iAMPCN: a deep-learning approach for identifying antimicrobial peptides and their functional activities.

Antimicrobial peptides (AMPs) are short peptides that play crucial roles in diverse biological processes and have various functional activities against target organisms. Due to the abuse of chemical antibiotics and microbial pathogens' increasing resistance to antibiotics, AMPs have the potential to be alternatives to antibiotics. As such, the identification of AMPs has become a widely discussed topic. A variety of computational approaches have been developed to identify AMPs based on machine learning algorithms. However, most of them are not capable of predicting the functional activities of AMPs, and those predictors that can specify activities only focus on a few of them. In this study, we first surveyed 10 predictors that can identify AMPs and their functional activities in terms of the features they employed and the algorithms they utilized. Then, we constructed comprehensive AMP datasets and proposed a new deep learning-based framework, iAMPCN (identification of AMPs based on CNNs), to identify AMPs and their related 22 functional activities. Our experiments demonstrate that iAMPCN significantly improved the prediction performance of AMPs and their corresponding functional activities based on four types of sequence features. Benchmarking experiments on the independent test datasets showed that iAMPCN outperformed a number of state-of-the-art approaches for predicting AMPs and their functional activities. Furthermore, we analyzed the amino acid preferences of different AMP activities and evaluated the model on datasets of varying sequence redundancy thresholds. To facilitate the community-wide identification of AMPs and their corresponding functional types, we have made the source codes of iAMPCN publicly available at https://github.com/joy50706/iAMPCN/tree/master. We anticipate that iAMPCN can be explored as a valuable tool for identifying potential AMPs with specific functional activities for further experimental validation.

Therapeutics for Vancomycin-Resistant Enterococcal Bloodstream Infections.

Vancomycin-resistant enterococci (VRE) are common causes of bloodstream infections (BSIs) with high morbidity and mortality rates. They are pathogens of global concern with a limited treatment pipeline. Significant challenges exist in the management of VRE BSI, including drug dosing, the emergence of resistance, and the optimal treatment for persistent bacteremia and infective endocarditis. Therapeutic drug monitoring (TDM) for antimicrobial therapy is evolving for VRE-active agents; however, there are significant gaps in the literature for predicting antimicrobial efficacy for VRE BSIs. To date, TDM has the greatest evidence for predicting drug toxicity for the three main VRE-active antimicrobial agents daptomycin, linezolid, and teicoplanin. This article presents an overview of the treatment options for VRE BSIs, the role of antimicrobial dose optimization through TDM in supporting clinical infection management, and challenges and perspectives for the future.

Virulence attributes of successful methicillin-resistant Staphylococcus aureus lineages.

Methicillin-resistant Staphylococcus aureus (MRSA) is a leading cause of severe and often fatal infections. MRSA epidemics have occurred in waves, whereby a previously successful lineage has been replaced by a more fit and better adapted lineage. Selection pressures in both hospital and community settings are not uniform across the globe, which has resulted in geographically distinct epidemiology. This review focuses on the mechanisms that trigger the establishment and maintenance of current, dominant MRSA lineages across the globe. While the important role of antibiotic resistance will be mentioned throughout, factors which influence the capacity of S. aureus to colonize and cause disease within a host will be the primary focus of this review. We show that while MRSA possesses a diverse arsenal of toxins including alpha-toxin, the success of a lineage involves more than just producing toxins that damage the host. Success is often attributed to the acquisition or loss of genetic elements involved in colonization and niche adaptation such as the arginine catabolic mobile element, as well as the activity of regulatory systems, and shift metabolism accordingly (e.g., the accessory genome regulator, agr). Understanding exactly how specific MRSA clones cause prolonged epidemics may reveal targets for therapies, whereby both core (e.g., the alpha toxin) and acquired virulence factors (e.g., the Panton-Valentine leukocidin) may be nullified using anti-virulence strategies.

Horizontal Gene Transfer, Fitness Costs and Mobility Shape the Spread of Antibiotic Resistance Genes into Experimental Populations of Acinetobacter Baylyi.

Horizontal gene transfer (HGT) is important for microbial evolution, but how evolutionary forces shape the frequencies of horizontally transferred genetic variants in the absence of strong selection remains an open question. In this study, we evolve laboratory populations of Acinetobacter baylyi (ADP1) with HGT from two clinically relevant strains of multidrug-resistant Acinetobacter baumannii (AB5075 and A9844). We find that DNA can cross the species barrier, even without strong selection, and despite substantial DNA sequence divergence between the two species. Our results confirm previous findings that HGT can drive the spread of antibiotic resistance genes (ARGs) without selection for that antibiotic, but not for all of the resistance genes present in the donor genome. We quantify the costs and benefits of horizontally transferred variants and use whole population sequencing to track the spread of ARGs from HGT donors into antibiotic-sensitive recipients. We find that even though most ARGs are taken up by populations of A. baylyi, the long-term fate of an individual gene depends both on its fitness cost and on the type of genetic element that carries the gene. Interestingly, we also found that an integron, but not its host plasmid, is able to spread in A. baylyi populations despite its strong deleterious effect. Altogether, our results show how HGT provides an evolutionary advantage to evolving populations by facilitating the spread of non-selected genetic variation including costly ARGs.

Polysaccharide-Targeting Lipid Nanoparticles to Kill Gram-Negative Bacteria.

The rapid increase and spread of Gram-negative bacteria resistant to many or all existing treatments threaten a return to the preantibiotic era. The presence of bacterial polysaccharides that impede the penetration of many antimicrobials and protect them from the innate immune system contributes to resistance and pathogenicity. No currently approved antibiotics target the polysaccharide regions of microbes. Here, describe monolaurin-based niosomes, the first lipid nanoparticles that can eliminate bacterial polysaccharides from hypervirulent Klebsiella pneumoniae, are described. Their combination with polymyxin B shows no cytotoxicity in vitro and is highly effective in combating K. pneumoniae infection in vivo. Comprehensive mechanistic studies have revealed that antimicrobial activity proceeds via a multimodal mechanism. Initially, lipid nanoparticles disrupt polysaccharides, then outer and inner membranes are destabilized and destroyed by polymyxin B, resulting in synergistic cell lysis. This novel lipidic nanoparticle system shows tremendous promise as a highly effective antimicrobial treatment targeting multidrug-resistant Gram-negative pathogens.

A Clinically Selected Staphylococcus aureus clpP Mutant Survives Daptomycin Treatment by Reducing Binding of the Antibiotic and Adapting a Rod-Shaped Morphology.

Daptomycin is a last-resort antibiotic used for the treatment of infections caused by Gram-positive antibiotic-resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA). Treatment failure is commonly linked to accumulation of point mutations; however, the contribution of single mutations to resistance and the mechanisms underlying resistance remain incompletely understood. Here, we show that a single nucleotide polymorphism (SNP) selected during daptomycin therapy inactivates the highly conserved ClpP protease and is causing reduced susceptibility of MRSA to daptomycin, vancomycin, and ß-lactam antibiotics as well as decreased expression of virulence factors. Super-resolution microscopy demonstrated that inactivation of ClpP reduced binding of daptomycin to the septal site and diminished membrane damage. In both the parental strain and the clpP strain, daptomycin inhibited the inward progression of septum synthesis, eventually leading to lysis and death of the parental strain while surviving clpP cells were able to continue synthesis of the peripheral cell wall in the presence of 10× MIC daptomycin, resulting in a rod-shaped morphology. To our knowledge, this is the first demonstration that synthesis of the outer cell wall continues in the presence of daptomycin. Collectively, our data provide novel insight into the mechanisms behind bacterial killing and resistance to this important antibiotic. Also, the study emphasizes that treatment with last-line antibiotics is selective for mutations that, like the SNP in clpP, favor antibiotic resistance over virulence gene expression.

The clinical application of beta-lactam antibiotic therapeutic drug monitoring in the critical care setting.

Critically ill patients have increased variability in beta-lactam antibiotic (beta-lactam) exposure due to alterations in their volume of distribution and elimination. Therapeutic drug monitoring (TDM) of beta-lactams, as a dose optimization and individualization tool, has been recommended to overcome this variability in exposure. Despite its potential benefit, only a few centres worldwide perform beta-lactam TDM. An important reason for the low uptake is that the evidence for clinical benefits of beta-lactam TDM is not well established. TDM also requires the availability of specific infrastructure, knowledge and expertise. Observational studies and systematic reviews have demonstrated that TDM leads to an improvement in achieving target concentrations, a reduction in potentially toxic concentrations and improvement of clinical and microbiological outcomes. However, a small number of randomized controlled trials have not shown a mortality benefit. Opportunities for improved study design are apparent, as existing studies are limited by their inclusion of heterogeneous patient populations, including patients that may not even have infection, small sample size, variability in the types of beta-lactams included, infections caused by highly susceptible bacteria, and varied sampling, analytical and dosing algorithm methods. Here we review the fundamentals of beta-lactam TDM in critically ill patients, the existing clinical evidence and the practical aspects involved in beta-lactam TDM implementation.

Oral ciprofloxacin activity against ceftriaxone-resistant Escherichia coli in an in vitro bladder infection model.

OBJECTIVES: Pharmacodynamic profiling of oral ciprofloxacin dosing for urinary tract infections caused by ceftriaxone-resistant Escherichia coli isolates with ciprofloxacin MIC ≥ 0.25 mg/L. BACKGROUND: Urine-specific breakpoints for ciprofloxacin do not exist. However, high urinary concentrations may promote efficacy in isolates with low-level resistance. METHODS: Ceftriaxone-resistant E. coli urinary isolates were screened for ciprofloxacin susceptibility. Fifteen representative strains were selected and tested using a dynamic bladder infection model. Oral ciprofloxacin dosing was simulated over 3 days (250 mg daily, 500 mg daily, 250 mg 12 hourly, 500 mg 12 hourly and 750 mg 12 hourly). The model was run for 96 h. Primary endpoint was change in bacterial density at 72 h. Secondary endpoints were follow-up change in bacterial density at 96 h and area-under-bacterial-kill-curve. Bacterial response was related to exposure (AUC0-24/MIC; Cmax/MIC). PTA was determined using Monte-Carlo simulation. RESULTS: Ninety-three clinical isolates demonstrated a trimodal ciprofloxacin MIC distribution (modal MICs at 0.016, 0.25 and 32 mg/L). Fifteen selected clinical isolates (ciprofloxacin MIC 0.25-512 mg/L) had a broad range of quinolone-resistance genes. Following ciprofloxacin exposure, E. coli ATCC 25922 (MIC 0.008 mg/L) was killed in all dosing experiments. Six isolates (MIC ≥ 16 mg/L) regrew in all experiments. Remaining isolates (MIC 0.25-8 mg/L) regrew variably after an initial period of killing, depending on simulated ciprofloxacin dose. A >95% PTA, using AUC0-24/MIC targets, supported 250 mg 12 hourly for susceptible isolates (MIC ≤ 0.25 mg/L). For isolates with MIC ≤ 1 mg/L, 750 mg 12 hourly promoted 3 log10 kill at the end of treatment (72 h), 1 log10 kill at follow-up (96 h) and 90% maximal activity (AUBKC0-96). CONCLUSIONS: Bladder infection modelling supports oral ciprofloxacin activity against E. coli with low-level resistance (ciprofloxacin MIC ≤ 1 mg/L) when using high dose therapy (750 mg 12 hourly).

Incidence and severity of cytomegalovirus infection in seropositive heart transplant recipients.

BACKGROUND: The frequency and significance of cytomegalovirus (CMV) infection in seropositive (R+) heart transplant recipients (HTR) is unclear, with preventative recommendations mostly extrapolated from other groups. We evaluated the incidence and severity of CMV infection in R+ HTR, to identify risk factors and describe outcomes. METHODS: R+ HTR from 2010 to 2019 were included. Antiviral prophylaxis was not routinely used, with clinically guided monitoring the local standard of care. The primary outcome was CMV infection within one-year post-transplant; secondary outcomes included other herpesvirus infections and mortality. RESULTS: CMV infection occurred in 27/155 (17%) R+ HTR. Patients with CMV had a longer hospitalization (27 vs. 20 days, unadjusted HR 1.02, 95% CI 1.00-1.02, p = .01), higher rate of intensive care readmission (26% vs. 9%, unadjusted HR 3.46, 1.46-8.20, p = .005), and increased mortality (33% vs. 8%, unadjusted HR 10.60, 4.52-24.88, p < .001). The association between CMV and death persisted after adjusting for multiple confounders (HR 24.19, 95% CI 7.47-78.30, p < .001). Valganciclovir prophylaxis was used in 35/155 (23%) and was protective against CMV (infection rate 4% vs. 27%, adjusted HR .07, .01-.72, p = .025), even though those receiving it were more likely to have received thymoglobulin (adjusted OR 10.5, 95% CI 2.01-55.0, p = .005). CONCLUSIONS: CMV infection is common in R+ HTR and is associated with a high burden of disease and increased mortality. Patients who received valganciclovir prophylaxis were less likely to develop CMV infection, despite being at higher risk. These findings support the routine use of antiviral prophylaxis following heart transplantation in all CMV R+ patients.

EHR-QC: A streamlined pipeline for automated electronic health records standardisation and preprocessing to predict clinical outcomes.

The adoption of electronic health records (EHRs) has created opportunities to analyse historical data for predicting clinical outcomes and improving patient care. However, non-standardised data representations and anomalies pose major challenges to the use of EHRs in digital health research. To address these challenges, we have developed EHR-QC, a tool comprising two modules: the data standardisation module and the preprocessing module. The data standardisation module migrates source EHR data to a standard format using advanced concept mapping techniques, surpassing expert curation in benchmarking analysis. The preprocessing module includes several functions designed specifically to handle healthcare data subtleties. We provide automated detection of data anomalies and solutions to handle those anomalies. We believe that the development and adoption of tools like EHR-QC is critical for advancing digital health. Our ultimate goal is to accelerate clinical research by enabling rapid experimentation with data-driven observational research to generate robust, generalisable biomedical knowledge.

Identification of a Class of Protein ADP-Ribosylating Sirtuins in Microbial Pathogens.

Sirtuins are an ancient family of NAD(+)-dependent deacylases connected with the regulation of fundamental cellular processes including metabolic homeostasis and genome integrity. We show the existence of a hitherto unrecognized class of sirtuins, found predominantly in microbial pathogens. In contrast to earlier described classes, these sirtuins exhibit robust protein ADP-ribosylation activity. In our model organisms, Staphylococcus aureus and Streptococcus pyogenes, the activity is dependent on prior lipoylation of the target protein and can be reversed by a sirtuin-associated macrodomain protein. Together, our data describe a sirtuin-dependent reversible protein ADP-ribosylation system and establish a crosstalk between lipoylation and mono-ADP-ribosylation. We propose that these posttranslational modifications modulate microbial virulence by regulating the response to host-derived reactive oxygen species.

Beta-Lactam Antibiotic Therapeutic Drug Monitoring in Critically Ill Patients: A Systematic Review and Meta-Analysis.

Therapeutic drug monitoring (TDM) of beta-lactam antibiotics is recommended to address the variability in exposure observed in critical illness. However, the impact of TDM-guided dosing on clinical outcomes remains unknown. We conducted a systematic review and meta-analysis on TDM-guided dosing and clinical outcomes (all-cause mortality, clinical cure, microbiological cure, treatment failure, hospital and intensive care unit length of stay, target attainment, antibiotic-related adverse events, and emergence of resistance) in critically ill patients with suspected or proven sepsis. Eleven studies (n = 1463 participants) were included. TDM-guided dosing was associated with improved clinical cure (relative risk, 1.17; 95% confidence interval [CI], 1.04 to 1.31), microbiological cure (RR, 1.14; 95% CI, 1.03 to 1.27), treatment failure (RR, 0.79; 95% CI, .66 to .94), and target attainment (RR, 1.85; 95% CI, 1.08 to 3.16). No associations with mortality and length of stay were found. TDM-guided dosing improved clinical and microbiological cure and treatment response. Larger, prospective, randomized trials are required to better assess the utility of beta-lactam TDM in critically ill patients.

Simulated Intravenous versus Inhaled Tobramycin with or without Intravenous Ceftazidime Evaluated against Hypermutable Pseudomonas aeruginosa via a Dynamic Biofilm Model and Mechanism-Based Modeling.

Acute exacerbations of chronic respiratory infections in patients with cystic fibrosis are highly challenging due to hypermutable Pseudomonas aeruginosa, biofilm formation and resistance emergence. We aimed to systematically evaluate the effects of intravenous versus inhaled tobramycin (TOB) with and without intravenous ceftazidime (CAZ). Two hypermutable P. aeruginosa isolates, CW30 (MICCAZ, 0.5 mg/liter; MICTOB, 2 mg/liter) and CW8 (MICCAZ, 2 mg/liter; MICTOB, 8 mg/liter), were investigated for 120 h in dynamic in vitro biofilm studies. Treatments were intravenous ceftazidime, 9 g/day (33% lung fluid penetration); intravenous tobramycin, 10 mg/kg of body every 24 h (50% lung fluid penetration); inhaled tobramycin, 300 mg every 12 h; and both ceftazidime-tobramycin combinations. Total and less susceptible planktonic and biofilm bacteria were quantified over 120 h. Mechanism-based modeling was performed. All monotherapies were ineffective for both isolates, with regrowth of planktonic (≥4.7 log10 CFU/ml) and biofilm (>3.8 log10 CFU/cm2) bacteria and resistance amplification by 120 h. Both combination treatments demonstrated synergistic or enhanced bacterial killing of planktonic and biofilm bacteria. With the combination simulating tobramycin inhalation, planktonic bacterial counts of the two isolates at 120 h were 0.47% and 36% of those for the combination with intravenous tobramycin; for biofilm bacteria the corresponding values were 8.2% and 13%. Combination regimens achieved substantial suppression of resistance of planktonic and biofilm bacteria compared to each antibiotic in monotherapy for both isolates. Mechanism-based modeling well described all planktonic and biofilm counts and indicated synergy of the combination regimens despite reduced activity of tobramycin in biofilm. Combination regimens of inhaled tobramycin with ceftazidime hold promise to treat acute exacerbations caused by hypermutable P. aeruginosa strains and warrant further investigation.

Mpeg1 is not essential for antibacterial or antiviral immunity, but is implicated in antigen presentation.

To control infections phagocytes can directly kill invading microbes. Macrophage-expressed gene 1 (Mpeg1), a pore-forming protein sometimes known as perforin-2, is reported to be essential for bacterial killing following phagocytosis. Mice homozygous for the mutant allele Mpeg1tm1Pod succumb to bacterial infection and exhibit deficiencies in bacterial killing in vitro. Here we describe a new Mpeg mutant allele Mpeg1tm1.1Pib on the C57BL/6J background. Mice homozygous for the new allele are not abnormally susceptible to bacterial or viral infection, and irrespective of genetic background show no perturbation in bacterial killing in vitro. Potential reasons for these conflicting findings are discussed. In further work, we show that cytokine responses to inflammatory mediators, as well as antibody generation, are also normal in Mpeg1tm1.1Pib/tm1.1Pib mice. We also show that Mpeg1 is localized to a CD68-positive endolysosomal compartment, and that it exists predominantly as a processed, two-chain disulfide-linked molecule. It is abundant in conventional dendritic cells 1, and mice lacking Mpeg1 do not present the model antigen ovalbumin efficiently. We conclude that Mpeg1 is not essential for innate antibacterial protection or antiviral immunity, but may play a focused role early in the adaptive immune response.

Oral fosfomycin activity against Klebsiella pneumoniae in a dynamic bladder infection in vitro model.

INTRODUCTION: The use of oral fosfomycin for urinary tract infections (UTIs) caused by non-Escherichia coli uropathogens is uncertain, including Klebsiella pneumoniae, the second most common uropathogen. METHODS: A multicompartment bladder infection in vitro model was used with standard media and synthetic human urine (SHU) to simulate urinary fosfomycin exposure after a single 3 g oral dose (fAUC0-72 16884 mg·h/L, t½ 5.5 h) against 15 K. pneumoniae isolates including ATCC 13883 (MIC 2 to >1024 mg/L) with a constant media inflow (20 mL/h) and 4-hourly voiding of each bladder. The impact of the media (CAMHB + G6P versus SHU) on fosfomycin MIC measurements, drug-free growth kinetics and regrowth after fosfomycin administration was assessed. A low and high starting inoculum (5.5 versus 7.5 log10 cfu/mL) was assessed in the bladder infection model. RESULTS: Compared with CAMHB, isolates in SHU had a slower growth rate doubling time (37.7 versus 24.1 min) and reduced growth capacity (9.0 ± 0.3 versus 9.4 ± 0.3 log10 cfu/mL), which was further restricted with increased inflow rate (40 mL/h) and more frequent voids (2-hourly). Regrowth was commonly observed in both media with emergence of fosfomycin resistance promoted by a high starting inoculum in CAMHB (MIC rise to ≥1024 mg/L in 13/14 isolates). Resistance was rarely detected in SHU, even with a high starting inoculum (MIC rise to ≥1024 mg/L in 2/14 isolates). CONCLUSIONS: Simulated in an in vitro UTI model, the regrowth of K. pneumoniae urinary isolates was inadequately suppressed following oral fosfomycin therapy. Efficacy was further reduced by a high starting inoculum.

Unstable chromosome rearrangements in Staphylococcus aureus cause phenotype switching associated with persistent infections.

Staphylococcus aureus small-colony variants (SCVs) are associated with unusually chronic and persistent infections despite active antibiotic treatment. The molecular basis for this clinically important phenomenon is poorly understood, hampered by the instability of the SCV phenotype. Here we investigated the genetic basis for an unstable S. aureus SCV that arose spontaneously while studying rifampicin resistance. This SCV showed no nucleotide differences across its genome compared with a normal-colony variant (NCV) revertant, yet the SCV presented the hallmarks of S. aureus linked to persistent infection: down-regulation of virulence genes and reduced hemolysis and neutrophil chemotaxis, while exhibiting increased survival in blood and ability to invade host cells. Further genome analysis revealed chromosome structural variation uniquely associated with the SCV. These variations included an asymmetric inversion across half of the S. aureus chromosome via recombination between type I restriction modification system (T1RMS) genes, and the activation of a conserved prophage harboring the immune evasion cluster (IEC). Phenotypic reversion to the wild-type-like NCV state correlated with reversal of the chromosomal inversion (CI) and with prophage stabilization. Further analysis of 29 complete S. aureus genomes showed strong signatures of recombination between hsdMS genes, suggesting that analogous CI has repeatedly occurred during S. aureus evolution. Using qPCR and long-read amplicon deep sequencing, we detected subpopulations with T1RMS rearrangements causing CIs and prophage activation across major S. aureus lineages. Here, we have discovered a previously unrecognized and widespread mechanism of reversible genomic instability in S. aureus associated with SCV generation and persistent infections.

Antibiotic resistance and host immune evasion in Staphylococcus aureus mediated by a metabolic adaptation.

Staphylococcus aureus is a notorious human bacterial pathogen with considerable capacity to develop antibiotic resistance. We have observed that human infections caused by highly drug-resistant S. aureus are more prolonged, complicated, and difficult to eradicate. Here we describe a metabolic adaptation strategy used by clinical S. aureus strains that leads to resistance to the last-line antibiotic, daptomycin, and simultaneously affects host innate immunity. This response was characterized by a change in anionic membrane phospholipid composition induced by point mutations in the phospholipid biosynthesis gene, cls2, encoding cardiolipin synthase. Single cls2 point mutations were sufficient for daptomycin resistance, antibiotic treatment failure, and persistent infection. These phenotypes were mediated by enhanced cardiolipin biosynthesis, leading to increased bacterial membrane cardiolipin and reduced phosphatidylglycerol. The changes in membrane phospholipid profile led to modifications in membrane structure that impaired daptomycin penetration and membrane disruption. The cls2 point mutations also allowed S. aureus to evade neutrophil chemotaxis, mediated by the reduction in bacterial membrane phosphatidylglycerol, a previously undescribed bacterial-driven chemoattractant. Together, these data illustrate a metabolic strategy used by S. aureus to circumvent antibiotic and immune attack and provide crucial insights into membrane-based therapeutic targeting of this troublesome pathogen.

Staphylococcus aureus induces cell-surface expression of immune stimulatory NKG2D ligands on human monocytes.

Staphylococcus aureus is among the leading causes of bacterial infections worldwide. The pathogenicity and establishment of S. aureus infections are tightly linked to its ability to modulate host immunity. Persistent infections are often associated with mutant staphylococcal strains that have decreased susceptibility to antibiotics; however, little is known about how these mutations influence bacterial interaction with the host immune system. Here, we discovered that clinical S. aureus isolates activate human monocytes, leading to cell-surface expression of immune stimulatory natural killer group 2D (NKG2D) ligands on the monocytes. We found that expression of the NKG2D ligand ULBP2 (UL16-binding protein 2) is associated with bacterial degradability and phagolysosomal activity. Moreover, S. aureus-induced ULBP2 expression was linked to altered host cell metabolism, including increased cytoplasmic (iso)citrate levels, reduced glycolytic flux, and functional mitochondrial activity. Interestingly, we found that the ability of S. aureus to induce ULBP2 and proinflammatory cytokines in human monocytes depends on a functional ClpP protease in S. aureus These findings indicate that S. aureus activates ULBP2 in human monocytes through immunometabolic mechanisms and reveal that clpP inactivation may function as a potential immune evasion mechanism. Our results provide critical insight into the interplay between the host immune system and S. aureus that has evolved under the dual selective pressure of host immune responses and antibiotic treatment. Our discovery of an immune stimulatory pathway consisting of human monocyte-based defense against S. aureus suggests that targeting the NKG2D pathway holds potential for managing persistent staphylococcal infections.

Search and Contain: Impact of an Integrated Genomic and Epidemiological Surveillance and Response Program for Control of Carbapenemase-producing Enterobacterales.

BACKGROUND: Multiresistant organisms (MROs) pose a critical threat to public health. Population-based programs for control of MROs such as carbapenemase-producing Enterobacterales (CPE) have emerged and evaluation is needed. We assessed the feasibility and impact of a statewide CPE surveillance and response program deployed across Victoria, Australia (population 6.5 million). METHODS: A prospective multimodal intervention including active screening, carrier isolation, centralized case investigation, and comparative pathogen genomics was implemented. We analyzed trends in CPE incidence and clinical presentation, risk factors, and local transmission over the program's first 3 years (2016-2018). RESULTS: CPE case ascertainment increased over the study period to 1.42 cases/100 000 population, linked to increased screening without a concomitant rise in active clinical infections (0.45-0.60 infections/100 000 population, P = .640). KPC-2 infection decreased from 0.29 infections/100 000 population prior to intervention to 0.03 infections/100 000 population in 2018 (P = .003). Comprehensive case investigation identified instances of overseas community acquisition. Median time between isolate referral and genomic and epidemiological assessment for local transmission was 11 days (IQR, 9-14). Prospective surveillance identified numerous small transmission networks (median, 2; range, 1-19 cases), predominantly IMP and KPC, with median pairwise distance of 8 (IQR, 4-13) single nucleotide polymorphisms; low diversity between clusters of the same sequence type suggested genomic cluster definitions alone are insufficient for targeted response. CONCLUSIONS: We demonstrate the value of centralized CPE control programs to increase case ascertainment, resolve risk factors, and identify local transmission through prospective genomic and epidemiological surveillance; methodologies are transferable to low-prevalence settings and MROs globally.

Pharmacokinetic/pharmacodynamic analysis of oral fosfomycin against Enterobacterales, Pseudomonas aeruginosa and Enterococcus spp. in an in vitro bladder infection model: impact on clinical breakpoints.

OBJECTIVES: Fosfomycin is an established treatment for uncomplicated urinary tract infections (UTIs), yet evidence supporting susceptibility breakpoints is limited. We examine the UTI susceptibility criteria. METHODS: Fosfomycin susceptibility, heteroresistance and in vitro growth in a bladder infection model, after a single 3 g dose of oral fosfomycin, were bridged to human pharmacokinetics with pharmacokinetic/pharmacodynamic and Monte Carlo analyses. Data from common uropathogens (24 Escherichia coli, 20 Klebsiella pneumoniae, 4 Enterobacter cloacae, 14 Pseudomonas aeruginosa, 8 Enterococcus faecalis and 8 Enterococcus faecium) were compared and analysed to ascertain species-specific PTA. RESULTS: Glucose-6-phosphate (G6P) increased MICs of E. coli, K. pneumoniae and E. cloacae (median 2-fold dilutions 3-5), but not of P. aeruginosa and Enterococcus. Atypical E. coli lacking G6P potentiation were killed in the bladder infection model despite high MICs (32-128 mg/L). Fosfomycin heteroresistance was uncommon in E. coli (MIC > 2 mg/L) but was detected in the majority of K. pneumoniae (MIC > 1 mg/L) and P. aeruginosa (MIC >8 mg/L). For these species, baseline heteroresistance was a strong predictor for treatment failure in the model. No heteroresistance was found in Enterococcus. The fAUC/MIC targets for stasis were 1935, 3393, 9968, 2738 and 283 for typical E. coli, K. pneumoniae, E. cloacae, P. aeruginosa and E. faecalis, respectively (synthetic human urine medium alone promoted a 1 log10 kill in E. faecium). A >95% PTA for stasis was only found at MIC ≤ epidemiological cut-off (ECOFF) for E. coli (4 mg/L). For other species, PTAs were low for WT populations. CONCLUSIONS: With the exception of E. coli, fosfomycin is a poor target for other uropathogen species. A reduction in oral fosfomycin UTI breakpoints is supported.