Severe Gram-negative Bacterial Infections Research Articles

Ceftazidime with avibactam for treating severe aerobic Gram-negative bacterial infections: technology evaluation to inform a novel subscription-style payment model.

To limit the use of antimicrobials without disincentivising the development of novel antimicrobials, there is interest in establishing innovative models that fund antimicrobials based on an evaluation of their value as opposed to the volumes used. The aim of this project was to evaluate the population-level health benefit of ceftazidime-avibactam in the NHS in England, for the treatment of severe aerobic Gram-negative bacterial infections when used within its licensed indications. The results were used to inform National Institute for Health and Care Excellence guidance in support of commercial discussions regarding contract value between the manufacturer and NHS England. The health benefit of ceftazidime-avibactam was first derived for a series of high-value clinical scenarios. These represented uses that were expected to have a significant impact on patients' mortality risks and health-related quality of life. Patient-level costs and health-related quality of life of ceftazidime-avibactam under various usage scenarios compared with alternative management strategies in the high-value clinical scenarios were quantified using decision modelling. Results were reported as incremental net health effects expressed in quality-adjusted life-years, which were scaled to 20-year population in quality-adjusted life-years using infection number forecasts based on data from Public Health England. The outcomes estimated for the high-value clinical scenarios were extrapolated to other expected uses for ceftazidime-avibactam. The clinical effectiveness of ceftazidime-avibactam relative to its comparators was estimated by synthesising evidence on susceptibility of the pathogens of interest to the antimicrobials in a network meta-analysis. In the base case, ceftazidime-avibactam was associated with a statistically significantly higher susceptibility relative to colistin (odds ratio 7.24, 95% credible interval 2.58 to 20.94). The remainder of the treatments were associated with lower susceptibility than colistin (odds ratio < 1). The results were sensitive to the definition of resistance and the studies included in the analysis. In the base case, patient-level benefit of ceftazidime-avibactam was between 0.08 and 0.16 quality-adjusted life-years, depending on the site of infection and the usage scenario. There was a high degree of uncertainty surrounding the benefits of ceftazidime-avibactam across all subgroups, and the results were sensitive to assumptions in the meta-analysis used to estimate susceptibility. There was substantial uncertainty in the number of infections that are suitable for treatment with ceftazidime-avibactam, so population-level results are presented for a range of scenarios for the current infection numbers, the expected increases in infections over time, and rates of emergence of resistance. The population-level benefit varied substantially across the scenarios, from 531 to 2342 quality-adjusted life-years over 20 years. This work has provided quantitative estimates of the value of ceftazidime-avibactam within its areas of expected usage within the NHS. Given existing evidence, the estimates of the value of ceftazidime-avibactam are highly uncertain. Future evaluations of antimicrobials would benefit from improvements to NHS data linkages, research to support appropriate synthesis of susceptibility studies, and application of routine data and decision modelling to assess enablement value. No registration of this study was undertaken. This award was funded by the National Institute for Health and Care Research (NIHR) Policy Research Programme (NIHR award ref: NIHR135592), conducted through the Policy Research Unit in Economic Methods of Evaluation in Health and Social Care Interventions, PR-PRU-1217-20401, and is published in full in Health Technology Assessment; Vol. 28, No. 73. See the NIHR Funding and Awards website for further award information.

Efficacy and Safety Factors Related to Plasma Concentration-Optimized Polymyxin B Therapy in Treating Carbapenem-Resistant Gram-Negative Bacterial Infections in China.

Polymyxin B (PMB)-based combination therapies are used to treat severe carbapenem-resistant gram-negative bacterial (CR-GNB) infections. This observational study investigated the relationship between clinical factors, including PMB concentration, and clinical efficacy and safety. Polymyxin B regimens were optimized through therapeutic drug monitoring (TDM) and area under the concentration-time curve (AUC). In all, 382 samples were tested from 130 patients. Logistic regression was used to analyze the relationships between variables with clinical efficacy and 30-day mortality factors were analyzed by Cox regression. The sensitivity and specificity of Cmin and AUC for the occurrence of acute kidney injury (AKI) were determined by ROC curve analysis. The clinical effectiveness of PMB was 65.4%. Multivariate logistic regression analysis revealed that lung infection, continuous renal replacement therapy, and C-reactive protein were independent factors significantly associated with efficacy. AKI occurred in 14.6% of the patients during treatment; age > 73 years (OR: 3.63; 95% CI: 1.035-12.727; P = 0.044), Cmin greater than 2.3 µg/mL (OR: 7.37; 95% CI: 1.571-34.580; P = 0.011), combined vancomycin (OR: 9.47; 95% CI: 1.732-51.731; P = 0.009), and combined piperacillin-tazobactam (OR: 21.87; 95% CI: 3.139-152.324; P = 0.002) were independent risk factors. The identified PMB cut-offs for predicting AKI were Cmin = 2.3 µg/mL and AUC = 82.0 mg h/L. Polymyxin B-based combination regimens are effective in treating CR-GNB infections, particularly bloodstream infections, but have shown unsatisfactory for lung infections. Cmin ≥ 2.3 µg /mL and AUC ≥ 82.0 mg h/L may increase PMB-associated AKI incidence. PMB dose should be adjusted based on TDM to ensure efficacy.

Cefiderocol for treating severe aerobic Gram-negative bacterial infections: technology evaluation to inform a novel subscription-style payment model.

To limit the use of antimicrobials without disincentivising the development of novel antimicrobials, there is interest in establishing innovative models that fund antimicrobials based on an evaluation of their value as opposed to the volumes used. The aim of this project was to evaluate the population-level health benefit of cefiderocol in the NHS in England, for the treatment of severe aerobic Gram-negative bacterial infections when used within its licensed indications. The results were used to inform the National Institute for Health and Care Excellence guidance in support of commercial discussions regarding contract value between the manufacturer and NHS England. The health benefit of cefiderocol was first derived for a series of high-value clinical scenarios. These represented uses that were expected to have a significant impact on patients' mortality risks and health-related quality of life. The clinical effectiveness of cefiderocol relative to its comparators was estimated by synthesising evidence on susceptibility of the pathogens of interest to the antimicrobials in a network meta-analysis. Patient-level costs and health outcomes of cefiderocol under various usage scenarios compared with alternative management strategies were quantified using decision modelling. Results were reported as incremental net health effects expressed in quality-adjusted life-years, which were scaled to 20-year population values using infection number forecasts based on data from Public Health England. The outcomes estimated for the high-value clinical scenarios were extrapolated to other expected uses for cefiderocol. Among Enterobacterales isolates with the metallo-beta-lactamase resistance mechanism, the base-case network meta-analysis found that cefiderocol was associated with a lower susceptibility relative to colistin (odds ratio 0.32, 95% credible intervals 0.04 to 2.47), but the result was not statistically significant. The other treatments were also associated with lower susceptibility than colistin, but the results were not statistically significant. In the metallo-beta-lactamase Pseudomonas aeruginosa base-case network meta-analysis, cefiderocol was associated with a lower susceptibility relative to colistin (odds ratio 0.44, 95% credible intervals 0.03 to 3.94), but the result was not statistically significant. The other treatments were associated with no susceptibility. In the base case, patient-level benefit of cefiderocol was between 0.02 and 0.15 quality-adjusted life-years, depending on the site of infection, the pathogen and the usage scenario. There was a high degree of uncertainty surrounding the benefits of cefiderocol across all subgroups. There was substantial uncertainty in the number of infections that are suitable for treatment with cefiderocol, so population-level results are presented for a range of scenarios for the current infection numbers, the expected increases in infections over time and rates of emergence of resistance. The population-level benefits varied substantially across the base-case scenarios, from 896 to 3559 quality-adjusted life-years over 20 years. This work has provided quantitative estimates of the value of cefiderocol within its areas of expected usage within the NHS. Given existing evidence, the estimates of the value of cefiderocol are highly uncertain. Future evaluations of antimicrobials would benefit from improvements to NHS data linkages; research to support appropriate synthesis of susceptibility studies; and application of routine data and decision modelling to assess enablement value. No registration of this study was undertaken. This award was funded by the National Institute for Health and Care Research (NIHR) Health Technology Assessment Policy Research Programme (NIHR award ref: NIHR135591), conducted through the Policy Research Unit in Economic Methods of Evaluation in Health and Social Care Interventions, PR-PRU-1217-20401, and is published in full in Health Technology Assessment; Vol. 28, No. 28. See the NIHR Funding and Awards website for further award information.

Capturing Value Attributes in the Economic Evaluation of Ceftazidime with Avibactam for Treating Severe Aerobic Gram-Negative Bacterial Infections in the United Kingdom.

Antimicrobial resistance remains a serious and growing threat to public health, both globally and in the UK, leading to diminishing effectiveness of antimicrobials. Despite a clear need for new antimicrobials, the clinical pipeline is insufficient, driven by high research and development costs and limited expected returns on investment. To counteract this, National Institute for Health and Care Excellence (NICE) and National Health Service (NHS) England have launched a reimbursement mechanism, de-linked from volume of sales, that aims to reduce economic risk by recognising the broader population-level value of antimicrobials. The objective of this study was to quantify the value of ceftazidime-avibactam for treating gram-negative infections in the UK considering some of these broader value elements unique to antimicrobials. A previously developed dynamic disease transmission and cost-effectiveness model was applied to assess the value of introducing ceftazidime-avibactam to UK treatment practice in the management of gram-negative hospital-acquired infections in line with the licenced indications for ceftazidime-avibactam. Model inputs were parameterised using sources aligned to the UK perspective. The introduction of ceftazidime-avibactam into a two-line treatment sequence saved over 2300 lives, leading to a gain of 27,600 life years and 22,000 quality-adjusted life years (QALY) at an additional cost of £17 million, over a ten-year transmission period. Ceftazidime-avibactam was associated with a net monetary benefit of £642 million at willingness to pay threshold of £30,000 per QALY; even at a lower threshold of £20,000 per QALY, the net monetary benefit is £422 million. Increasing the diversity of antimicrobial treatments through the introduction of an additional antimicrobial, in this instance ceftazidime-avibactam, was associated with substantial clinical and economic benefits, when considering broader population-level value. Despite revealing considerable benefits, the value of ceftazidime-avibactam is only partially reflected in this analysis. Further efforts are required to fully operationalise the spectrum, transmission, enablement, diversity and insurance (STEDI) value framework and accurately reflect the population-level value of antimicrobials.

First Detection of mcr-9 in a Multidrug-Resistant Escherichia coli of Animal Origin in Italy Is Not Related to Colistin Usage on a Pig Farm.

The emergence of colistin resistance raises growing concerns because of its use as a last-resort antimicrobial for the treatment of severe gram-negative bacterial infections in humans. Plasmid-borne mobile colistin resistance genes (mcr) are particularly worrisome due to their high propensity to spread. An mcr-9-positive Escherichia coli was isolated from a piglet in Italy, representing the first isolation of this gene from an E. coli of animal origin in the country. Whole genome sequencing (WGS) revealed that mcr-9 was borne by an IncHI2 plasmid carrying several other resistance genes. The strain was indeed phenotypically resistant to six different antimicrobial classes, including 3rd and 4th generation cephalosporins. Despite the presence of mcr-9, the isolate was susceptible to colistin, probably because of a genetic background unfavourable to mcr-9 expression. The lack of colistin resistance, coupled with the fact that the farm of origin had not used colistin in years, suggests that mcr-9 in such a multidrug-resistant strain can be maintained thanks to the co-selection of neighbouring resistance genes, following usage of different antimicrobials. Our findings highlight how a comprehensive approach, integrating phenotypical testing, targeted PCR, WGS-based techniques, and information on antimicrobial usage is crucial to shed light on antimicrobial resistance.

4F-Indole Enhances the Susceptibility of Pseudomonas aeruginosa to Aminoglycoside Antibiotics.

Infections caused by multidrug-resistant bacteria are becoming increasingly serious. The aminoglycoside antibiotics have been widely used to treat severe Gram-negative bacterial infections. Here, we reported that a class of small molecules, namely, halogenated indoles, can resensitize Pseudomonas aeruginosa PAO1 to aminoglycoside antibiotics such as gentamicin, kanamycin, tobramycin, amikacin, neomycin, ribosomalin sulfate, and cisomicin. We selected 4F-indole as a representative of halogenated indoles to investigate its mechanism and found that the two-component system (TCS) PmrA/PmrB inhibited the expression of multidrug efflux pump MexXY-OprM, allowing kanamycin to act intracellularly. Moreover, 4F-indole inhibited the biosynthesis of several virulence factors, such as pyocyanin, type III secretion system (T3SS), and type VI secretion system (T6SS) exported effectors, and reduced the swimming and twitching motility by suppressing the expression of flagella and type IV pili. This study suggests that the combination of 4F-indole and kanamycin can be more effective against P. aeruginosa PAO1 and affect its multiple physiological activities, providing a novel insight into the reactivation of aminoglycoside antibiotics. IMPORTANCE Infections caused by Pseudomonas aeruginosa have become a major public health crisis. Its resistance to existing antibiotics causes clinical infections that are hard to cure. In this study, we found that halogenated indoles in combination with aminoglycoside antibiotics could be more effective than antibiotics alone against P. aeruginosa PAO1 and preliminarily revealed the mechanism of the 4F-indole-induced regulatory effect. Moreover, the regulatory effect of 4F-indole on different physiological behaviors of P. aeruginosa PAO1 was analyzed by combined transcriptomics and metabolomics. We explain that 4F-indole has potential as a novel antibiotic adjuvant, thus slowing down the further development of bacterial resistance.

A Survey of Current Activities and Technologies Used to Detect Carbapenem Resistance in Bacteria Isolated from Companion Animals at Veterinary Diagnostic Laboratories-United States, 2020.

Carbapenems are antimicrobial drugs reserved for the treatment of severe multidrug-resistant Gram-negative bacterial infections. Carbapenem-resistant organisms (CROs) are an urgent public health threat and have been made reportable to public health authorities in many jurisdictions. Recent reports of CROs in companion animals and veterinary settings suggest that CROs are a One Health problem. However, standard practices of U.S. veterinary diagnostic laboratories (VDLs) to detect CROs are unknown. We assessed the capacity of VDLs to characterize carbapenem resistance in isolates from companion animals. Among 74 VDLs surveyed in 42 states, 23 laboratories (31%) from 22 states responded. Most (22/23, 96%) included ≥1 carbapenem on their primary antimicrobial susceptibility testing panel, and approximately one-third (9/23, 39%) performed phenotypic carbapenemase production testing or molecular identification of carbapenemase genes. Overall, 35% (8/23) of VDLs across eight states reported they would notify public health if a CRO was detected. Most (17/21, 81%) VDLs were not aware of CRO reporting mandates, and some expressed uncertainty about whether the scope of known mandates included CROs from veterinary sources. Although nearly all surveyed VDLs tested for carbapenem resistance, fewer had the capacity for mechanism testing or awareness of public health reporting requirements. Addressing these gaps is critical to monitoring CRO incidence and trends in veterinary medicine, preventing spread in veterinary settings, and mounting an effective One Health response. Improved collaboration and communication between public health and veterinary medicine is critical to inform infection control practices in veterinary settings and conduct a public health response when resistant isolates are detected.

Dose Optimization of Colistin: A Systematic Review

Colistin is considered a last treatment option for multi-drug and extensively resistant Gram-negative infections. We aimed to assess the available data on the dosing strategy of colistin. A systematic review was performed to identify all published studies on the dose optimization of colistin. Grey literature and electronic databases were searched. Data were collected in a specified form and the quality of the included articles was then assessed using the Newcastle-Ottawa scale for cohort studies, the Cochrane bias tool for randomized clinical trials (RCT), and the Joanna Briggs Institute (JBI) critical checklist for case reports. A total of 19 studies were included, of which 16 were cohort studies, one was a RCT, and two were case reports. A total of 18 studies proposed a dosing regimen for adults, while only one study proposed a dosing schedule for pediatric populations. As per the available evidence, a loading dose of 9 million international units (MIU) of colistin followed by a maintenance dose of 4.5 MIU every 12 h was considered the most appropriate dosing strategy to optimize the safety and efficacy of treatment and improve clinical outcomes. This review supports the administration of a loading dose followed by a maintenance dose of colistin in severe and life-threatening multi-drug Gram-negative bacterial infections.

Amelioration of renal cortex histological alterations by aqueous garlic extract in gentamicin induced renal toxicity in albino rats: a histological and immunohistochemical study

ABSTRACT Background: Aminoglycosides, particularly gentamicin, endure crucial antibiotics in the armamentarium for severe Gram-negative bacterial infections through their significant risk for nephrotoxicity. Co-administration of several applicant nephroprotective agents has been investigated at the preclinical level. Garlic was proved to be an oxidative stress combatant with unique antioxidant potential. Aim of the work: To assess renal cortex structural changes due to gentamicin treatment and the role of the aqueous garlic extract (AGE) in ameliorating these changes. Material and methods: Thirty-two male albino rats were assigned into four equal groups. Group I (Control group) received 0.9% NaCl solution through oral gavage in the same volume as in AGE-treated group. Group II (AGE treated group) received AGE by oral gavage at 250 mg/kg/day. Group III (Gentamicin treated group) received Gentamicin at 80 mg/kg/day intraperitoneally. Group IV (Gentamicin and AGE cotreated group) received both gentamicin and AGE. The duration of the treatment was 21 days. Specimens of renal cortex of all groups were processed for light microscopic examination. Specimens were additionally prepared for electron microscopic examination. Morphometric study and statistical analysis were performed. Results: Examination of the renal cortex in the gentamicin treated rats showed both proximal and distal tubular necrosis, vacuolation, desquamation and interstitial mononuclear cell infiltration. Masson’s trichrome staining revealed intense deposition of collagen fibers. Strong positive immunoreaction for caspase-3 was observed. Ultrastructurally, the glomerulus showed thickened basement membrane, destructed endothelium. Proximal and distal convoluted tubular cells exhibited vacuolations, distorted mitochondria and nuclear chromatin condensation with loss of microvilli. AGE ameliorated these changes. Conclusion: Aqueous garlic extract ameliorates the histological changes caused by gentamicin in the rat renal cortex.

Septic shock after liver transplantation successfully treated with endotoxin and cytokine adsorption continuous renal replacement therapy: a case report and literature review

Endotoxins and cytokines play an important role in multiple organ failure pathogenesis in patients with severe Gram-negative bacterial infection. We present a clinical case where an oXiris hemofilter was used for continuous renal replacement therapy (CRRT) treatment in a patient with septic shock after liver transplantation. A 35-year-old man with a 20-year history of hepatitis B presented with jaundice, loss of appetite, and decreased urine output. He was diagnosed with decompensated cirrhosis with acute-on-chronic liver failure, and liver transplantation was indicated. The day after surgery, he developed hyperthermia, hypotension, anuria, and a progressive increase in blood inflammatory markers and creatinine. Combined with the donor source and blood culture results, septic shock after transplantation was considered. The patient was immediately treated with endotoxin and cytokine adsorption CRRT (oXiris hemofilter) with tigecycline, caspofungin, and ganciclovir as anti-infectives. After 48 hours on CRRT, his blood pressure gradually stabilized, the CLIF Consortium Acute-on-Chronic Liver Failure score decreased from 63 to 43. Procalcitonin, endotoxin, and the inflammatory factors interleukin (IL)-6 and IL-10 also decreased gradually. The patient’s liver and kidney functions were completely restored. Our experience suggests that oXiris CRRT combined with antibacterial therapy is an effective treatment for septic shock after liver transplantation.

Prescribing Patterns of Aminoglycoside Antibiotics in a Teaching Hospital in Sudan

Background: Aminoglycoside antimicrobials are widely used for the treatment of severe gram-negative bacterial infections. Their inappropriate use may result in treatment failure, development of resistant pathogens, and toxicity. Drug utilization evaluation of aminoglycosides can improve rational use. Purpose: This study intended to assess the prescribing patterns and potential problems associated with gentamicin and amikacin use. Methods: This prospective observational study was conducted at Soba University Hospital, Khartoum, Sudan. Patients’ demographics, clinical and prescription information were collected using a data collection form. Laboratory data of patients were collected from the patients' files. Ideal body weight was calculated to assess dose appropriateness, and adverse drug reactions were observed. Results: A total of 200 patients were included in the present study, two-thirds of the patients were prescribed gentamicin, while the remainder were given amikacin. Dosing was inappropriate in (60.6%) and (76.5%) of patients on gentamicin and amikacin respectively. Conventional dosing was used more commonly (67%) than extended dosing (33%). All indications for the use of these drugs were found to be appropriate. In (72%) of cases, both gentamicin and amikacin were prescribed based on microbiological sensitivity testing. Auditory function monitoring was only performed for neonates and infants. Nephrotoxicity was detected in 7 patients (9.3%), and ototoxicity in one patient (1.3%). Conclusion: Although prescription and dosing based on ideal body weight and microbiological data were reasonably common, higher compliance with recommended practice is needed in addition to therapeutic drug monitoring in order to ensure safe and effective treatment, and reduced antibiotic resistance.

A new active peptide from Neptunea arthritica cumingii exerts protective effects against gentamicin-induced sensory-hair cell injury in zebrafish

Gentamicin is commonly used for effective treatment of severe Gram-negative bacterial infections. However, its use is being increasingly restricted owing to the ototoxic effects attributed to it. Gentamicin-induced ototoxicity is thought to be related with apoptosis induced by reactive oxygen species (ROS). In this study, we found a novel active peptide from Neptunea arthritica cumingii with otoprotective effects and no significant embryotoxic effects. The combined application of gentamicin and this novel active peptide helped sensory-hair cells to protect themselves from lethal ROS accumulation. This, in turn, reduced the expression of three genes (caspase-3, caspase-9, Bax), and thereby, the sensory-hair cell apoptosis promoted by ROS accumulation upon gentamicin administration. Our findings provided new insights into the prevention of gentamicin-induced hearing loss.

Impact of Disease on Amikacin Pharmacokinetics and Dosing in Children

Amikacin is widely used to treat severe Gram-negative bacterial infections. Its peak concentration in plasma is associated with treatment efficacy. Amikacin pharmacokinetics (PK) is influenced by disease conditions, in addition to other patient characteristics. In this retrospective study, we evaluated the impact of clinical characteristics and disease condition on amikacin PK in children with burn injuries and those with cancer. Amikacin PK data from 66 children with burn injuries and 112 children with cancer were analyzed. A population PK model was developed using the nonlinear mixed-effects modeling approach. Models were developed using NONMEM 7.3 (ICON Development Solutions, LLC, Ellicott City, MD). Data processing and visualization was performed using R packages. The amikacin PK data were best described by a 2-compartment model. The parameters were estimated with mean values (95% confidence intervals) as follows: central volume of distribution (V1), 5.70 L (4.64-6.76 L); central clearance, 2.12 L/h (1.79-2.46 L/h); peripheral volume of distribution (V2), 4.79 L (2.36-7.22 L); and distribution clearance (Q), 0.71 L/h (0.25-1.16 L/h). The final model identified the disease condition as a significant covariate and indicated 55% (28%-82%) higher central clearance and 17% (1%-34%) higher V1 in burn patients compared with cancer patients. Volume of distribution was significantly influenced by age and body weight. Clearance was significantly influenced by age, body weight, and creatinine clearance. Using the final PK model, we developed a workflow for selecting optimal dosing strategies for 3 representative pediatric patient profiles. Disease condition was significant in influencing amikacin PK in children. To reach the same target concentrations (64 mg/L peak concentration) with a daily-dose plan, burn patients need higher doses than cancer patients. Future investigations are needed to explore the impact of other diseases on amikacin disposition in children, and to prospectively validate the proposed dosing strategy.

Curative Treatment of Severe Gram-Negative Bacterial Infections by a New Class of Antibiotics Targeting LpxC.

ABSTRACTThe infectious diseases caused by multidrug-resistant bacteria pose serious threats to humankind. It has been suggested that an antibiotic targeting LpxC of the lipid A biosynthetic pathway in Gram-negative bacteria is a promising strategy for curing Gram-negative bacterial infections. However, experimental proof of this concept is lacking. Here, we describe our discovery and characterization of a biphenylacetylene-based inhibitor of LpxC, an essential enzyme in the biosynthesis of the lipid A component of the outer membrane of Gram-negative bacteria. The compound LPC-069 has no known adverse effects in mice and is effective in vitro against a broad panel of Gram-negative clinical isolates, including several multiresistant and extremely drug-resistant strains involved in nosocomial infections. Furthermore, LPC-069 is curative in a murine model of one of the most severe human diseases, bubonic plague, which is caused by the Gram-negative bacterium Yersinia pestis. Our results demonstrate the safety and efficacy of LpxC inhibitors as a new class of antibiotic against fatal infections caused by extremely virulent pathogens. The present findings also highlight the potential of LpxC inhibitors for clinical development as therapeutics for infections caused by multidrug-resistant bacteria.

Global Characterization of Protein Secretion from Human Macrophages Following Non-canonical Caspase-4/5 Inflammasome Activation

Gram-negative bacteria are associated with a wide spectrum of infectious diseases in humans. Inflammasomes are cytosolic protein complexes that are assembled when the cell encounters pathogens or other harmful agents. The non-canonical caspase-4/5 inflammasome is activated by Gram-negative bacteria-derived lipopolysaccharide (LPS) and by endogenous oxidized phospholipids. Protein secretion is a critical component of the innate immune response. Here, we have used label-free quantitative proteomics to characterize global protein secretion in response to non-canonical inflammasome activation upon intracellular LPS recognition in human primary macrophages. Before proteomics, the total secretome was separated into two fractions, enriched extracellular vesicle (EV) fraction and rest-secretome (RS) fraction using size-exclusion centrifugation. We identified 1048 proteins from the EV fraction and 1223 proteins from the RS fraction. From these, 640 were identified from both fractions suggesting that the non-canonical inflammasome activates multiple, partly overlapping protein secretion pathways. We identified several secreted proteins that have a critical role in host response against severe Gram-negative bacterial infection. The soluble secretome (RS fraction) was highly enriched with inflammation-associated proteins upon intracellular LPS recognition. Several ribosomal proteins were highly abundant in the EV fraction upon infection, and our data strongly suggest that secretion of translational machinery and concomitant inhibition of translation are important parts of host response against Gram-negative bacteria sensing caspase-4/5 inflammasome. Intracellular recognition of LPS resulted in the secretion of two metalloproteinases, adisintegrin and metalloproteinase domain-containing protein 10 (ADAM10) and MMP14, in the enriched EV fraction. ADAM10 release was associated with the secretion of TNF, a key inflammatory cytokine, and M-CSF, an important growth factor for myeloid cells probably through ADAM10-dependent membrane shedding of these cytokines. Caspase-4/5 inflammasome activation also resulted in secretion of danger-associated molecules S100A8 and prothymosin-α in the enriched EV fraction. Both S100A8 and prothymosin-α are ligands for toll-like receptor 4 recognizing extracellular LPS, and they may contribute to endotoxic shock during non-canonical inflammasome activation.

Genetic Variants Contributing to Colistin Cytotoxicity: Identification of TGIF1 and HOXD10 Using a Population Genomics Approach.

Colistin sulfate (polymixin E) is an antibiotic prescribed with increasing frequency for severe Gram-negative bacterial infections. As nephrotoxicity is a common side effect, the discovery of pharmacogenomic markers associated with toxicity would benefit the utility of this drug. Our objective was to identify genetic markers of colistin cytotoxicity that were also associated with expression of key proteins using an unbiased, whole genome approach and further evaluate the functional significance in renal cell lines. To this end, we employed International HapMap lymphoblastoid cell lines (LCLs) of Yoruban ancestry with known genetic information to perform a genome-wide association study (GWAS) with cellular sensitivity to colistin. Further association studies revealed that single nucleotide polymorphisms (SNPs) associated with gene expression and protein expression were significantly enriched in SNPs associated with cytotoxicity (p ≤ 0.001 for gene and p = 0.015 for protein expression). The most highly associated SNP, chr18:3417240 (p = 6.49 × 10−8), was nominally a cis-expression quantitative trait locus (eQTL) of the gene TGIF1 (transforming growth factor β (TGFβ)-induced factor-1; p = 0.021) and was associated with expression of the protein HOXD10 (homeobox protein D10; p = 7.17 × 10−5). To demonstrate functional relevance in a murine colistin nephrotoxicity model, HOXD10 immunohistochemistry revealed upregulated protein expression independent of mRNA expression in response to colistin administration. Knockdown of TGIF1 resulted in decreased protein expression of HOXD10 and increased resistance to colistin cytotoxicity. Furthermore, knockdown of HOXD10 in renal cells also resulted in increased resistance to colistin cytotoxicity, supporting the physiological relevance of the initial genomic associations.

Finding ways to solve or prevent aminoglycoside-induced ototoxicity?

Aminoglycoside (AG), which was first discovered in 1944, is widely used in treating severe Gram-negative bacterial infections (1). Gentamicin, kanamycin, amikacin and neomycin are the major AGs used nowadays, and vancomycin is the drug of choice for methicillin-resistant Staphylococcus aureus (MRSA) infection. Although other antibiotics can also target Gram-negative bacteria such as penicillins and cephalosporins, AGs remain to be the popular choice because of their economical and clinical advantages including low cost, rapid bactericidal activity and low incidences of resistance (2). However, the adverse effects of reversible nephrotoxicity and irreversible ototoxicity limit their use to short term therapy (3,4). Precaution is needed for treating senior patients who are prone to have hearing, vestibular and renal dysfunctions. Identification of the predisposing factors of such toxicities will allow us to maximize the benefit of using AGs and greatly improve the therapeutic outcome.

Optimal methodology is important for optimal pharmacokinetic studies, therapeutic drug monitoring and patient care.

The present paper by Pai et al. [1] is a well-motivated attempt to improve dosage regimens for continuous intravenous meropenem administration in morbidly obese patients with stable or unstable renal function who have severe Gram-negative bacterial infections. The desire to employ continuous intravenous infusion of time-dependent antibiotics to maximize their therapeutic effect is laudable. The authors appear to have used a nomogram based on a formula they previously described for meropenem clearance [2]. They then correlated meropenem clearance with creatinine clearance (CLCR). In the present paper by Pai et al. [1], they obtained what are described as steady-state serum samples of meropenem in the obese patients they studied. They made a one-compartment pharmacokinetic model of meropenem. They then calculated individual Bayesian estimates of the meropenem central volume of distribution (Vc) and clearance (CL) in each obese patient. From this, they developed a dosage nomogram for various categories of renal function, designed for their obese patients. This is a step in the right direction, since assays for meropenem are not commonly available. However, in their previous paper [2], their target steady-state concentration was 8–12 lg/mL. Figure 2 in their previous paper [2] describes the serum concentrations observed in validating the previous nomogram. They ranged from about 2 to about 19 lg/mL. Counting the points in that plot, about 47 % were below the target range of 8–12 lg/mL, about 29 % were within it and about 26 % were above it. This seems to be the evidence they used to validate their earlier nomogram [2]. In that paper, they stated that they gave a loading dose, which they described as essential. However, in the present paper by Pai et al. [1], I could not find mention of a loading dose. In Figs. 1 and 2 of the present paper by Pai et al. [1], the assumed steady-state serum concentrations range from about 2 to about 100 lg/mL. The authors report 41.1 % of serum concentrations as being over 16 lg/mL (the apparent upper limit of their target range), 76.1 % as being at least 8 lg/mL (so only 35 % were within the target range of 8–16 lg/mL), 97.4 % being at least 4 lg/mL (so 21 % within 4–8 lg/mL) and 99.9 % being at least 2 lg/mL (or 2.5 % within 2–4 lg/mL). So only 35 % were within 8–16 lg/mL, while 65 % were outside the target range. The very large range of serum concentrations found actually suggests that either (1) there was large interpatient variability; (2) the serum concentrations might not have been at steady state; or (3) the previous nomogram was not very well suited to the present obese patients. The authors seem not to have seriously considered an upper limit of serum concentrations but mostly to have set a lower therapeutic threshold as the target concentration. If this is so, then one apparently might give almost any large enough dosage regimen without consideration of toxicity. While the incidence of toxicity appears to be small, seizures have been reported, and one might consider setting some upper constraint on the serum concentrations to be achieved. The authors state that ‘‘the base structural model provided an excellent fit to the data’’, as in their Fig. 2. & Roger Jelliffe jelliffe@usc.edu; http://www.lapk.org

Pharmacokinetics and pharmacodynamics of continuous infusion meropenem in overweight, obese, and morbidly obese patients with stable and unstable kidney function: a step toward dose optimization for the treatment of severe gram-negative bacterial infections.

Meropenem is an anti-Gram-negative antimicrobial, the time-dependent activity of which may be maximized through administration by continuous infusion. The objectives of this study were to characterize the pharmacokinetics of continuous infusion meropenem in relation to body size and Cockcroft-Gault estimated creatinine clearance (CLCR) in overweight and obese patients with stable and unstable kidney function with the intent of creating a nomogram for optimal dosing. Patients from a single institution with a body mass index ≥25kg/m(2) receiving meropenem by continuous infusion with measurement of meropenem steady-state concentrations (C ss) were identified. Individual Bayesian estimates of meropenem volume of distribution of the central compartment (V c) and clearance (CL) were calculated and relationships to body size descriptors and CLCR estimated using these body size descriptors were defined by regression. Kidney function stability was defined based on median absolute deviation, stratification by the ratio of maximum to minimum serum creatinine (SCr) and individual patient-level regression of SCr over time. The influence of kidney function stability on meropenem CL estimation by CLCR was tested. A total of 375 patients (77.9% male) with 846 C ss values (62.4% of patients with ≥2 measurements) were identified. The median daily dose of meropenem and frequency of infusion bag changes were 2000mg/day and four times per day, respectively. The meropenem C ss values were ≥16, ≥8, ≥4, and ≥2mg/L for 41.1, 76.1, 97.4, and 99.9% of observations, respectively. The median (range) age, weight, and BMI were 66 (24-90) years, 90 (70-250)kg, and 30.8 (25.1-81.6)kg/m(2), respectively. The mean [standard deviation (SD)] serum creatinine at baseline was 1.57 (1.37)mg/dL. The mean (SD) V c was 28.1 (1.36)L and not related to body size, while CL was 8.85 (6.40)L/h and best related to CLCR estimated using adjusted body weight (ABW). The meropenem CL to CLCR relationship was not significantly impacted by the presence or absence of kidney function stability. The user-friendly dosing nomogram based on CLCR estimated using ABW showed that optimal drug exposure [Css≥minimum inhibitory concentration (MIC)] may be obtained even against multi-drug resistant (MDR) pathogens when considering dosages up to 1250mg every 6h by continuous infusion. Meropenem CL is best estimated using CLCR with ABW in patients with a BMI ≥25kg/m(2) and this relationship is not altered by unstable kidney function. Application of our dosing nomogram may improve the care of overweight and obese patients with severe MDR Gram-negative infections treated with meropenem by continuous infusion.

Delineating the Biosynthesis of Gentamicin X2, the Common Precursor of the Gentamicin C Antibiotic Complex

SummaryGentamicin C complex is a mixture of aminoglycoside antibiotics used worldwide to treat severe Gram-negative bacterial infections. Despite its clinical importance, the enzymology of its biosynthetic pathway has remained obscure. We report here insights into the four enzyme-catalyzed steps that lead from the first-formed pseudotrisaccharide gentamicin A2 to gentamicin X2, the last common intermediate for all components of the C complex. We have used both targeted mutations of individual genes and reconstitution of portions of the pathway in vitro to show that the secondary alcohol function at C-3″ of A2 is first converted to an amine, catalyzed by the tandem operation of oxidoreductase GenD2 and transaminase GenS2. The amine is then specifically methylated by the S-adenosyl-l-methionine (SAM)-dependent N-methyltransferase GenN to form gentamicin A. Finally, C-methylation at C-4″ to form gentamicin X2 is catalyzed by the radical SAM-dependent and cobalamin-dependent enzyme GenD1.