymatic activity and assess its role in protecting C. difficile against oxygen exposure. Insertional inactivation of sor rendered C. difficile more sensitive to superoxide, indicating that SOR contributes to antioxidant defense. Heterologous C. difficile sor expression in Escherichia coli conferred protection against superoxide-dependent growth inhibition, and the corresponding cell lysates showed superoxide scavenging activity. Finally, a C. difficile SOR mutant exhibited global proteome changes under oxygen stress when compared to the parent strain. Collectively, our data establish the enzymatic activity of C. difficile SOR, confirm its role in protection against oxidative stress, and demonstrate SOR's broader impacts on the C. difficile vegetative cell proteome.IMPORTANCEClostridioides difficile is an important pathogen strongly associated with healthcare settings and capable of causing severe diarrheal disease. While considered a strict anaerobe in vitro, C. difficile has been shown to tolerate low levels of oxygen in the mammalian host. Among other well-characterized antioxidant proteins, the C. difficile genome encodes a predicted superoxide reductase (SOR), an understudied component of antioxidant defense in pathogens. The significance of the research reported herein is the characterization of SOR's enzymatic activity, including confirmation of its role in protecting C. difficile against oxidative stress. This furthers our understanding of C. difficile pathogenesis and presents a potential new avenue for targeted therapies.

Evaluating teachers' pedagogical content knowledge in implementing classroom-based assessment: A case study among esl secondary school teachers in Selangor, Malaysia.

The Malaysian Education Blueprint (PPPM) 2013-2025 has spurred significant reforms in the Primary School Standard Curriculum (KSSR) and Secondary School Standard Curriculum (KSSM), particularly concerning classroom-based assessment (CBA). CBA evaluates students' understanding and progress, informs instruction, and enhances the learning outcomes. Teachers with robust pedagogical content knowledge (PCK) are better equipped to design and implement effective CBA strategies that accurately assess students' comprehension and growth, provide personalised feedback, and guide instruction. This study aims to investigate the relationship between PCK and CBA among English as a Second Language (ESL) secondary school teachers in Selangor, Malaysia. A 5-point Likert-scale questionnaire was administered to 338 teachers across 27 regional secondary schools in Selangor. The Covariance-based structural equation modelling (SEM) was used to analyse the data. The findings revealed that the secondary school teachers demonstrated a high level of PCK, with content knowledge (CK) obtaining the highest mean, followed by pedagogical knowledge (PK) and pedagogical content knowledge (PCK). The CBA practices among these teachers were also found to be high. SEM analysis showed a positive association between PK and CBA practices and between PCK and CBA. However, no positive association was observed between CK and CBA practices. In order to enhance teachers' PCK and ensure the effective implementation of CBA, which is crucial for student learning outcomes in Malaysian ESL secondary schools, it is recommended that continuous professional development opportunities be provided, specifically focusing on PCK and CBA.

Prevalence of diagnostically-discrepant Clostridioides difficile clinical specimens: insights from longitudinal surveillance.

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.

Low-toxin Clostridioides difficile RT027 strains exhibit robust virulence.

Clostridioides difficile is a leading cause of healthcare-associated infections worldwide. Currently, there is a lack of consensus for an optimal diagnostic method for C. difficile infection (CDI). Multi-step diagnostic algorithms use enzyme immunosorbent analysis (EIA)-based detection of C. difficile toxins TcdA/TcdB in stool, premised on the rationale that EIA toxin-negative (Tox-) patients have less severe disease and shorter diarrhoea duration. The aim of this study was to characterize toxigenic (i.e. tcdA/tcdB-positive) C. difficile strains isolated from diarrheic patient stool with an EIA Tox- (i.e. "discrepant") CDI diagnostic test result. Recovered strains were DNA fingerprinted (ribotyped), subjected to multiple toxin, genome and proteome evaluations, and assessed for virulence. Overall, of 1243 C. difficile-positive patient stool specimens from Southern Arizona hospitals, 31% were discrepant. For RT027 (the most prevalent ribotype)-containing specimens, 34% were discrepant; the corresponding RT027 isolates were cytotoxic to cultured fibroblasts, but their total toxin levels were comparable to, or lower than, the historic low-toxin-producing C. difficile strain CD630. Nevertheless, these low-toxin RT027 strains (LT-027) exhibited similar lethality to a clade-matched high-toxin RT027 strain in Golden Syrian hamsters, and heightened colonization and persistence in mice. Genomics and proteomics analyses of LT-027 strains identified unique genes and altered protein abundances, respectively, relative to high-toxin RT027 strains. Collectively, our data highlight the robust virulence of LT-027 C. difficile, provide a strong argument for reconsidering the clinical significance of a Tox- EIA result, and underscore the potential limitations of current diagnostic protocols.

Surface-displayed glycopolymers of Clostridioides difficile.

Clostridioides difficile is a leading cause of the healthcare-associated disease C. difficile infection (CDI), which has an annual US burden of over 200 000 cases. CDI mitigation strategies have been complicated by the emergence, and widespread distribution, of phylogenetically diverse lineages, as well as pathogen recalcitrance to genetic manipulation. In this review, we highlight past and current efforts to elucidate C. difficile surface glycopolymer biology since these molecules are essential for colonization, disease, and immunity elicitation, and may therefore have potential as CDI anti-infective targets.

Phylogenomic analysis of Clostridioides difficile ribotype 106 strains reveals novel genetic islands and emergent phenotypes.

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.

Anti-virulence strategies for Clostridioides difficile infection: advances and roadblocks.

Clostridioides difficile infection (CDI) is a common healthcare- and antibiotic-associated diarrheal disease. If mis-diagnosed, or incompletely treated, CDI can have serious, indeed fatal, consequences. The clinical and economic burden imposed by CDI is great, and the US Centers for Disease Control and Prevention has named the causative agent, C. difficile (CD), as an Urgent Threat To US healthcare. CDI is also a significant problem in the agriculture industry. Currently, there are no FDA-approved preventives for this disease, and the only approved treatments for both human and veterinary CDI involve antibiotic use, which, ironically, is associated with disease relapse and the threat of burgeoning antibiotic resistance. Research efforts in multiple laboratories have demonstrated that non-toxin factors also play key roles in CDI, and that these are critical for disease. Specifically, key CD adhesins, as well as other surface-displayed factors have been shown to be major contributors to host cell attachment, and as such, represent attractive targets for anti-CD interventions. However, research on anti-virulence approaches has been more limited, primarily due to the lack of genetic tools, and an as-yet nascent (but increasingly growing) appreciation of immunological impacts on CDI. The focus of this review is the conceptualization and development of specific anti-virulence strategies to combat CDI. Multiple laboratories are focused on this effort, and the field is now at an exciting stage with numerous products in development. Herein, however, we focus only on select technologies (Figure 1) that have advanced near, or beyond, pre-clinical testing (not those that are currently in clinical trial), and discuss roadblocks associated with their development and implementation.

MerTK negatively regulates Staphylococcus aureus induced inflammatory response via Toll-like receptor signaling in the mammary gland.

Mastitis is the most commonly diagnosed infectious disease reducing milk yield and quality and is accompanied by mammary tissue damage in both humans and animals. Mastitis incurs welfare and economic costs as well as environmental concerns regarding treatment. Staphylococcus aureus (S. aureus) is a prevalent Gram-positive bacteria and a major cause of mastitis, however, pathogenesis of the intrinsic anti-inflammatory response in mammary tissues is still principally unknown. Our aim, in combatting the S. aureus induced inflammatory response in mammary tissues, was to elucidate the intrinsic anti-inflammatory role of MerTK signaling. Here, we demonstrate that Mer receptor tyrosine kinase (MerTK) regulates an intrinsic negative feedback to balance the over-reaction of the host defense system. S. aureus elicits toll-like receptors 2 and 6 (TLR2/TLR6) signaling pathways, subsequently recruiting TRAF6, whose ubiquitination is intricate to the downstream signaling including MAPKs and NF-κB. We observed that TLR2/TLR6 activation, in response to S. aureus, was concomitant with induced MerTK activation, leading to raised expression of suppressor of cytokine signaling 1 and 3 (SOCS1, SOCS3) in wild type mice mammary tissues and epithelial cells. Meanwhile, S. aureus infection in MerTK-/- mice showed significant increased phosphorylation of p65, IκBα, p38, JNK and ERK along with production of pro-inflammatory cytokines. Moreover, MerTK-/- evidently inhibited S. aureus induced phosphorylation of STAT1 and subsequent SOCS1/SOCS3 expression which are pivotal in the negative feedback mechanism for targeting TRAF6 to inhibit the TLR2/TLR6 mediated immune response. Taken together, our findings demonstrate the importance of MerTK in the regulation of the intrinsic feedback during the inflammatory response induced by S. aureus through STAT1/SOCS1/SOCS3 in mice mammary tissues and mice mammary epithelial cells (MMECs).

An Engineered Synthetic Biologic Protects Against Clostridium difficile Infection.

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.

Surface-layer protein A (SlpA) is a major contributor to host-cell adherence of Clostridium difficile.

Clostridium difficile is a leading cause of antibiotic-associated diarrhea, and a significant etiologic agent of healthcare-associated infections. The mechanisms of attachment and host colonization of C. difficile are not well defined. We hypothesize that non-toxin bacterial factors, especially those facilitating the interaction of C. difficile with the host gut, contribute to the initiation of C. difficile infection. In this work, we optimized a completely anaerobic, quantitative, epithelial-cell adherence assay for vegetative C. difficile cells, determined adherence proficiency under multiple conditions, and investigated C. difficile surface protein variation via immunological and DNA sequencing approaches focused on Surface-Layer Protein A (SlpA). In total, thirty-six epidemic-associated and non-epidemic associated C. difficile clinical isolates were tested in this study, and displayed intra- and inter-clade differences in attachment that were unrelated to toxin production. SlpA was a major contributor to bacterial adherence, and individual subunits of the protein (varying in sequence between strains) mediated host-cell attachment to different extents. Pre-treatment of host cells with crude or purified SlpA subunits, or incubation of vegetative bacteria with anti-SlpA antisera significantly reduced C. difficile attachment. SlpA-mediated adherence-interference correlated with the attachment efficiency of the strain from which the protein was derived, with maximal blockage observed when SlpA was derived from highly adherent strains. In addition, SlpA-containing preparations from a non-toxigenic strain effectively blocked adherence of a phylogenetically distant, epidemic-associated strain, and vice-versa. Taken together, these results suggest that SlpA plays a major role in C. difficile infection, and that it may represent an attractive target for interventions aimed at abrogating gut colonization by this pathogen.