Methodological and Reporting Quality of Noninferiority Randomized Controlled Trials Comparing Antiretroviral Therapies: A Systematic Review.

BACKGROUND: It is unclear whether the reporting quality of antiretroviral (ARV) noninferiority (NI) randomized controlled trials (RCTs) has improved since the CONSORT guideline release in 2006. The primary objective of this systematic review was assessing the methodological and reporting quality of ARV NI-RCTs. We also assessed reporting quality by funding source and publication year. METHODS: We searched Medline, Embase, and Cochrane Central from inception to 14 November 2022. We included NI-RCTs comparing ≥2 ARV regimens used for human immunodeficiency virus treatment or prophylaxis. We used the Cochrane Risk of Bias 2.0 tool to assess risk of bias. Screening and data extraction were performed blinded and in duplicate. Descriptive statistics were used to summarize data; statistical tests were 2 sided, with significance defined as P < .05. The systematic review was prospectively registered (PROSPERO CRD42022328586), and not funded. RESULTS: We included 160 articles reporting 171 trials. Of these articles, 101 (63.1%) did not justify the NI margin used, and 28 (17.5%) did not provide sufficient information for sample size calculation. Eighty-nine of 160 (55.6%) reported both intention-to-treat and per-protocol analyses, while 118 (73.8%) described missing data handling. Ten of 171 trials (5.9%) reported potentially misleading results. Pharmaceutical industry-funded trials were more likely to be double-blinded (28.1% vs 10.3%; P = .03) and to describe missing data handling (78.5% vs 59.0%; P = .02). The overall risk of bias was low in 96 of 160 studies (60.0%). CONCLUSIONS: ARV NI-RCTs should improve NI margin justification, reporting of intention-to-treat and per-protocol analyses, and missing data handling to increase CONSORT adherence.

Establishment of a Novel Rat Model of Gram-Negative Periprosthetic Joint Infection Using Cementless Hip Hemiarthroplasty.

BACKGROUND: Gram-negative periprosthetic joint infections (GN-PJIs) present unique challenges. Our aim was to establish a clinically representative GN-PJI model that recapitulates biofilm formation in vivo. We also hypothesized that biofilm formation on the implant surface would affect its ability to osseointegrate. METHODS: Three-dimensionally-printed medical-grade titanium hip implants were used to replace the femoral heads of male Sprague-Dawley rats. GN-PJI was induced using 2 bioluminescent Pseudomonas aeruginosa strains: a reference strain (PA14-lux) and a mutant biofilm-defective strain (ΔflgK-lux). Infection was monitored in real time using an in vivo imaging system (IVIS) and magnetic resonance imaging (MRI). Bacterial loads were quantified utilizing the viable colony count. Biofilm formation at the bone-implant interface was visualized using field-emission scanning electron microscopy (FE-SEM). Implant stability, as an outcome, was directly assessed by quantifying osseointegration using microcomputed tomography, and indirectly assessed by identifying gait-pattern changes. RESULTS: Bioluminescence detected by the IVIS was focused on the hip region and demonstrated localized infection, with greater ability of PA14-lux to persist in the model compared with the ΔflgK-lux strain, which is defective in biofilm formation. This was corroborated by MRI, as PA14-lux induced relatively larger implant-related abscesses. Biofilm formation at the bone-implant interface induced by PA14-lux was visualized using FE-SEM versus defective-biofilm formation by ΔflgK-lux. Quantitatively, the average viable colony count of the sonicated implants, in colony-forming units/mL, was 3.77 × 108 for PA14-lux versus 3.65 × 103 for ΔflgK-lux, with a 95% confidence interval around the difference of 1.45 × 108 to 6.08 × 108 (p = 0.0025). This difference in the ability to persist in the model was reflected significantly on implant osseointegration, with a mean intersection surface of 4.1 × 106 ± 1.99 × 106 µm2 for PA14-lux versus 6.44 × 106 ± 2.53 × 106 µm2 for ΔflgK-lux and 7.08 × 106 ± 1.55 × 106 µm2 for the noninfected control (p = 0.048). CONCLUSIONS: To our knowledge, this proposed, novel in vivo biofilm-based model is the most clinically representative for GN-PJI to date, since animals can bear weight on the implant, poor osseointegration was associated with biofilm formation, and localized PJI was assessed by various modalities. CLINICAL RELEVANCE: This model will allow for more reliable testing of novel biofilm-targeting therapeutics.

Clr-f expression regulates kidney immune and metabolic homeostasis.

The C-type lectin-related protein, Clr-f, encoded by Clec2h in the mouse NK gene complex (NKC), is a member of a family of immune regulatory lectins that guide immune responses at distinct tissues of the body. Clr-f is highly expressed in the kidney; however, its activity in this organ is unknown. To assess the requirement for Clr-f in kidney health and function, we generated a Clr-f-deficient mouse (Clr-f-/-) by targeted deletions in the Clec2h gene. Mice lacking Clr-f exhibited glomerular and tubular lesions, immunoglobulin and C3 complement protein renal deposits, and significant abdominal and ectopic lipid accumulation. Whole kidney transcriptional profile analysis of Clr-f-/- mice at 7, 13, and 24 weeks of age revealed a dynamic dysregulation in lipid metabolic processes, stress responses, and inflammatory mediators. Examination of the immune contribution to the pathologies of Clr-f-/- mouse kidneys identified elevated IL-12 and IFNγ in cells of the tubulointerstitium, and an infiltrating population of neutrophils and T and B lymphocytes. The presence of these insults in a Rag1-/-Clr-f-/- background reveals that Clr-f-/- mice are susceptible to a T and B lymphocyte-independent renal pathogenesis. Our data reveal a role for Clr-f in the maintenance of kidney immune and metabolic homeostasis.

Potentiation of Aminoglycoside Lethality by C4-Dicarboxylates Requires RpoN in Antibiotic-Tolerant Pseudomonas aeruginosa.

Antibiotic tolerance contributes to the inability of standard antimicrobial therapies to clear the chronic Pseudomonas aeruginosa lung infections that often afflict patients with cystic fibrosis (CF). Metabolic potentiation of bactericidal antibiotics with carbon sources has emerged as a promising strategy to resensitize tolerant bacteria to antibiotic killing. Fumarate (FUM), a C4-dicarboxylate, has been recently shown to resensitize tolerant P. aeruginosa to killing by tobramycin (TOB), an aminoglycoside antibiotic, when used in combination (TOB+FUM). Fumarate and other C4-dicarboxylates are taken up intracellularly by transporters regulated by the alternative sigma factor RpoN. Once in the cell, FUM is metabolized, leading to enhanced electron transport chain activity, regeneration of the proton motive force, and increased TOB uptake. In this work, we demonstrate that a ΔrpoN mutant displays impaired FUM uptake and, consequently, nonsusceptibility to TOB+FUM treatment. RpoN was also found to be essential for susceptibility to other aminoglycoside and C4-dicarboxylate combinations. Importantly, RpoN loss-of-function mutations have been documented to evolve in the CF lung, and these loss-of-function alleles can also result in TOB+FUM nonsusceptibility. In a mixed-genotype population of wild-type and ΔrpoN cells, TOB+FUM specifically killed cells with RpoN function and spared the cells that lacked RpoN function. Unlike C4-dicarboylates, both d-glucose and l-arginine were able to potentiate TOB killing of ΔrpoN stationary-phase cells. Our findings raise the question of whether TOB+FUM will be a suitable treatment option in the future for CF patients infected with P. aeruginosa isolates that lack RpoN function.

Pseudomonas aeruginosa Biofilm Antibiotic Resistance Gene ndvB Expression Requires the RpoS Stationary-Phase Sigma Factor.

Chronic, biofilm-based bacterial infections are exceptionally difficult to eradicate due to the high degree of antibiotic recalcitrance exhibited by cells in biofilm communities. In the opportunistic pathogen Pseudomonas aeruginosa, biofilm recalcitrance is multifactorial and arises in part from the preferential expression of resistance genes in biofilms compared to exponential-phase planktonic cells. One such mechanism involves ndvB, which we have previously shown to be expressed specifically in biofilms. In this study, we investigated the regulatory basis of this lifestyle-specific expression by developing an unstable green fluorescent protein (GFP) transcriptional reporter to observe the expression pattern of ndvB We found that in addition to its expression in biofilms, ndvB was upregulated in planktonic cells as they enter stationary phase. The transcription of ndvB in both growth phases was shown to be dependent on the stationary-phase sigma factor RpoS, and mutation of a putative RpoS binding site in the ndvB promoter abolished the activity of the promoter in stationary-phase cells. Overall, we have expanded our understanding of the temporal expression of ndvB in P. aeruginosa and have uncovered a regulatory basis for its growth phase-dependent expression.IMPORTANCE Bacterial biofilms are more resistant to antibiotics than free-living planktonic cells, and understanding the mechanistic basis of this resistance can inform treatments of biofilm-based infections. In addition to chemical and structural barriers that can inhibit antibiotic entry, the upregulation of specific genes in biofilms contributes to the resistance. We investigated this biofilm-specific gene induction by examining expression patterns of ndvB, a gene involved in biofilm resistance of the opportunistic pathogen Pseudomonas aeruginosa We characterized ndvB expression in planktonic and biofilm growth conditions with an unstable green fluorescent protein (GFP) reporter and found that the expression of ndvB in biofilms is dependent on the stationary-phase sigma factor RpoS. Overall, our results support the physiological similarity between biofilms and stationary-phase cells and suggest that the induction of some stationary-phase genes in biofilms may contribute to their increased antibiotic resistance.

Targeting the alternative sigma factor RpoN to combat virulence in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a Gram-negative, opportunistic pathogen that infects immunocompromised and cystic fibrosis patients. Treatment is difficult due to antibiotic resistance, and new antimicrobials are needed to treat infections. The alternative sigma factor 54 (σ54, RpoN), regulates many virulence-associated genes. Thus, we evaluated inhibition of virulence in P. aeruginosa by a designed peptide (RpoN molecular roadblock, RpoN*) which binds specifically to RpoN consensus promoters. We expected that RpoN* binding to its consensus promoter sites would repress gene expression and thus virulence by blocking RpoN and/or other transcription factors. RpoN* reduced transcription of approximately 700 genes as determined by microarray analysis, including genes related to virulence. RpoN* expression significantly reduced motility, protease secretion, pyocyanin and pyoverdine production, rhamnolipid production, and biofilm formation. Given the effectiveness of RpoN* in vitro, we explored its effects in a Caenorhabditis elegans-P. aeruginosa infection model. Expression of RpoN* protected C. elegans in a paralytic killing assay, whereas worms succumbed to paralysis and death in its absence. In a slow killing assay, which mimics establishment and proliferation of an infection, C. elegans survival was prolonged when RpoN* was expressed. Thus, blocking RpoN consensus promoter sites is an effective strategy for abrogation of P. aeruginosa virulence.

PA3225 Is a Transcriptional Repressor of Antibiotic Resistance Mechanisms in Pseudomonas aeruginosa.

The tssABC1 locus is part of the Hcp secretion island I (HSI-I) type VI secretion system (T6SS) in Pseudomonas aeruginosa Previous work implicated the tssC1 gene in P. aeruginosa biofilm-specific antibiotic resistance, and tssC1 is preferentially expressed in biofilms compared to planktonic cells. Using a DNA-dependent protein pulldown approach, we discovered that PA3225, an uncharacterized LysR-type transcriptional regulator, specifically bound to the tssABC1 upstream regulatory region. The deletion of PA3225 led to a 2-fold decrease in tssA1 expression levels in planktonic cells compared to the wild type, and tssA1 expression was slightly reduced in ΔPA3225 biofilms compared to wild-type biofilms. Intriguingly, further investigations revealed that the ΔPA3225 mutant was less susceptible to multiple, structurally unrelated antibiotics with various mechanisms of action when grown planktonically. The ΔPA3225 mutant was additionally more resistant to ciprofloxacin when grown in a biofilm. The decreased antibiotic susceptibility of the ΔPA3225 strain was linked to the transcriptional upregulation of the MexAB-OprM efflux pump. By using transcriptome sequencing (RNA-seq), other PA3225-regulated genes were identified, and the products of these genes, such as the putative ABC transporter PA3228, may also contribute to antibiotic resistance.

Molecular mechanisms of biofilm-based antibiotic resistance and tolerance in pathogenic bacteria.

Biofilms are surface-attached groups of microbial cells encased in an extracellular matrix that are significantly less susceptible to antimicrobial agents than non-adherent, planktonic cells. Biofilm-based infections are, as a result, extremely difficult to cure. A wide range of molecular mechanisms contribute to the high degree of recalcitrance that is characteristic of biofilm communities. These mechanisms include, among others, interaction of antimicrobials with biofilm matrix components, reduced growth rates and the various actions of specific genetic determinants of antibiotic resistance and tolerance. Alone, each of these mechanisms only partially accounts for the increased antimicrobial recalcitrance observed in biofilms. Acting in concert, however, these defences help to ensure the survival of biofilm cells in the face of even the most aggressive antimicrobial treatment regimens. This review summarises both historical and recent scientific data in support of the known biofilm resistance and tolerance mechanisms. Additionally, suggestions for future work in the field are provided.

Optimized tetramer analysis reveals Ly49 promiscuity for MHC ligands.

Murine Ly49 receptors, which are expressed mainly on NK and NKT cells, interact with MHC class I (MHC-I) molecules with varying specificity. Differing reports of Ly49/MHC binding affinities may be affected by multiple factors, including cis versus trans competition and species origin of the MHC-I L chain (ß2-microglobulin). To determine the contribution of each of these factors, Ly49G, Ly49I, Ly49O, Ly49V, and Ly49Q receptors from the 129 mouse strain were expressed individually on human 293T cells or the mouse cell lines MHC-I-deficient C1498, H-2(b)-expressing MC57G, and H-2(k)-expressing L929. The capacity to bind to H-2D(b)- and H-2K(b)-soluble MHC-I tetramers containing either human or murine ß2-microglobulin L chains was tested for all five Ly49 receptors in all four cell lines. We found that most of these five inhibitory Ly49 receptors show binding for one or both self-MHC-I molecules in soluble tetramer binding assays when three conditions are fulfilled: 1) lack of competing cis interactions, 2) tetramer L chain is of mouse origin, and 3) Ly49 is expressed in mouse and not human cell lines. Furthermore, Ly49Q, the single known MHC-I receptor on plasmacytoid dendritic cells, was shown to bind H-2D(b) in addition to H-2K(b) when the above conditions were met, suggesting that Ly49Q functions as a pan-MHC-Ia receptor on plasmacytoid dendritic cells. In this study, we have optimized the parameters for soluble tetramer binding analyses to enhance future Ly49 ligand identification and to better evaluate specific contributions by different Ly49/MHC-I pairs to NK cell education and function.