Postdoctoral Fellow, University of British Columbia / Microbiology and Immunology
Anti-biofilm peptides as antibiotic adjuvants to treat drug-resistant pathogens
One of the greatest health crises facing the world today are bacterial infections that have evolved resistance to commonly used clinical antibiotics; compounds generally targeting cellular processes. A promising approach to reduce selective pressure for resistance development are compounds that aim to disarm bacterial invaders rather than killing them. The stringent stress response has recently been identified as such a potential drug target. We have found that stringent response mutants of Pseudomonas aeruginosa PAO1 and LESB58 were viable in a cutaneous mouse abscess model of high-density, chronic infections, however, they led to significantly reduced abscesses and cutaneous lesions. Intriguingly, our experiments revealed that treatment of P. aeruginosa infections with anti-biofilm peptides, which promote degradation of the stringent response nucleotide mediator ppGpp, also significantly reduced abscess sizes while improving animal welfare. Conversely, a stringent response mutant complemented with relA (a ppGpp synthase) reverted back to wild-type phenotype and was able to withstand peptide treatment, providing evidence that the stringent stress response is indeed targeted in vivo. Next we investigated the potential of using synthetic peptides in combination with antibiotics to treat bacterial abscesses. Treatment of LESB58 infections with a combination of ciprofloxacin and peptide (1018/DJK-5) significantly decreased bacterial burden inside the abscess by more than 100-fold compared to saline-treated controls while stand-alone treatment caused only a 5- to 10-fold reduction. Our findings show that stringent response inhibitors are promising new therapeutic agents and work synergistically in vivo, which could dramatically reduce the use of prescribed antibiotics to treat high-density bacterial infections.
Abstract: Cutaneous abscess infections are difficult to treat with current therapies and alternatives to conventional antibiotics are needed. Understanding the regulatory mechanisms that govern abscess pathology should reveal therapeutic interventions for these recalcitrant infections. Here we demonstrated that the stringent stress response employed by bacteria to cope and adapt to environmental stressors was essential for the formation of lesions, but not bacterial growth, in a methicillin resistant Staphylococcus aureus (MRSA) cutaneous abscess mouse model. To pharmacologically confirm the role of the stringent response in abscess formation, a cationic peptide that causes rapid degradation of the stringent response mediator, guanosine tetraphosphate (ppGpp), was employed. The therapeutic application of this peptide strongly inhibited lesion formation in mice infected with Gram-positive MRSA and Gram-negative Pseudomonas aeruginosa. Overall, we provide insights into the mechanisms governing abscess formation and a paradigm for treating multidrug resistant cutaneous abscesses.
Pub.: 24 Sep '16, Pinned: 28 Jun '17
Abstract: The β-lactam antibiotic temocillin (6-α-methoxy-ticarcillin) shows stability to most extended spectrum β-lactamases, but is considered inactive against Pseudomonas aeruginosa. Mutations in the MexAB-OprM efflux system, naturally occurring in cystic fibrosis (CF) isolates, have been previously shown to reverse this intrinsic resistance. In the present study, we measured temocillin activity in a large collection (n = 333) of P. aeruginosa CF isolates. 29% of the isolates had MICs ≤ 16 mg/L (proposed clinical breakpoint for temocillin). Mutations were observed in mexA or mexB in isolates for which temocillin MIC was ≤512 mg/L (nucleotide insertions or deletions, premature termination, tandem repeat, nonstop, and missense mutations). A correlation was observed between temocillin MICs and efflux rate of N-phenyl-1-naphthylamine (MexAB-OprM fluorescent substrate) and extracellular exopolysaccharide abundance (contributing to a mucoid phenotype). OpdK or OpdF anion-specific porins expression decreased temocillin MIC by ~1 two-fold dilution only. Contrarily to the common assumption that temocillin is inactive on P. aeruginosa, we show here clinically-exploitable MICs on a non-negligible proportion of CF isolates, explained by a wide diversity of mutations in mexA and/or mexB. In a broader context, this work contributes to increase our understanding of MexAB-OprM functionality and help delineating how antibiotics interact with MexA and MexB.
Pub.: 17 Jan '17, Pinned: 28 Jun '17
Abstract: Only a few, relatively cumbersome animal models enable long-term Gram-negative bacterial infections that mimic human situations, where untreated infections can last for weeks. Here, we describe a simple murine cutaneous abscess model that enables chronic or progressive infections, depending on the subcutaneously injected bacterial strain. In this model, Pseudomonas aeruginosa cystic fibrosis epidemic isolate LESB58 caused localized high-density skin and soft tissue infections and necrotic skin lesions for up to 10 days but did not disseminate in either CD-1 or C57BL/6 mice. The model was adapted for use with four major Gram-negative nosocomial pathogens, Acinetobacter baumannii, Klebsiella pneumoniae, Enterobacter cloacae, and Escherichia coli This model enabled noninvasive imaging and tracking of lux-tagged bacteria, the influx of activated neutrophils, and production of reactive oxygen-nitrogen species at the infection site. Screening antimicrobials against high-density infections showed that local but not intravenous administration of gentamicin, ciprofloxacin, and meropenem significantly but incompletely reduced bacterial counts and superficial tissue dermonecrosis. Bacterial RNA isolated from the abscess tissue revealed that Pseudomonas genes involved in iron uptake, toxin production, surface lipopolysaccharide regulation, adherence, and lipase production were highly upregulated whereas phenazine production and expression of global activator gacA were downregulated. The model was validated for studying virulence using mutants of more-virulent P. aeruginosa strain PA14. Thus, mutants defective in flagella or motility, type III secretion, or siderophore biosynthesis were noninvasive and suppressed dermal necrosis in mice, while a strain with a mutation in the bfiS gene encoding a sensor kinase showed enhanced invasiveness and mortality in mice compared to controls infected with wild-type P. aeruginosa PA14.IMPORTANCE More than two-thirds of hospital infections are chronic or high-density biofilm infections and difficult to treat due to adaptive, multidrug resistance. Unfortunately, current models of chronic infection are technically challenging and difficult to track without sacrificing animals. Here we describe a model of chronic subcutaneous infection and abscess formation by medically important nosocomial Gram-negative pathogens that is simple and can be used for tracking infections by imaging, examining pathology and immune responses, testing antimicrobial treatments suitable for high-density bacterial infections, and studying virulence. We propose that this mouse model can be a game changer for modeling hard-to-treat Gram-negative bacterial chronic and skin infections.
Pub.: 02 Mar '17, Pinned: 28 Jun '17