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Thursday, December 23, 2010
The Moskowitz research program at MGHfC focuses on chronic airway infection and host-pathogen interactions in cystic fibrosis (CF), encompassing the fields of microbiology, biochemistry, immunology, epithelial biology, bacterial and eukaryotic genetics, and pulmonary medicine. The overall goal is to understand how the most common CF airway pathogen, Pseudomonas aeruginosa, establishes a chronic infection, and also to understand and prevent antibiotic resistance, a problem that complicates the treatment of CF airway infection. Such infections are strongly associated with progressive CF lung disease, the predominant cause of CF mortality. Antibiotics are key agents in the CF therapeutic arsenal. However, CF pathogens are notoriously difficult to eradicate because they develop marked resistance in response to repeated antibiotic exposure. Moreover, some P. aeruginosa resistance mechanisms such as altered bacterial permeability may be associated with cross-resistance (i.e., decreased efficacy of antibiotics in diverse mechanistic classes). These clinical and microbiological realities have shaped three long-term objectives: (1) to define mechanisms of antibiotic resistance in P. aeruginosa and determine those most relevant to CF lung infection and disease progression; (2) to determine how antibiotics and antibiotic resistance influence bacterial virulence and host-pathogen interactions; and (3) to develop therapeutic strategies, such as improved susceptibility testing, novel combinations of antibiotics, or resistance inhibitors, that may overcome or minimize these resistance mechanisms. As part of the translational research goal of this third objective, Dr. Moskowitz is involved in clinical trials of inhaled antibiotics to treat CF airway infection.
Resistance to polymxins (colistin and polymyxin B sulfate; collectively, Pm) is emerging as an important problem in CF and also in critical care medicine, where P. aeruginosa is strongly associated with ventilator-associated pneumonia. Pm belongs to a large family of antimicrobial peptides, including the defensins and alpha-helical peptides found in the white blood cells of vertebrates, which work by punching holes in the bacterial cell wall. Pm resistance mechanisms reduce bacterial permeability by modifying the cell wall, and thus may decrease susceptibility to host antimicrobial peptides as well as to other antibiotics.1 To identify mechanisms of Pm resistance in P. aeruginosa, Dr. Moskowitz and his collaborators are studying Pm-resistantstrains of P. aeruginosa isolated from CF patients treated with colistin, using a variety of state-of-the-art techniques such as whole genome sequencing. Current collaborators on this project include Drs. Niels Høiby and Helle Krogh Johansen (University of Copenhagen), Dr. Lisa Saiman (Columbia University), and Drs. Rajinder Kaul and Michael Jacobs (University of Washington).
Another project uses zebrafish embryos as a model of P. aeruginosa systemic infection to study the role of antibiotic resistance genes and other bacterial virulence determinants in host-pathogen interactions. The optically transparent and genetically tractable zebrafish embryo is being used as an in vivo model of systemic P. aeruginosa infection in the Moskowitz lab. Despite lacking adaptive immunity at this developmental stage, zebrafish embryos are highly resistant to P. aeruginosa infection, but as in humans, phagocyte depletion dramatically increases their susceptibility. We have shown that P. aeruginosa lacking a functional Type III secretion system (T3SS) has decreased virulence, which can be restored through phagocyte depletion.2 This suggests that the T3SS influences virulence through its effects on phagocytes. Neutrophils and macrophages in zebrafish embryos rapidly phagocytose and kill P. aeruginosa (Figure), suggesting that both cell types play a role in protection against infection. Our ongoing work takes advantage of the real-time visualization capabilities and genetic tractability of the zebrafish embryoinfection model to elucidate the molecular and cellular details of P. aeruginosa pathogenesis and to explore how the antibiotic resistance of this organism may be induced in vivo. Collaborators on this project include Dr. Lalita Ramakrishnan (University of Washington) and Dr. Simon Dove (Harvard Medical School).
A third laboratory project is to develop improved methods for detection of antibiotic resistance (susceptibility testing) for CF pathogens. In previous work, Dr. Moskowitz and his collaborators developed and refined methods of biofilm susceptibility testing, and conducted a multi-center randomized controlled trial to examine the microbiological effectiveness of such testing compared to conventional testing.3 Current work focuses on developing more reliable methods of Pm susceptibility testing.4 Dr. Lisa Saiman (Columbia University) is a collaborator on this project. A related project involves in vitro pharmacokinetic (PK) and pharmacodynamic (PD) modeling of interactions between antibiotics and P. aeruginosa biofilms. Conventional susceptibility testing measure the ability of a fixed concentration of a single antibiotic to inhibit actively dividing bacteria. In reality, antibiotic treatment of CF airway infection exposes bacterial populations to multiple, concurrently administered drugs at concentrations that vary owing to distribution within and elimination from the body. In addition, P. aeruginosa in the CF airway form self-adherent biofilm communities with much greater antibiotic resistance than measured through conventional testing. The Moskowitz lab has developed an in vitro system in which P. aeruginosa grown as biofilms is exposed to one or more antibiotics at concentrations that vary or cycle over time. The purpose of this system is to model drug-pathogen interactions that occur when antibiotics are used to treat biofilm infection, and to mimic the appearance and disappearance of these drugs in the systemic circulation and at the site of action. Conditions such as bacterial inoculum, nutrient availability, and antibiotic dosing and clearance are adjusted within the in vitro system to simulate in vivo conditions based on actual patient data using patient bacterial samples. The long-term goal is to combine state-of-the-art biofilm culture methods, in vitroPK-PD modeling, and computer-based PK-PD modeling in order to predict antimicrobial and anti-biofilm effects of antibiotic dosing regimens. Current collaborators on the biofilm PK-PD project include Drs. Jane Burns (Seattle Children’s Hospital) and Dr. George Drusano (Ordway Research Institute, Albany, NY).
More information about these projects can be found on the Moskowitz lab website, at http://www2.massgeneral.org/MoskowitzLab/index.htm.
Figure. P. aeruginosa infection of a transgenic zebrafish embryo engineered so that the phagocytes express a green fluorescent protein (eGFP; bright in neutrophils, dull in macrophages). In these images, green phagocytes in the ventral tail of an embryo are seen engulfing bacterial cells that express a red fluorescent protein. (Photos by J. Muse Davis and Mark Brannon)
1. Moskowitz, S.M., R.K. Ernst, and S.I. Miller, PmrAB, a two-component regulatory system of Pseudomonas aeruginosa that modulates resistance to cationic antimicrobial peptides and addition of aminoarabinose to lipid A. J Bacteriol, 2004. 186(2): p. 575-9.2. Brannon, M.K., et al., Pseudomonas aeruginosa Type III secretion system interacts with phagocytes to modulate systemic infection of zebrafish embryos. Cell Microbiol, 2009. 11(5): p. 755-768.3. Moskowitz, S.M., et al., Randomized trial of biofilm testing to select antibiotics for cystic fibrosis airway infection. Pediatr Pulmonol, 2010.4. Moskowitz, S.M., et al., Colistin susceptibility testing: evaluation of reliability for cystic fibrosis isolates of Pseudomonas aeruginosa and Stenotrophomonas maltophilia. J Antimicrob Chemother, 2010. 65(7): p. 1416-23.
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