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      Ca-EDTA restores the activity of ceftazidime-avibactam or aztreonam against carbapenemase-producing Klebsiella pneumoniae infections

      research-article
      1 , 2 , 3 , 4 , 5 , 6 , 47 , , 2 , 3 , 46 , 7 , 46 , 8 , 9 , 10 , ∗∗ , 11 , 12 , 4 , 13 , 14 , 15 , 16 , 17 , 18 , 2 , 3 , 2 , 3 , 2 , 3 , 2 , 3 , 2 , 3 , 2 , 3 , 19 , 20 , 6 , 21 , 22 , 23 , 24 , 25 , 2 , 26 , 6 , 27 , 28 , 29 , 30 , 31 , 24 , 32 , 33 , 34 , 2 , 31 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 46 , 42 , 43 , 44 , 46 , 42 , 43 , 44 , 45 , ∗∗∗ , 2 , 3 , ∗∗∗∗ , 5 , 6 , ∗∗∗∗∗
      iScience
      Elsevier
      Health sciences, Medicine, Medical specialty, Immunology, Medical microbiology, Biological sciences, Microbiology

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          Summary

          Developing an effective therapy to overcome carbapenemase-positive Klebsiella pneumoniae (CPKp) is an important therapeutic challenge that must be addressed urgently. Here, we explored a Ca-EDTA combination with aztreonam or ceftazidime-avibactam in vitro and in vivo against diverse CPKp clinical isolates. The synergy testing of this study demonstrated that novel aztreonam-Ca-EDTA or ceftazidime-avibactam-Ca-EDTA combination was significantly effective in eliminating planktonic and mature biofilms in vitro, as well as eradicating CPKp infections in vivo. Both combinations revealed significant therapeutic efficacies in reducing bacterial load in internal organs and protecting treated mice from mortality. Conclusively, this is the first in vitro and in vivo study to demonstrate that novel aztreonam-Ca-EDTA or ceftazidime-avibactam-Ca-EDTA combinations provide favorable efficacy and safety for successful eradication of carbapenemase-producing Klebsiella pneumoniae planktonic and biofilm infections.

          Graphical abstract

          Highlights

          • Carbapenemase-positive Klebsiella pneumoniae (CPKp) possess therapeutic challenges

          • Ca-EDTA paired with aztreonam/ceftazidime-avibactam can eliminate CPKp in vitro

          • These combinations reduce bacterial load, but they also protect mice from mortality

          • Promising efficacy and safety have been demonstrated with these combinations

          Abstract

          Health sciences; Medicine; Medical specialty; Immunology; Medical microbiology; Biological sciences; Microbiology

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          Most cited references40

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          Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis

          (2022)
          Summary Background Antimicrobial resistance (AMR) poses a major threat to human health around the world. Previous publications have estimated the effect of AMR on incidence, deaths, hospital length of stay, and health-care costs for specific pathogen–drug combinations in select locations. To our knowledge, this study presents the most comprehensive estimates of AMR burden to date. Methods We estimated deaths and disability-adjusted life-years (DALYs) attributable to and associated with bacterial AMR for 23 pathogens and 88 pathogen–drug combinations in 204 countries and territories in 2019. We obtained data from systematic literature reviews, hospital systems, surveillance systems, and other sources, covering 471 million individual records or isolates and 7585 study-location-years. We used predictive statistical modelling to produce estimates of AMR burden for all locations, including for locations with no data. Our approach can be divided into five broad components: number of deaths where infection played a role, proportion of infectious deaths attributable to a given infectious syndrome, proportion of infectious syndrome deaths attributable to a given pathogen, the percentage of a given pathogen resistant to an antibiotic of interest, and the excess risk of death or duration of an infection associated with this resistance. Using these components, we estimated disease burden based on two counterfactuals: deaths attributable to AMR (based on an alternative scenario in which all drug-resistant infections were replaced by drug-susceptible infections), and deaths associated with AMR (based on an alternative scenario in which all drug-resistant infections were replaced by no infection). We generated 95% uncertainty intervals (UIs) for final estimates as the 25th and 975th ordered values across 1000 posterior draws, and models were cross-validated for out-of-sample predictive validity. We present final estimates aggregated to the global and regional level. Findings On the basis of our predictive statistical models, there were an estimated 4·95 million (3·62–6·57) deaths associated with bacterial AMR in 2019, including 1·27 million (95% UI 0·911–1·71) deaths attributable to bacterial AMR. At the regional level, we estimated the all-age death rate attributable to resistance to be highest in western sub-Saharan Africa, at 27·3 deaths per 100 000 (20·9–35·3), and lowest in Australasia, at 6·5 deaths (4·3–9·4) per 100 000. Lower respiratory infections accounted for more than 1·5 million deaths associated with resistance in 2019, making it the most burdensome infectious syndrome. The six leading pathogens for deaths associated with resistance (Escherichia coli, followed by Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa) were responsible for 929 000 (660 000–1 270 000) deaths attributable to AMR and 3·57 million (2·62–4·78) deaths associated with AMR in 2019. One pathogen–drug combination, meticillin-resistant S aureus, caused more than 100 000 deaths attributable to AMR in 2019, while six more each caused 50 000–100 000 deaths: multidrug-resistant excluding extensively drug-resistant tuberculosis, third-generation cephalosporin-resistant E coli, carbapenem-resistant A baumannii, fluoroquinolone-resistant E coli, carbapenem-resistant K pneumoniae, and third-generation cephalosporin-resistant K pneumoniae. Interpretation To our knowledge, this study provides the first comprehensive assessment of the global burden of AMR, as well as an evaluation of the availability of data. AMR is a leading cause of death around the world, with the highest burdens in low-resource settings. Understanding the burden of AMR and the leading pathogen–drug combinations contributing to it is crucial to making informed and location-specific policy decisions, particularly about infection prevention and control programmes, access to essential antibiotics, and research and development of new vaccines and antibiotics. There are serious data gaps in many low-income settings, emphasising the need to expand microbiology laboratory capacity and data collection systems to improve our understanding of this important human health threat. Funding Bill & Melinda Gates Foundation, Wellcome Trust, and Department of Health and Social Care using UK aid funding managed by the Fleming Fund.
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            Microtiter dish biofilm formation assay.

            Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings. In fact, life in a biofilm probably represents the predominate mode of growth for microbes in most environments. Mature biofilms have a few distinct characteristics. Biofilm microbes are typically surrounded by an extracellular matrix that provides structure and protection to the community. Microbes growing in a biofilm also have a characteristic architecture generally comprised of macrocolonies (containing thousands of cells) surrounded by fluid-filled channels. Biofilm-grown microbes are also notorious for their resistance to a range of antimicrobial agents including clinically relevant antibiotics. The microtiter dish assay is an important tool for the study of the early stages in biofilm formation, and has been applied primarily for the study of bacterial biofilms, although this assay has also been used to study fungal biofilm formation. Because this assay uses static, batch-growth conditions, it does not allow for the formation of the mature biofilms typically associated with flow cell systems. However, the assay has been effective at identifying many factors required for initiation of biofilm formation (i.e, flagella, pili, adhesins, enzymes involved in cyclic-di-GMP binding and metabolism) and well as genes involved in extracellular polysaccharide production. Furthermore, published work indicates that biofilms grown in microtiter dishes do develop some properties of mature biofilms, such a antibiotic tolerance and resistance to immune system effectors. This simple microtiter dish assay allows for the formation of a biofilm on the wall and/or bottom of a microtiter dish. The high throughput nature of the assay makes it useful for genetic screens, as well as testing biofilm formation by multiple strains under various growth conditions. Variants of this assay have been used to assess early biofilm formation for a wide variety of microbes, including but not limited to, pseudomonads, Vibrio cholerae, Escherichia coli, staphylococci, enterococci, mycobacteria and fungi. In the protocol described here, we will focus on the use of this assay to study biofilm formation by the model organism Pseudomonas aeruginosa. In this assay, the extent of biofilm formation is measured using the dye crystal violet (CV). However, a number of other colorimetric and metabolic stains have been reported for the quantification of biofilm formation using the microtiter plate assay. The ease, low cost and flexibility of the microtiter plate assay has made it a critical tool for the study of biofilms.
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              Agents that increase the permeability of the outer membrane.

              The outer membrane of gram-negative bacteria provides the cell with an effective permeability barrier against external noxious agents, including antibiotics, but is itself a target for antibacterial agents such as polycations and chelators. Both groups of agents weaken the molecular interactions of the lipopolysaccharide constituent of the outer membrane. Various polycations are able, at least under certain conditions, to bind to the anionic sites of lipopolysaccharide. Many of these disorganize and cross the outer membrane and render it permeable to drugs which permeate the intact membrane very poorly. These polycations include polymyxins and their derivatives, protamine, polymers of basic amino acids, compound 48/80, insect cecropins, reptilian magainins, various cationic leukocyte peptides (defensins, bactenecins, bactericidal/permeability-increasing protein, and others), aminoglycosides, and many more. However, the cationic character is not the sole determinant required for the permeabilizing activity, and therefore some of the agents are much more effective permeabilizers than others. They are useful tools in studies in which the poor permeability of the outer membrane poses problems. Some of them undoubtedly have a role as natural antibiotic substances, and they or their derivatives might have some potential as pharmaceutical agents in antibacterial therapy as well. Also, chelators (such as EDTA, nitrilotriacetic acid, and sodium hexametaphosphate), which disintegrate the outer membrane by removing Mg2+ and Ca2+, are effective and valuable permeabilizers.
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                Author and article information

                Contributors
                Journal
                iScience
                iScience
                iScience
                Elsevier
                2589-0042
                28 June 2023
                21 July 2023
                28 June 2023
                : 26
                : 7
                : 107215
                Affiliations
                [1 ]Department of Infectious Diseases and Infection Control, Yamagata Prefectural Central Hospital, Yamagata, Japan
                [2 ]Department of Microbiology, Faculty of Medicine, Chulalongkorn University, King Chulalongkorn Memorial Hospital, Thai Red Cross Society, Bangkok, Thailand
                [3 ]Center of Excellence in Antimicrobial Resistance and Stewardship Research, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
                [4 ]School of Medicine, Faculty of Health and Medical Sciences, The University of Western Australia, Nedlands, WA, Australia
                [5 ]Biofilms and Antimicrobial Resistance Consortium of ODA Receiving Countries, The University of Sheffield, Sheffield, UK
                [6 ]Pathogen Hunter’s Research Team, Department of Infectious Diseases and Infection Control, Yamagata Prefectural Central Hospital, Yamagata, Japan
                [7 ]Molly Wardaguga Research Centre, Charles Darwin University, Brisbane, QLD, Australia
                [8 ]Department of Infection, Immunity & Cardiovascular Disease, University of Sheffield Medical School, UK
                [9 ]Mae Fah Luang University Hospital, Chiang Rai, Thailand
                [10 ]School of Integrative Medicine, Mae Fah Luang University, Chiang Rai, Thailand
                [11 ]Department of Emergency Medicine, Center of Excellence, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand
                [12 ]Institute of Clinical Sciences, Department of Surgery, Sahlgrenska Academy, Gothenburg University, 40530 Gothenburg, Sweden
                [13 ]Department of Biology, Faculty of Science, Mahidol University, Bangkok, Thailand
                [14 ]Department of Computer Science, Faculty of Science, Kasetsart University, Bangkok, Thailand
                [15 ]Center of Excellence in Systems Biology, Research Affairs, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
                [16 ]Department of Biochemistry, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
                [17 ]Department of Clinical Science, University of Bergen, Bergen, Norway
                [18 ]Department of Clinical Microbiology and Applied Technology, Faculty of Medical Technology, Mahidol University, Bangkok, Thailand
                [19 ]Department of Physiology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
                [20 ]Center of Excellence for Microcirculation, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
                [21 ]Department of Dermatology. Faculty of Medicine Siriraj Hospital. Mahidol University, Bangkok, Thailand
                [22 ]Center of Excellence in Materials and Bio-Interfaces, Faculty of Science, Chulalongkorn University, Bangkok, Thailand
                [23 ]Department of Clinical Sciences and Public Health, Faculty of Veterinary Science, Mahidol University, Nakhon Pathom, Thailand
                [24 ]Division of Nephrology, Department of Medicine, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
                [25 ]Division of General Internal Medicine-Nephrology Department of Medicine, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
                [26 ]Translational Research in Inflammation and Immunology Research Unit (TRIRU), Department of Microbiology, Chulalongkorn University, Bangkok, Thailand
                [27 ]Department of Chemical and Biological Engineering, The University of Sheffield, Sheffield, UK
                [28 ]Department of Clinical Microbiology, Christian Medical College, Vellore, India
                [29 ]School of Pharmacy & Technology Management, SVKM’s Narsee Monjee Institute of Management Studies (NMIMS), Hyderabad 509301, India
                [30 ]Department of Microbiology and Immunology, University of Otago, Dunedin, Otago 9010, New Zealand
                [31 ]Center of Excellence in Immunology and Immune-Mediated Diseases, Chulalongkorn University, Bangkok 10330, Thailand
                [32 ]Center of Excellence in Kidney Metabolic Disorders, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
                [33 ]Dialysis Policy and Practice Program (DiP3), School of Global Health, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
                [34 ]Peritoneal Dialysis Excellence Center, King Chulalongkorn Memorial Hospital, Bangkok, Thailand
                [35 ]Office of Research Affairs, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
                [36 ]Division of Bacteriology, School of Medicine, Jichi Medical University, Tochigi, Japan
                [37 ]Laboratory of Environmental Hygiene, Department of Health Science, School of Allied Health Sciences, Kitasato University, Kitasato, Sagamihara-Minami, Kanagawa 252-0373, Japan
                [38 ]Yamagata Prefectural University of Health Sciences, Kamiyanagi, Yamagata 990-2212, Japan
                [39 ]Institute for Medical Microbiology, Immunology and Hygiene, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany
                [40 ]German Centre for Infection Research, Partner site Bonn-Cologne, Cologne, Germany
                [41 ]Center for Molecular Medicine Cologne, Faculty of Medicine and University Hospital Cologne, University of Cologne, 50935 Cologne, Germany
                [42 ]Telethon Kids Institute, University of Western Australia, Nedlands, WA 6009, Australia
                [43 ]Centre for Cell Therapy and Regenerative Medicine, Medical School, The University of Western Australia, Nedlands, WA 6009, Australia
                [44 ]Department of Respiratory and Sleep Medicine, Perth Children’s Hospital, Nedlands, WA 6009, Australia
                [45 ]School of Public Health, Curtin University, Bentley, WA 6102, Australia
                Author notes
                []Corresponding author dhammika.l@ 123456chula.ac.th
                [∗∗ ]Corresponding author parichart.hon@ 123456mfu.ac.th
                [∗∗∗ ]Corresponding author anthony.kicic@ 123456telethonkids.org.au
                [∗∗∗∗ ]Corresponding author tanittha.c@ 123456chula.ac.th
                [∗∗∗∗∗ ]Corresponding author abeshu@ 123456icloud.com
                [46]

                These authors contributed equally

                [47]

                Lead contact

                Article
                S2589-0042(23)01292-0 107215
                10.1016/j.isci.2023.107215
                10366478
                37496674
                63fb671a-3078-4164-acbf-cc6b73868b9a
                © 2023 The Author(s)

                This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

                History
                : 11 April 2023
                : 12 May 2023
                : 22 June 2023
                Categories
                Article

                health sciences,medicine,medical specialty,immunology,medical microbiology,biological sciences,microbiology

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