Drug-resistant Gram-negative Bacterial Infections Research Articles

Development of scalable processes to prepare a key chiral, nonracemic intermediate en route to LpxC inhibitors for gram-negative infections

Deaths resulting from drug-resistant gram-negative bacterial infections are a growing public health concern. Pyridone methylsulfone hydroxamic acid LpxC inhibitors, such as 1, are being developed for the treatment of serious gram-negative infections. Carboxylic acid 2 is a key intermediate in the synthesis of analogs of type 1. The current synthesis of 2 is unsuitable as a manufacturing process due to safety concerns and high cost. Two scalable and potentially lower cost processes have been developed, one based on chromatographic resolution of a novel intermediate and a second based on a classical resolution of the key intermediate 3. The advantages of these new chemical approaches are illustrated in the process details described in this letter.

New Predictive Equation for the Estimation of Plasma Concentrations of Formed Colistin in Patients Treated With Colistimethate Sodium for Multidrug-Resistant Gram-Negative Bacterial Infections.

The clinical use of colistin methanesulphonate (CMS) is limited by potential nephrotoxicity. The selection of an efficient and safe CMS dose for individual patients is complicated by the narrow therapeutic window and high interpatient pharmacokinetic variability. In this study, a simple predictive equation for estimating the plasma concentration of formed colistin in patients with multidrug and extremely drug-resistant gram-negative bacterial infections was developed. The equation was derived from the largest clinical cohort of patients undergoing therapeutic drug monitoring (TDM) of colistin for over 8 years in a tertiary Spanish hospital. All variables associated with C ss,avg were selected in a multiple linear regression model that was validated in a second cohort of 40 patients. Measured C ss,avg values were compared with those predicted by our model and a previous published algorithm for critically ill patients. In total, 276 patients were enrolled [the mean age was 67.2 (13.7) years, 203 (73.6%)] were male, and the mean (SD) C ss,avg was 1.12 (0.98) mg/L. Age, gender, estimated glomerular filtration rate, CMS dose and frequency, and concomitant drugs were included in the model. In the external validation, the previous algorithm appeared to yield more optimized colistin plasma concentrations when all types of C ss,avg values (high and low) were considered, while our equation yielded a more optimized prediction in the subgroup of patients with low colistin plasma concentrations (C ss,avg <1.5 mg/L). The proposed equation may help clinicians to better use CMS among a wide variety of patients, to maximize efficacy and prevent nephrotoxicity. A further prospective PK study is warranted to externally validate this algorithm.

Leveraging Large Data, Statistics, and Machine Learning to Predict the Emergence of Resistant E. coli Infections.

Drug-resistant Gram-negative bacterial infections, on average, increase the length of stay (LOS) in U.S. hospitals by 5 days, translating to approximately $15,000 per patient. We used statistical and machine-learning models to explore the relationship between antibiotic usage and antibiotic resistance over time and to predict the clinical and financial costs associated with resistant E. coli infections. We acquired data on antibiotic utilization and the resistance/sensitivity of 4776 microbial cultures at a Kaiser Permanente facility from April 2013 to December 2019. The ARIMA (autoregressive integrated moving average), neural networks, and random forest time series algorithms were employed to model antibiotic resistance trends. The models' performance was evaluated using mean absolute error (MAE) and root mean squared error (RMSE). The best performing model was then used to predict antibiotic resistance rates for the year 2020. The ARIMA model with cefazolin, followed by the one with cephalexin, provided the lowest RMSE and MAE values without signs of overfitting across training and test datasets. The study showed that reducing cefazolin usage could decrease the rate of resistant E. coli infections. Although piperacillin/tazobactam did not perform as well as cefazolin in our time series models, it performed reasonably well and, due to its broad spectrum, might be a practical target for interventions in antimicrobial stewardship programs (ASPs), at least for this particular facility. While a more generalized model could be developed with data from multiple facilities, this study acts as a framework for ASP clinicians to adopt statistical and machine-learning approaches, using region-specific data to make effective interventions.

The enhancement effect of small molecule Lyb24 reveals AzoR as a novel target of polymyxin B

Given the important role of polymyxin B (PB) in the treatment of drug-resistant Gram-negative bacterial infections, the emergence of PB resistance poses a serious threat to public health. Adjuvant development is a supplementary strategy that can compensate for the lack of novel antibiotics by protecting PB. In this study, we found a small molecule named Lyb24 that showed weak antibacterial activity (minimum inhibitory concentration ≥ 10 μg/ml) but potentiated and revitalized the efficacy of PB against Gram-negative pathogens, including mcr-1- and mgrB-deletion-mediated PB-resistant strains. Our results showed that Lyb24 inhibits the translational levels of genes associated with the modification of lipid A. In addition, Lyb24 increases the permeability, disrupts the integrity and induces the depolarization of the membrane. We further found that both Lyb24 and PB could directly bind to AzoR and inhibit its activity. Structural analysis showed that Lyb24 binds to the isoalloxazine ring of flavin mononucleotide (FMN) through pi-pi stacking and loop η4 of AzoR. A pneumonia model was used to confirm that the activity against clinical PB-resistant Klebsiella pneumoniae was enhanced due to Lyb24 on PB. In conclusion, we provide a potential therapeutic regimen by combining Lyb24 and PB to treat Gram-negative-resistant bacterial infections. Our findings not only explain the synergistic effect of Lyb24, but also expand our knowledge on the mechanism of action of PB.

Priorities and Progress in Gram-negative Bacterial Infection Research by the Antibacterial Resistance Leadership Group.

Addressing the treatment and prevention of antibacterial-resistant gram-negative bacterial infections is a priority area of the Antibacterial Resistance Leadership Group (ARLG). The ARLG has conducted a series of observational studies to define the clinical and molecular global epidemiology of carbapenem-resistant and ceftriaxone-resistant Enterobacterales, carbapenem-resistant Pseudomonas aeruginosa, and carbapenem-resistant Acinetobacter baumannii, with the goal of optimizing the design and execution of interventional studies. One ongoing ARLG study aims to better understand the impact of fluoroquinolone-resistant gram-negative gut bacteria in neutropenic patients, which threatens to undermine the effectiveness of fluoroquinolone prophylaxis in these vulnerable patients. The ARLG has conducted pharmacokinetic studies to inform the optimal dosing of antibiotics that are important in the treatment of drug-resistant gram-negative bacteria, including oral fosfomycin, intravenous minocycline, and a combination of intravenous ceftazidime-avibactam and aztreonam. In addition, randomized clinical trials have assessed the safety and efficacy of step-down oral fosfomycin for complicated urinary tract infections and single-dose intravenous phage therapy for adult patients with cystic fibrosis who are chronically colonized with P. aeruginosa in their respiratory tract. Thus, the focus of investigation in the ARLG has evolved from improving understanding of drug-resistant gram-negative bacterial infections to positively affecting clinical care for affected patients through a combination of interventional pharmacokinetic and clinical studies, a focus that will be maintained moving forward.

Development of an antibody fused with an antimicrobial peptide targeting Pseudomonas aeruginosa: A new approach to prevent and treat bacterial infections.

The increase in emerging drug resistant Gram-negative bacterial infections is a global concern. In addition, there is growing recognition that compromising the microbiota through the use of broad-spectrum antibiotics can impact long term patient outcomes. Therefore, there is the need to develop new bactericidal strategies to combat Gram-negative infections that would address these specific issues. In this study, we report and characterize one such approach, an antibody-drug conjugate (ADC) that combines (i) targeting the surface of a specific pathogenic organism through a monoclonal antibody with (ii) the high killing activity of an antimicrobial peptide. We focused on a major pathogenic Gram-negative bacterium associated with antibacterial resistance: Pseudomonas aeruginosa. To target this organism, we designed an ADC by fusing an antimicrobial peptide to the C-terminal end of the VH and/or VL-chain of a monoclonal antibody, VSX, that targets the core of P. aeruginosa lipopolysaccharide. This ADC demonstrates appropriately minimal levels of toxicity against mammalian cells, rapidly kills P. aeruginosa strains, and protects mice from P. aeruginosa lung infection when administered therapeutically. Furthermore, we found that the ADC was synergistic with several classes of antibiotics. This approach described in this study might result in a broadly useful strategy for targeting specific pathogenic microorganisms without further augmenting antibiotic resistance.

Interactions of polymyxin B1 with the gram-negative inner membrane: A simulation study.

The rise of drug resistant Gram-negative bacterial infections in recent decades has led to increased use of polymyxins, a class of antimicrobial peptides, as a ‘last resort’ treatment. In addition, the recent emergence of polymyxin-resistant strains threatens to bring about an era in which bacteria are resistant to all known antibiotics. It is thus necessary to develop new polymyxin-based antibiotics, but a lack of understanding of how polymyxins work at the molecular level may hinder such efforts. One question that remains open is how do polymyxins interact with the Gram-negative inner membrane to cause cell lysis? It has recently been suggested that polymyxins may target small quantities of the complex glycolipid lipopolysaccharide (LPS) that are present in the inner membrane prior to LPS transport to the outer membrane. In this work, we used a multiscale molecular dynamics (MD) approach including long-timescale, coarse-grained (CG) and atomistic simulations to investigate the interactions of polymyxin B1 (PMB1) with bacterial inner membrane models in the presence of small quantities of LPS. We observed that PMB1 is inserted into the inner membrane and localises at the LPS-phospholipid interface, sampling a wide range of interfacial environments. Our CG simulations also indicate that PMB1-LPS interactions are long-lived and numerous. In the presence of membrane proteins, a small number of PMB1 molecules were observed to form interactions with native inner membrane proteins, reducing the overall probability of PMB1 molecules remaining bound to LPS. Overall, this work indicates that PMB1 can target LPS molecules in the inner membrane, supporting recent hypotheses on its mode of action, while also providing new insights into the effect of membrane proteins upon PMB1-inner membrane interactions.

Chinese consensus guidelines for therapeutic drug monitoring of polymyxin B, endorsed by the Infection and Chemotherapy Committee of the Shanghai Medical Association and the Therapeutic Drug Monitoring Committee of the Chinese Pharmacological Society.

Polymyxin B, which is a last-line antibiotic for extensively drug-resistant Gram-negative bacterial infections, became available in China in Dec. 2017. As dose adjustments are based solely on clinical experience of risk toxicity, treatment failure, and emergence of resistance, there is an urgent clinical need to perform therapeutic drug monitoring (TDM) to optimize the use of polymyxin B. It is thus necessary to standardize operating procedures to ensure the accuracy of TDM and provide evidence for their rational use. We report a consensus on TDM guidelines for polymyxin B, as endorsed by the Infection and Chemotherapy Committee of the Shanghai Medical Association and the Therapeutic Drug Monitoring Committee of the Chinese Pharmacological Society. The consensus panel was composed of clinicians, pharmacists, and microbiologists from different provinces in China and Australia who made recommendations regarding target concentrations, sample collection, reporting, and explanation of TDM results. The guidelines provide the first-ever consensus on conducting TDM of polymyxin B, and are intended to guide optimal clinical use.

Detection of E. coli IncX1 Plasmid-Mediated mcr-5.1 Gene in an Indian Hospital Sewage Water Using Shotgun Metagenomic Sequencing: A First Report.

Colistin is used against a multitude of multidrug-resistant and extremely drug-resistant Gram-negative bacterial infections. The emergence of colistin resistance is highly concerning as it may lead to the failure of this last-resort antibiotic. Since the identification of first mobile colistin resistance (mcr) genes, several variants of mcr genes have been reported, but still there are limited studies detecting mcr genes in hospital sewage water. The prevalence of mcr in the hospital environment is extremely hazardous putting health care workers, patients, and visitors at a higher risk of exposure. It may lead to a multidrug-resistant bacterial infection outbreak. In this study, we report mcr-5.1 gene in an Indian hospital sewage water using shotgun metagenomics, as a first report. The mcr-5.1 gene in the metagenome has been explored using RGI, ABRicate, NCBI database, CARD, and Resfinder. This mcr-5.1 gene harbored by Escherichia coli is a plasmid-mediated gene carried by an IncX1 plasmid pSGMCR103. The bioinformatics analysis revealed the genetic environment of mcr-5.1 gene, which consisted of mobile element protein, ChrB domain protein, putative major facilitator superfamily type transporter, and a hypothetical protein.

Prevalence of carbapenemase-producing Klebsiella pneumoniae at a tertiary care hospital in Kathmandu, Nepal

AimAlthough carbapenem is the last-resort drug for treating drug-resistant Gram-negative bacterial infections, prevalence of carbapenem-resistant bacteria has substantially increased worldwide owing to irrational use of antibiotics particularly in developing countries like Nepal. Therefore, this study was aimed to determine the prevalence of carbapenemase-producing K. pneumoniae and to detect the carbapenemase genes (blaNDM-2 and blaOXA-48) in at a tertiary care hospital in Nepal.Materials and methodsA hospital-based cross-sectional study was carried out from June 2018 to January 2019 at the Microbiology Laboratory of Annapurna Neurological Institute and Allied Sciences, Kathmandu, Nepal. Different clinical samples were collected and cultured in appropriate growth media. Biochemical tests were performed for the identification of K. pneumoniae. Antibiotic susceptibility testing (AST) was performed by the Kirby–Bauer disc diffusion method. The modified Hodge test (MHT) was performed to detect carbapenemase producers. The plasmid was extracted by the modified alkaline hydrolysis method. Carbapenemase-producing K. pneumoniae were further confirmed by detecting blaNDM-2 and blaOXA-48 genes by PCR using specific forward and reverse primers followed by gel electrophoresis.ResultsOut of the total 720 samples, 38.9% (280/720) were culture positive. K. pneumoniae was the most predominant isolate 31.4% (88/280). Of 88 K. pneumoniae isolates, 56.8% (50/88) were multi-drug resistant (MDR), and 51.1% (45/88) were MHT positive. Colistin showed the highest sensitivity (100%; 88/88), followed by tigecycline (86.4%; 76/88). blaNDM-2 and blaOXA-48 genes were detected in 24.4% (11/45) and 15.5% (7/45) of carbapenemase-producing K. pneumoniae isolates, respectively.ConclusionThe rate of MDR and carbapenemase production was high in the K. pneumoniae isolates. Colistin and tigecycline could be the drug of choice for the empirical treatments of MDR and carbapenemase-producing K. pneumoniae. Our study provides a better understanding of antibiotic resistance threat and enables physicians to select the most appropriate antibiotics.

The effects of vitamin E on colistin-induced nephrotoxicity in treatment of drug-resistant gram-negative bacterial infections: A randomized clinical trial

IntroductionNephrotoxicity remains a major long-standing concern for colistin, and it is critical to find agents that can prevent it. The present study aims to investigate the effect of vitamin E on the prevention of colistin-induced nephrotoxicity based on its antioxidant and free radical scavenging properties. MethodsA randomized clinical trial was designed for 52 patients taking colistin. These patients were categorized into two groups of equal size, receiving colistin or colistin plus vitamin E (α-Tocopherol). Vitamin E with doses of 400 units was administrated daily either orally or by a nasogastric tube if needed. The incidence of Acute Kidney Injury (AKI) and its duration was recorded based on RIFLE criteria. ResultsThe Incidence of AKI based on RIFLE criteria was 42.3% and 46.2% in intervention and control groups, respectively. The analysis showed no significant difference in the prevalence of AKI for the two groups (P = 0.78). There was no significant difference in the duration of AKI neither (P = 0.83). ConclusionAlthough vitamin E is a powerful biological antioxidant, the effects of Vitamin E prophylaxis on colistin-induced nephrotoxicity was not taken into consideration in this study.

Carbapenem Consumption and Rate of carbapenemresistant gram-negative bacteria: results from the Sicilian Surveillance System.

The results of the present study highlight the need for comprehensive strategies targeting the appropriate use of antibiotics and infection control measures. In the last decades, multidrug-resistant gram-negative bacteria have been increasing and they are involved in severe healthcare associated infections. In treating drug-resistant gram-negative bacterial infections, carbapenems are generally administered as a last choice. However, the rate of carbapenemresistant bacteria is constantly increasing the last years. The aim of the present study is to describe the relationship between the rate of carbapenem-resistant gram-negative bacteria and antibiotic consumption intensity. In 2017, the Sicilian Region implemented a regional surveillance system to describe and analyze consumption of antibiotics in hospital settings, as well as prevalence of antibiotic resistant microorganisms. Resistance data were retrospectively collected from routinary clinical antimicrobial susceptibility tests. Resistance rates (RRs) of carbapenems were calculated and Spearman's correlation analysis was performed to investigate the relationship between annual antibiotic consumption and rate of carbapenem resistance. In 2015, the overall prevalence of carbapenem-resistant Klebsiella pneumoniae isolates was 43.9 %, and was positively correlated with beta-lactam consumption (R=0.529, p<0.01), and with carbapenem consumption (R=0.364, p<0.05). In 2016, the overall prevalence of carbapenem-resistant Klebsiella pneumoniae isolates was 53.8 %, and was positively correlated with carbapenem antibiotic consumption (R=0.364, p<0.05). In 2017, the overall prevalence of carbapenem-resistant Klebsiella pneumoniae isolates was 58.7 %, and was significantly positively correlated with carbapenem antibiotic consumption (R=0.427, p<0.05). In 2015, the overall prevalence of carbapenem-resistant Escherichia coli isolates was 6.5 %, and was significantly positively correlated with antibiotic consumption for the ATC class J01 (i.e., antibacterial for systemic use) (R=0.402, p<0.05).

Molecular detection of the New Delhi metallo-β-lactamase clinical variant with double mutation- V88L and M154L in Escherichia coli isolates from South India

Carbapenem is usually prescribed as a last resort of antibiotic for treating drug resistant Gram-negative bacterial infections. However, the rise in carbapenem resistance due to the emergence of Zn2+ dependent New Delhi metallo-β lactamase (NDM) variants imposes a huge threat to global health. In Indian context, focusing on molecular epidemiology and underpinning the genetic insights of such continuously evolving superbug is perquisite to guide any form of control strategies or therapeutic interventions. Herein, we investigated the occurrence of NDM variants in Escherichia coli (n = 70) isolated from clinical samples. PCR assay targeting blaNDM gene detected 5 E. coli isolates as NDM positive. Sequencing analyses of blaNDM region of the plasmid DNA determined two types of variants viz., NDM-1 (n = 3) and NDM-5 (n = 2). Notably, NDM-5 variants harbored V88L and M154L mutations, which are known to increase the fitness of NDM-5 variant towards host immune system and zinc starvation. On further characterization, three NDM variants showed the ESBL and AmpC β-lactamase genes, along with 3 different plasmid replicon types (L/M, Y, FIC). Virulence profiling showed traT gene as the most predominant gene among the variants. The emergence of NDM variants especially NDM-5 like variants with critical mutations in enterobacteriaceae increases the significant menaces to the global healthcare. The fact that these NDM variants harboring other antibiotic resistance markers, along with plasmid replicons and virulence genes can be transmitted underlines the gravity of this public health issue.

Cefiderocol for Extensively Drug-Resistant Gram-Negative Bacterial Infections: Real-world Experience From a Case Series and Review of the Literature.

Cefiderocol is a new siderophore cephalosporin with activity against carbapenem-resistant gram-negative bacteria. Data on its clinical efficacy are limited to complicated urinary tract infections. We present a series of 3 patients successfully treated with cefiderocol for complicated health care–associated infections and review published case reports.

Molecular epidemiology of mcr gene group

Colistin and polymyxin B are the “last reserve” antimicrobials for the treatment of extensively drug-resistant Gram-negative bacterial infections. The rapidly increasing prevalence of polymyxin resistance mediated by the mcr gene localized on plasmid DNA currently poses a high epidemiological threat. In order to control a distribution of mcr genes, it is necessary to develop highly accurate, highly sensitive and easy-to-use diagnostic tools. This paper provides a review of the most relevant studies on the molecular epidemiology as well as current approaches to microbiological and molecular detection of mcr group genes.

Intra-Vesical Colistin for Pseudomonas aeruginosa Urinary Tract Infections.

The case report is one of the few in the literature on the intravesical instillation of colistin to address lower urinary tract infection.Isolated intravesical colistin instillation was effective in a patient with anuria.

Exploiting a conjugative CRISPR/Cas9 system to eliminate plasmid harbouring the mcr-1 gene from Escherichia coli

The transfer of multi-drug-resistance plasmids by bacterial conjugation is largely responsible for the development of drug resistance in bacteria, and causes serious problems in the treatment of infectious diseases. Since the first discovery of plasmid-borne colistin resistance gene mcr-1 was reported in late 2016, this gene has been found in a great number of Escherichia coli and other Gram-negative pathogens separated from different types of sources worldwide. The elimination of plasmids carrying mcr-1 and restoration of polymyxin sensitivity has very important clinical significance because polymyxins are frequently used as last-resort antibiotics to treat extensively drug-resistant Gram-negative bacterial infections. A host-independent conjugative plasmid was constructed in this study, and an engineered CRISPR/Cas9 system was used to remove plasmid harbouring mcr-1 from bacteria. This study found that this conjugative plasmid can not only be used as a new tool to remove resistance plasmids and sensitize the recipient bacteria to antibiotics, but can also make the recipient cell acquire immunity against mcr-1. This strategy provides a novel method to counteract the ever-worsening spread of mcr-1 among bacterial pathogens.

Targeted Antibiotic Delivery: Selective Siderophore Conjugation with Daptomycin Confers Potent Activity against Multidrug Resistant Acinetobacter baumannii Both in Vitro and in Vivo.

In order to address the dire need for new antibiotics to treat specific strains of drug resistant Gram-negative bacterial infections, a mixed ligand analog of the natural Acinetobacter baumannii selective siderophore, fimsbactin, was coupled to daptomycin, a Gram-positive only antibiotic. The resulting conjugate 11 has potent activity against multidrug resistant strains of A. baumannii both in vitro and in vivo. The study also indicates that conjugation of siderophores to "drugs" that are much larger than the siderophore (iron transport agent) itself facilitates active uptake that circumvents the normal permeability problems in Gram-negative bacteria. The results demonstrate the ability to extend activity of a normally Gram-positive only antibiotic to create a potent and targeted Gram-negative antibiotic using a bacterial iron transport based sideromycin Trojan horse strategy.

Predicting allosteric mutants that increase activity of a major antibiotic resistance enzyme.

The CTX-M family of beta lactamases mediate broad-spectrum antibiotic resistance and are present in the majority of drug-resistant Gram-negative bacterial infections worldwide. Allosteric mutations that increase catalytic rates of these drug resistance enzymes have been identified in clinical isolates but are challenging to predict prospectively. We have used molecular dynamics simulations to predict allosteric mutants increasing CTX-M9 drug resistance, experimentally testing top mutants using multiple antibiotics. Purified enzymes show an increase in catalytic rate and efficiency, while mutant crystal structures show no detectable changes from wild-type CTX-M9. We hypothesize that increased drug resistance results from changes in the conformational ensemble of an acyl intermediate in hydrolysis. Machine-learning analyses on the three top mutants identify changes to the binding-pocket conformational ensemble by which these allosteric mutations transmit their effect. These findings show how molecular simulation can predict how allosteric mutations alter active-site conformational equilibria to increase catalytic rates and thus resistance against common clinically used antibiotics.

Colistin efficacy in the treatment of multidrug-resistant and extremelydrug-resistant gram-negative bacterial infections.

Colistin is used as a salvage therapy for multidrug-resistant and extremely drug-resistant gram-negative bacterial infections. Our aim was to evaluate colistin efficiency and toxicity in the treatment of these resistant gram-negative bacterial infections. This is a retrospective study carried out in a tertiary care hospital during 2011-2013. Study data were collected from the medical records and consultations of the infectious diseases clinic. The study group included 158 patients with nosocomial infections and 136 (86.1%) of them were hospitalized in the ICU. Respiratory tract infections were the most commonly observed ones (n = 103, 65.2%). The most frequently isolated microorganism was Acinetobacter baumannii (72.2%). A total of 98 (62.0%) patients had clinical cure. There was no statistically significant difference between monotherapy (n = 3/6, 50.0%) and combination therapies (n = 95/152, 62.5%) according to clinical response. Underlying ultimately fatal disease, previous renal disease, and total parenteral nutrition were independent risk factors for poor clinical response. Nephrotoxicity developed in 80 (50.6%) patients and clinical cure was statistically unrelated with nephrotoxicity. Colistin may be used as an effective agent for multidrug-resistant and extremely drug-resistant gram-negative bacterial infections with close monitoring of renal functions, especially for older and critically ill patients.