Health System Change for Alzheimer's Disease-Modifying Therapies in Canada: Beginning the Discussion.

Alzheimer's disease (AD) is a neurodegenerative disorder that accounts for 60%-70% of patients with dementia, and it is estimated that over one million Canadians will be living with dementia by 2030. Disease-modifying therapies (DMTs) targeting the underlying pathophysiology of AD are currently in development. Several models have demonstrated that the potential arrival of Alzheimer's DMTs will most likely overwhelm the already-constrained Canadian healthcare system. Canada does not have a strategy to address the extensive requirements of using DMTs, including providing an early diagnosis of AD, confirming DMT eligibility via amyloid biomarkers, and conducting ongoing treatment monitoring. Thus, a multidisciplinary group of experts involved in AD care in Canada gathered to review (1) the current barriers to diagnosis and management of AD; (2) how existing clinic models, including those used in multiple sclerosis (MS), could be applied to address key barriers in AD; and (3) how to design and implement optimal care pathways in the future. The actions outlined in this review will help clinicians and healthcare systems improve readiness to integrate the use of disease-modifying therapies in Alzheimer's disease, if such therapies are approved in Canada.

A Model Predicting Healthcare Capacity Gaps For Alzheimer's Disease-Modifying Treatment in Canada.

BACKGROUND: Alzheimer's disease (AD) is experienced by > 600,000 Canadians. Disease-modifying therapies (DMTs) for earlier stages of disease are in development. Existing health system capacity constraints and the need for biomarker-driven diagnostics to confirm DMT eligibility are concerning. This study aimed to characterize the capacity gap related to early AD (eAD) treatment with DMTs in Canada. METHODS: A capacity model was developed to simulate the flow of a patient from screening to treatment for eAD to quantify the gap between available and required healthcare resources and qualify the bottlenecks restricting the patient journey at a provincial and national level. The model inputs (epidemiological, human resource, and clinical) were evidence-based, healthcare professional-, and patient advocate-informed. RESULTS: The model estimated that nationally < 2% of patients would have access to the required healthcare resources for treatment with a DMT. Eligibility assessment represented the step with the largest capacity gap across all provinces, with a wait list of about 382,000 Canadians one year following DMT introduction. The top three resource gaps included AD specialist time and positron emission tomography and magnetic resonance imaging exam slots. Sensitivity analysis showed that full reliance on cerebrospinal fluid for eligibility testing increased capacity for assessment by about 47,000 patients. CONCLUSION: This model highlights that the Canadian health system is critically under-resourced to diagnose, assess, and treat patients with eAD with DMT. It underscores an urgent need for national policy and provincial resource allocation to close the gap.

Determining the potential use of biosurfactants in preventing endodontic infections.

Microbial biofilms play a dominant role in the failure of endodontic therapies. Bacterial adhesion is the first step in the establishment of biofilms, activating the host immune response leading to tissue damage. Biosurfactants are microbe-derived tensioactive molecules with latent anti-adhesive and anti-microbial activity. This study reports the extraction and characterization of a biosurfactant from Lactobacillus (L.) plantarum (Lp-BS) and investigates its anti-microbial and anti-adhesive properties compared to rhamnolipid, a commercially available biosurfactant. Lp-BS, extracted from L. plantarum during the growth phase, was characterized as a glycoprotein, able to reduce surface tension and emulsify non-polar liquids. Proteomic analysis of Lp-BS identified three bacterial adhesin-like proteins, suggesting roles in hindering bacterial adhesion. Lp-BS did not show significant anti-microbial activity against endodontic pathogens from the Streptococcus (Strep.) anginosus group or Enterococcus (Ent.) faecalis at 50 mg/ml. However, anti-adhesive activity on abiotic surfaces was observed against both Strep. anginosus and Strep. intermedius. Rhamnolipid exhibited strong anti-microbial activity, with minimum inhibitory concentrations of 0.097 mg/ml against Strep. anginosus, and 0.048 mg/ml against Strep. constellatus and Strep. intermedius, in addition to a marked anti-adhesive activity. These findings offer preliminary evidence for the potential application of biosurfactants as an anti-microbial and/or anti-adhesive pharmacotherapy in endodontics.

Immunoglobulin D enhances immune surveillance by activating antimicrobial, proinflammatory and B cell-stimulating programs in basophils.

Immunoglobulin D (IgD) is an enigmatic antibody isotype that mature B cells express together with IgM through alternative RNA splicing. Here we report active T cell-dependent and T cell-independent IgM-to-IgD class switching in B cells of the human upper respiratory mucosa. This process required activation-induced cytidine deaminase (AID) and generated local and circulating IgD-producing plasmablasts reactive to respiratory bacteria. Circulating IgD bound to basophils through a calcium-mobilizing receptor that induced antimicrobial, opsonizing, inflammatory and B cell-stimulating factors, including cathelicidin, interleukin 1 (IL-1), IL-4 and B cell-activating factor (BAFF), after IgD crosslinking. By showing dysregulation of IgD class-switched B cells and 'IgD-armed' basophils in autoinflammatory syndromes with periodic fever, our data indicate that IgD orchestrates an ancestral surveillance system at the interface between immunity and inflammation.

A Leukocyte Immune-Type Receptor Subset Is a Marker of Antiviral Cytotoxic Cells in Channel Catfish, Ictalurus punctatus.

Channel catfish, Ictalurus punctatus, leukocyte immune type receptors (LITRs) represent a multigene family that encodes Ig superfamily proteins that mediate activating or inhibitory signaling. In this study, we demonstrate the use of mAb CC41 to monitor viral cytotoxic responses in catfish and determine that CC41 binds to a subset of LITRs on the surface of catfish clonal CTLs. Homozygous gynogenetic catfish were immunized with channel catfish virus (CCV)-infected MHC-matched clonal T cells (G14D-CCV), and PBL were collected at various times after immunization for flow cytometric analyses. The percentage of CC41(+) cells was significantly increased 5 d after primary immunization with G14D-CCV and at 3 d after a booster immunization as compared with control fish only injected with G14D. Moreover, CC41(+) cells magnetically isolated from the PBL specifically killed CCV-infected targets as measured by (51)Cr release assays and expressed messages for CD3γδ, perforin, and at least one of the CD4-like receptors as analyzed by RNA flow cytometry. When MLC effector cells derived from a G14D-CCV-immunized fish were preincubated with CC41 mAb, killing of G14D-CCV targets was reduced by ∼40%, suggesting that at least some LITRs have a role in target cell recognition and/or cytotoxicity. The availability of a LITR-specific mAb has allowed, to our knowledge for the first time, functional characterization of LITRs in an autologous system. In addition, the identification of an LITR subset as a cytotoxic cell marker will allow for more effective monitoring of catfish immune responses to pathogens.

Modulation of Candida albicans virulence by bacterial biofilms on titanium surfaces.

Whilst Candida albicans occurs in peri-implant biofilms, its role in peri-implantitis remains unclear. This study therefore examined the virulence of C. albicans in mixed-species biofilms on titanium surfaces. Biofilms of C. albicans (Ca), C. albicans with streptococci (Streptococcus sanguinis, S. mutans) (Ca-Ss-Sm) and those incorporating Porphyromonas gingivalis (Ca-Pg and Ca-Ss-Sm-Pg) were developed. Expression of C. albicans genes associated with adhesion (ALS1, ALS3, HWP1) and hydrolytic enzymes (SAP2, SAP4, SAP6, PLD1) was measured and hyphal production by C. albicans quantified. Compared with Ca biofilms, significant (p<0.05) up-regulation of ALS3, HWP1, SAP2 and SAP6, and hyphal production occurred in biofilms containing streptococci (Ca-Ss-Sm). In Ca-Pg biofilms, down-regulation of HWP1 and SAP4 expression, with reduced hyphal production occurred. Ca-Ss-Sm-Pg biofilms had increased hyphal proportions and up-regulation of ALS3, SAP2 and SAP6. In conclusion, C. albicans expressed virulence factors in biofilms that could contribute to peri-implantitis, but this was dependent on associated bacterial species.

Development of the Perinatal Depression Inventory (PDI)-14 using item response theory: a comparison of the BDI-II, EPDS, PDI, and PHQ-9.

The objective of this study is to develop a simple, brief, self-report perinatal depression inventory that accurately measures severity in a number of populations. Our team developed 159 Likert-scale perinatal depression items using simple sentences with a fifth-grade reading level. Based on iterative cognitive interviewing (CI), an expert panel improved and winnowed the item pool based on pre-determined criteria. The resulting 67 items were administered to a sample of 628 pregnant and 251 postpartum women with different levels of depression at private and public sector obstetrics clinics, together with the Beck Depression Inventory (BDI-II), Edinburg Postpartum Depression Scale (EPDS), and the Patient Health Questionnaire (PHQ-9), as well as Module A of the Structured Clinical Interview for DSM-IV Diagnoses (SCID). Responses were evaluated using Item Response Theory (IRT). The Perinatal Depression Inventory (PDI)-14 items are highly informative regarding depression severity and function similarly and informatively across pregnant/postpartum, white/non-white, and private-clinic/public-clinic populations. PDI-14 scores correlate well with the PHQ-9, EPDS, and BDI-II, but the PDI-14 provides a more precise measure of severity using far fewer words. The PDI-14 is a brief depression assessment that excels at accurately measuring depression severity across a wide range of severity and perinatal populations.

Spectrum of mutations in a cohort of UK patients with ADA deficient SCID: Segregation of genotypes with specific ethnicities.

Severe combined immunodeficiency (SCID) arises from a number of different genetic defects, one of the most common being mutations in the gene encoding adenosine deaminase (ADA). In the UK, ADA deficient SCID compromises approximately 20% of all known cases of SCID. We carried out a retrospective analysis of the ADA gene in 46 known ADA deficient SCID patients on whom DNA had been stored. Here, we report a high frequency of two previously reported mutations and provide a link between the mutations and patient ethnicity within our patient cohort. We also report on 9 novel mutations that have been previously unreported.

Virulence and pathogenicity of Candida albicans is enhanced in biofilms containing oral bacteria.

This study examined the influence of bacteria on the virulence and pathogenicity of candidal biofilms. Mature biofilms (Candida albicans-only, bacteria-only, C. albicans with bacteria) were generated on acrylic and either analysed directly, or used to infect a reconstituted human oral epithelium (RHOE). Analyses included Candida hyphae enumeration and assessment of Candida virulence gene expression. Lactate dehydrogenase (LDH) activity and Candida tissue invasion following biofilm infection of the RHOE were also measured. Candida hyphae were more prevalent (p < 0.05) in acrylic biofilms also containing bacteria, with genes encoding secreted aspartyl-proteinases (SAP4/SAP6) and hyphal-wall protein (HWP1) up-regulated (p < 0.05). Candida adhesin genes (ALS3/EPA1), SAP6 and HWP1 were up-regulated in mixed-species biofilm infections of RHOE. Multi-species infections exhibited higher hyphal proportions (p < 0.05), up-regulation of IL-18, higher LDH activity and tissue invasion. As the presence of bacteria in acrylic biofilms promoted Candida virulence, consideration should be given to the bacterial component when managing denture biofilm associated candidoses.

Screening of neonatal UK dried blood spots using a duplex TREC screening assay.

PURPOSE: Severe Combined Immunodeficiency (SCID) is considered to be a paediatric emergency and unless identified promptly can be life-threatening. Frequently, infants are not diagnosed with SCID until they have become seriously ill with infection leading to treatment complications and a poorer prognosis. We aimed to test a newly available commercial duplex assay to measure T cell receptor excision circles (TRECs) to establish if this would be suitable for newborn screening for SCID in the UK. METHODS: Over 5000 anonymous retrospective dried blood spots (DBS) were used alongside 18 confirmed SCID positive DBS with a newly available duplex assay to measure TRECs levels and control gene levels. We also included testing of premature babies and babies from neonatal intensive care units (NICU) as these have been shown to have high false positive rates in other TREC screening assays. RESULTS: All 18 SCID DBS samples were successfully identified as SCID positives in the study. The number of presumptive positives detected was dependent on the TREC cut-off threshold settings. When analysed with five different TRECs cut-off values (20, 25, 30, 35 and 40 TREC copies/µl blood) the presumptive positive rate ranged from 0.04 to 1.00 % of samples tested. Premature infants and neonates from NICU did not show high presumed false positive rates in this assay. CONCLUSIONS: The study demonstrated that this duplex assay kit will identify all newborns with SCID as presumptive positives. The data also shows that with suitable TREC cut-off settings the number of presumptive positives from non-SCID newborns will be manageable in the context of a national screening service.

Identification and characterization of TCRγ and TCRδ chains in channel catfish, Ictalurus punctatus.

Channel catfish, Ictalurus punctatus, T cell receptors (TCR) γ and δ were identified by mining of expressed sequence tag databases, and full-length sequences were obtained by 5'-RACE and RT-PCR protocols. cDNAs for each of these TCR chains encode typical variable (V), diversity (D), joining (J), and constant (C) regions. Three TCRγ V families, seven TCRγ J sequences, and three TCRγ C sequences were identified from sequencing of cDNA. Primer walking on bacterial artificial chromosomes (BACs) confirmed that the TRG locus contained seven TRGJ segments and indicated that the locus consists of (Vγ3-Jγ6-Cγ2)-(Vγ1n-Jγ7-Cγ3)-(Vγ2-Jγ5-Jγ4-Jγ3-Jγ2-Jγ1-Cγ1). In comparison for TCRδ, two V families, four TCRδ D sequences, one TCRδ J sequence, and one TCRδ C sequence were identified by cDNA sequencing. Importantly, the finding that some catfish TCRδ cDNAs contain TCR Vα-D-Jδ rearrangements and some TCRα cDNAs contain Vδ-Jα rearrangements strongly implies that the catfish TRA and TRD loci are linked. Finally, primer walking on BACs and Southern blotting suggest that catfish have four TRDD gene segments and a single TRDJ and TRDC gene. As in most vertebrates, all three reading frames of each of the catfish TRDD segments can be used in functional rearrangements, and more than one TRDD segment can be used in a single rearrangement. As expected, catfish TCRδ CDR3 regions are longer and more diverse than TCRγ CDR3 regions, and as a group they utilize more nucleotide additions and contain more nucleotide deletions than catfish TCRγ rearrangements.

Novel Nystatin A1 derivatives exhibiting low host cell toxicity and antifungal activity in an in vitro model of oral candidosis.

Opportunistic oral infections caused by Candida albicans are frequent problems in immunocompromised patients. Management of such infections is limited due to the low number of antifungal drugs available, their relatively high toxicity and the emergence of antifungal resistance. Given these issues, our investigations have focused on novel derivatives of the antifungal antibiotic Nystatin A1, generated by modifications at the amino group of this molecule. The aims of this study were to evaluate the antifungal effectiveness and host cell toxicity of these new compounds using an in vitro model of oral candidosis based on a reconstituted human oral epithelium (RHOE). Initial studies employing broth microdilution, revealed that against planktonic C. albicans, Nystatin A1 had lower minimal inhibitory concentration than novel derivatives. However, Nystatin A1 was also markedly more toxic against human keratinocyte cells. Interestingly, using live/dead staining to assess C. albicans and tissue cell viability after RHOE infection, Nystatin A1 derivatives were more active against Candida with lower toxicity to epithelial cells than the parent drug. Lactate dehydrogenase activity released by the RHOE indicated a fourfold reduction in tissue damage when certain Nystatin derivatives were used compared with Nystatin A1. Furthermore, compared with Nystatin A1, colonisation of the oral epithelium by C. albicans was notably reduced by the new polyenes. In the absence of antifungal agents, confocal laser scanning microscopy showed that C. albicans extensively invaded the RHOE. However, the presence of the novel derivatives greatly reduced or totally prevented this fungal invasion.

TLR7 in channel catfish (Ictalurus punctatus) is expressed in the endolysosome and is stimulated by synthetic ssRNA analogs, imiquimod, and resiquimod.

Toll-like receptors (TLRs) are pivotal pattern recognition receptors (PRRs) and key mediators of innate immunity. Despite the significance of channel catfish (Ictalurus punctatus) in comparative immunology and aquaculture, its 20 TLR genes remain largely functionally uncharacterized. In this study, our aim was to determine the catfish TLR7 agonists, signaling potential, and cellular localization. Using a mammalian reporter system, we identified imiquimod and resiquimod, typical ssRNA analogs, as potent catfish TLR7 agonists. Notably, unlike grass carp TLR7, catfish TLR7 lacks the ability to respond to poly (I:C). Confocal microscopy revealed predominant catfish TLR7 expression in lysosomes, co-localizing with the endosomal chaperone protein, UNC93B1. Furthermore, imiquimod stimulation elicited robust IFNb transcription in peripheral blood leukocytes isolated from adult catfish. These findings underscore the conservation of TLR7 signaling in catfish, reminiscent of mammalian TLR7 responses. Our study sheds light on the functional aspects of catfish TLR7 and contributes to a better understanding of its role in immune defense mechanisms.

Exploring synergistic and antagonistic interactions in phage-antibiotic combinations against ESKAPE pathogens.

In the era of antimicrobial resistance, phage-antibiotic combinations offer a promising therapeutic option, yet research on their synergy and antagonism is limited. This study aims to assess these interactions, focusing on protein synthesis inhibitors and cell envelope-active agents against multidrug-resistant bacterial strains. We evaluated synergistic and antagonistic interactions in multidrug-resistant Staphylococcus aureus, Enterococcus faecium, and Pseudomonas aeruginosa strains. Phages were combined with protein synthesis inhibitors [linezolid (LZD), minocycline (MIN), gentamicin (GEN), and azithromycin (AZM)] or cell envelope-active agents [daptomycin (DAP), ceftaroline (CPT), and cefepime (FEP)]. Modified checkerboard minimum inhibitory concentration assays and 24-h time-kill analyses were conducted, alongside one-step growth curves to analyze phage growth kinetics. Statistical comparisons used one-way analysis of variance (ANOVA) and the Tukey test (P < 0.05). In the checkerboard and 24-h time-kill analyses (TKA) of S. aureus and E. faecium, phage-LZD and phage-MIN combinations were antagonistic (FIC > 4) while phage-DAP and phage-CPT were synergistic (FIC 0.5) (ANOVA range of mean differences 0.52-2.59 log10 CFU/mL; P < 0.001). For P. aeruginosa, phage-AZM was antagonistic (FIC > 4), phage-GEN was additive (FIC = 1), and phage-FEP was synergistic (ANOVA range of mean differences 1.04-1.95 log10 CFU/mL; P < 0.001). Phage growth kinetics were altered in the presence of LZD and MIN against S. aureus and in the presence of LZD against a single E. faecium strain (HOU503). Our findings indicate that select protein synthesis inhibitors may induce phage-antibiotic antagonism. However, this antagonism may not solely stem from changes in phage growth kinetics, warranting further investigation into the complex interplay among strains, phage attributes, and antibiotic mechanisms affecting bacterial inhibition.IMPORTANCEIn the face of escalating antimicrobial resistance, combining phages with antibiotics offers a promising avenue for treating infections unresponsive to traditional antibiotics. However, while studies have explored synergistic interactions, less attention has been given to potential antagonism and its impact on phage growth kinetics. This research evaluates the interplay between phages and antibiotics, revealing both synergistic and antagonistic patterns across various bacterial strains and shedding light on the complex dynamics that influence treatment efficacy. Understanding these interactions is crucial for optimizing combination therapies and advancing phage therapy as a viable solution for combating antimicrobial resistance.

The Use of a Synthetic Hybrid-Scale Fiber Matrix to Treat Difficult-to-Heal Wounds.

INTRODUCTION: Complex and chronic wounds are often difficult to treat, and current advanced therapies have their limitations. A synthetic hybrid-scale fiber matrix could be a viable option in treating these wounds, as previous clinical studies utilizing the matrix have shown positive results in treating chronic ulcers and surgical wounds. METHODS: Patients with difficult-to-treat wounds of varying etiologies were treated with a synthetic hybrid-scale fiber matrix (Restrata®, Acera Surgical, Inc., St. Louis, Missouri). The wound bed was debrided and prepared, and the synthetic matrix was prepared and applied to the wound. Wounds were monitored for healing progress. Additional applications of the synthetic matrix were used based on clinician discretion. RESULTS: Six patients with wounds of varying etiologies were assessed, including a 30-year recalcitrant wound. All wounds achieved significant healing, with four of the six wounds (67%) achieving complete closure in an average of 57.8 ± 27.0 days (8.3 ± 3.9 weeks). CONCLUSIONS: The study found that the synthetic matrix was effective, resulting in improved healing across various etiologies, including cancer resection and amputation. The clinical results presented here suggest that the synthetic hybrid-scale fiber matrix may be an optimal alternative in treating difficult-to-heal wounds.

A subset of leukocyte immune-type receptors (LITRs) regulates phagocytosis in channel catfish (Ictalurus punctatus) leukocytes.

Channel catfish, Ictalurus punctatus, leukocyte immune-type receptors (LITRs) constitute a large family of paired, immunoregulatory receptors unique to teleosts. A role for LITRs in phagocytosis has been proposed based on studies in mammalian cell lines; however, LITR-mediated phagocytosis has not been examined in the catfish model. In this study, we use two anti-LITR monoclonal antibodies, CC41 and 125.2, to contrast the effects of crosslinking subsets of inhibitory and activating LITRs. Briefly, LITRs expressed by catfish γδ T cells, αß T cells, and macrophage cell lines were crosslinked using mAb-conjugated fluorescent microbeads, and bead uptake was evaluated by flow cytometry and confirmed by confocal microscopy. A clear difference in the uptake of 125.2- and CC41-conjugated beads was observed. Crosslinking LITRs with mAb 125.2 resulted in efficient bead internalization, while mAb CC41 crosslinking of inhibitory LITRs resulted predominantly in a capturing phenotype. Pretreating catfish macrophages with mAb CC41 resulted in a marked decrease in LITR-mediated phagocytosis of 125.2-conjugated beads. Overall, these findings provide insight into fish immunobiology and validate LITRs as regulators of phagocytosis in catfish macrophages and γδ T cells.

Compositional shifts within the denture-associated bacteriome in pneumonia - an analytical cross-sectional study.

Introduction. Bacterial pneumonia is a common cause of morbidity and mortality in elderly individuals. While the incidence of edentulism is falling, approximately 19 % of the UK population wear a full or partial removable denture. Despite advances in denture biomaterials, the majority of dentures are fabricated using polymethyl-methacrylate. Growing evidence suggests that colonization of the oral cavity by putative respiratory pathogens predisposes individuals to respiratory infection, by translocation of these microorganisms along the respiratory tract.Hypothesis/Gap Statement. We hypothesized that denture surfaces provide a susceptible colonization site for putative respiratory pathogens, and thus could increase pneumonia risk in susceptible individuals.Aim. This study aimed to characterize the bacterial community composition of denture-wearers in respiratory health compared with individuals with a confirmed diagnosis of pneumonia.Methodology. This was an analytical cross-sectional study, comparing frail elderly individuals without respiratory infection (n=35) to hospitalized patients with pneumonia (n=26). The primary outcome was the relative abundance of putative respiratory pathogens identified by 16S rRNA metataxonomic sequencing, with quantitative PCR used to identified Streptococcus pneumoniae.Results. There was a statistically significant increase in the overall relative abundance of putative respiratory pathogens (P<0.0001), with a greater than 20-fold increase in the bioburden of these microorganisms. In keeping with these findings, there were significant shifts in bacterial community diversity (Chao index, P=0.0003) and richness (Inverse Simpson index P<0.0001) in the denture-associated microbiota of pneumonia patients compared with control subjects.Conclusion. Within the limitations of this study, our evidence supports the role of denture acrylic biomaterials as a potential colonization site for putative respiratory pathogens, which may lead to an increased risk of pneumonia in susceptible individuals. These findings support prior observational studies which have found denture-wearers to be at increased risk of respiratory infection. Further research is needed to confirm the sequence of colonization and translocation to examine potential causal relationships.

Phage-Antibiotic Cocktail Rescues Daptomycin and Phage Susceptibility against Daptomycin-Nonsusceptible Enterococcus faecium in a Simulated Endocardial Vegetation Ex Vivo Model.

Enterococcus faecium is a difficult-to-treat pathogen with emerging resistance to most clinically available antibiotics. Daptomycin (DAP) is the standard of care, but even high DAP doses (12 mg/kg body weight/day) failed to eradicate some vancomycin-resistant strains. Combination DAP-ceftaroline (CPT) may increase ß-lactam affinity for target penicillin binding proteins (PBP); however, in a simulated endocardial vegetation (SEV) pharmacokinetic/pharmacodynamic (PK/PD) model, DAP-CPT did not achieve therapeutic efficacy against a DAP-nonsusceptible (DNS) vancomycin-resistant E. faecium (VRE) isolate. Phage-antibiotic combinations (PAC) have been proposed for resistant high-inoculum infections. We aimed to identify PAC with maximum bactericidal activity and prevention/reversal of phage and antibiotic resistance in an SEV PK/PD model against DNS isolate R497. Phage-antibiotic synergy (PAS) was evaluated with modified checkerboard MIC and 24-h time-kill analyses (TKA). Human-simulated antibiotic doses of DAP and CPT with phages NV-497 and NV-503-01 were then evaluated in 96-h SEV PK/PD models against R497. Synergistic and bactericidal activity was identified with the PAC of DAP-CPT combined with phage cocktail NV-497-NV-503-01, demonstrating a significant reduction in viability down to 3-log10 CFU/g (-Δ, 5.77-log10 CFU/g; P < 0.001). This combination also demonstrated isolate resensitization to DAP. Evaluation of phage resistance post-SEV demonstrated prevention of phage resistance for PACs containing DAP-CPT. Our results provide novel data highlighting bactericidal and synergistic activity of PAC against a DNS E. faecium isolate in a high-inoculum ex vivo SEV PK/PD model with subsequent DAP resensitization and prevention of phage resistance. IMPORTANCE Our study supports the additional benefit of standard-of-care antibiotics combined with a phage cocktail compared to antibiotic alone against a daptomycin-nonsusceptible (DNS) E. faecium isolate in a high-inoculum simulated endocardial vegetation ex vivo PK/PD model. E. faecium is a leading cause of hospital-acquired infections and is associated with significant morbidity and mortality. Daptomycin is considered the first-line therapy for vancomycin-resistant E. faecium (VRE), but the highest published doses have failed to eradicate some VRE isolates. The addition of a ß-lactam to daptomycin may result in synergistic activity, but previous in vitro data demonstrate that daptomycin plus ceftaroline failed to eradicate a VRE isolate. Phage therapy as an adjunct to antibiotic therapy has been proposed as a salvage therapy for high-inoculum infections; however, pragmatic clinical comparison trials for endocarditis are lacking and difficult to design, reinforcing the timeliness of such analysis.

Treatment of methicillin-resistant Staphylococcus aureus infections with a minimal inhibitory concentration of 2 µg/mL to vancomycin: old (trimethoprim/sulfamethoxazole) versus new (daptomycin or linezolid) agents.

BACKGROUND: Guidelines recommend that agents other than vancomycin be considered for some types of infection due to methicillin-resistant Staphylococcus aureus (MRSA) when the minimum inhibitory concentration (MIC) to vancomycin is 2 µg/mL or more. Alternative therapeutic options include daptomycin and linezolid, 2 relatively new and expensive drugs, and trimethoprim/sulfamethoxazole (TMP/SMX), an old and inexpensive agent. OBJECTIVE: To compare the clinical efficacy and potential cost savings associated with use of TMP/SMX compared to linezolid and daptomycin. METHODS: A retrospective study was conducted at Detroit Medical Center. For calendar year 2009, unique adults (age >18 years) with infections due to MRSA with an MIC to vancomycin of 2 µg/mL were included if they received 2 or more doses of TMP/SMX and/or daptomycin and/or linezolid. Data were abstracted from patient charts and pharmacy records. RESULTS: There were 328 patients included in the study cohort: 143 received TMP/SMX alone, 89 received daptomycin alone, 75 received linezolid alone, and 21 patients received a combination of 2 or more of these agents. In univariate analysis, patients who received TMP/SMX alone had significantly better outcomes, including in-hospital (p = 0.003) and 90-day mortality (p < 0.001) compared to patients treated with daptomycin or linezolid. Patients receiving TMP/SMX were also younger (p < 0.001), had fewer comorbid conditions (p < 0.001), had less severe acute severity of illness (p < 0.001), and received appropriate therapy more rapidly (p = 0.001). In multivariate models the association between TMP/SMX treatment and mortality was no longer significant. Antimicrobial cost savings associated with using TMP/SMX averaged $2067.40 per patient. CONCLUSIONS: TMP/SMX monotherapy compared favorably to linezolid and daptomycin in terms of treatment efficacy and mortality. Use of TMP/SMX instead of linezolid or daptomycin could potentially significantly reduce antibiotic costs. TMP/SMX should be considered for the treatment of MRSA infection with MIC of 2 µg/mL to vancomycin.

Identification of two IgD+ B cell populations in channel catfish, Ictalurus punctatus.

Channel catfish Ictalurus punctatus express two Ig isotypes: IgM and IgD. Although catfish IgM has been extensively studied at the functional and structural levels, much less is known about IgD. In this study, IgM(+)/IgD(+) and IgM(-)/IgD(+) catfish B cell populations were identified through the use of anti-IgM and anti-IgD mAbs. Catfish IgM(+)/IgD(+) B cells are small and agranular. In contrast, IgM(-)/IgD(+) B cells are larger and exhibit a plasmablast morphology. The use of cell sorting, flow cytometry, and RT-PCR demonstrated that IgD(+) B cell expression varies among individuals. For example, some catfish have <5% IgM(-)/IgD(+) B cells in their PBLs, whereas in others the IgM(-)/IgD(+) B cell population can represent as much as 72%. Furthermore, IgD expressed by IgM(-)/IgD(+) B cells preferentially associates with IgL σ. Comparatively, IgM(+)/IgD(+) B cells can express any of the four catfish IgL isotypes. Also, transfection studies show that IgD functions as a typical BCR, because Igδ-chains associate with CD79a and CD79b molecules, and all membrane IgD transcripts from sorted IgM(-)/IgD(+) B cells contain viable VDJ rearrangements, with no bias in family member usage. Interestingly, all secreted IgD transcripts from IgM(+)/IgD(+) and IgM(-)/IgD(+) B cells were V-less and began with a leader spliced to Cδ1. Importantly, transfection of catfish clonal B cells demonstrated that this leader mediated IgD secretion. Together, these findings imply that catfish IgM(-)/IgD(+) B cells likely expand in response to certain pathogens and that the catfish IgD Fc-region, as has been suggested for human IgD, may function as a pattern recognition molecule.