Amyes, Sebastian

Hamouda, Ahmed

Findlay, Jacqueline

2012-10-04T13:15:02Z

2012-10-04T13:15:02Z

2012-06-22

Klebsiella pneumoniae is a common cause of nosocomial and community-acquired infections, and the increasing incidence and prevalence of antibiotic resistant strains is proving to be particularly problematic to clinicians. K. pneumoniae is capable of employing a multitude of mechanisms by which to confer resistance to most available antibiotics. The carbapenem antibiotics are usually reserved for the treatment of complicated or multidrug resistant (MDR) K. pneumoniae infections. The recent emergence of not only MDR but also pan-drug resistant (PDR) K. pneumoniae strains has signified that it is now more important than ever to understand the mechanisms by which these strains confer resistance so that we may find ways to combat or hinder this progression. This project aimed to investigate the regulation of the transcriptional activator RamA, its ability to confer a MDR phenotype, and the mechanisms employed by K. pneumoniae to confer levels of carbapenem resistance sufficient to result in therapy failure. The analysis of a panel of K. pneumoniae strains, containing both RamA expressers and non-expressers, demonstrated that the overexpression of RamA was sufficient to confer an MDR phenotype. Two compounds, chlorpromazine (CPZ) and tigecycline, were shown to act as inducers of ramA, romA and acrA transcription. CPZ exhibited synergy with the antibiotics chloramphenicol, norfloxacin and tetracycline, all of which are known substrates of the AcrAB efflux pump. The current lack of novel classes of antimicrobials in development indicate a potential for a compound, such as CPZ, to be developed and exploited for clinical use. The ability of both CPZ and tigecycline to cause mutations within ramR however, indicate that both compounds may have the ability to select for efflux mutants as a result of their ability to upregulate ramA, which in turn causes the upregulation of the AcrAB efflux pump. The regulation of RamA by the upstream gene ramR, which encodes a TetR family protein was investigated in K. pneumoniae isolates. Sequencing of the ramR genes revealed that strains exhibiting an MDR phenotype commonly contained mutations within their gene sequences. The complementation of a wildtype ramR into a strain containing a 32 amino acid deletion within its ramR, was shown to increase susceptibility to various antibiotics of different classes, and additionally downregulate the expression of ramA, romA and acrA. CPZ, ciprofloxacin and tigecycline K. pneumoniae mutants were shown to exhibit increased MICs to a broad spectrum of antibiotics with respect to their parent strains, and possess mutations within their ramR genes. Complementation of the wildtype ramR resulted in partial reversion to the parental phenotypes, indicating another mechanism must also be involved in conferring the MDR phenotypes. These studies indicated that RamR plays an important role as a negative regulator of RamA, but also that it is not the sole regulator. The development of reduced susceptibility to the carbapenems was investigated in two clinical strains of K. pneumoniae, K1 and K2, isolated from the urine of a single patient at different stages of antibiotic therapy. The strains were shown to exhibit similar resistance phenotypes with the exception of their susceptibilities to the carbapenems. PCR and phenotypic analyses revealed that neither strain contained any carbapenemases or AmpC enzymes, but both contained OXA-1, SHV-1 TEM-1 and CTX-M-15. Analysis of their OMP profiles indicated that both strains lacked OmpK35, and K2 additionally lacked OmpK36. Mutation studies showed that the phenotype and OMP profile exhibited by K2 could be achieved in K1 via single step mutations using ertapenem, imipenem or meropenem. Susceptibility testing of CTXM- 15 clinical strains showed that strains containing CTX-M-15 showed reduced activity against ertapenem in the presence of clavulanic acid. These studies indicated a potential role for CTX-M-15 in conferring reduced susceptibility to the carbapenems when found in conjunction with altered permeability and active efflux. The mechanisms of antibiotic resistance employed by K. pneumoniae are numerous and complex. This work highlights several of these mechanisms and, more importantly, how they can work in synergy with one another to devastating consequences.

http://hdl.handle.net/1842/6473

en

The University of Edinburgh

Findlay J, Schneiders T, Amyes SGB. (2008) The effect of chlorpromazine on the multidrug resistance regulator RamA. The 48th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington DC, USA (25 – 28th October) Abstract no. 1943

Findlay J, Schneiders T. (2009) The differential effect of mutations in RamR, in mediating antibiotic susceptibility in Klebsiella pneumoniae. The 19th European Congress on Clinical Microbiology and Infectious Diseases, Helsinki, Finland (16 – 19th May) Abstract no. P1483

Findlay J, Schneiders T. (2009) RamR: A dual regulator of antibiotic susceptibility and biofilm formation in Klebsiella pneumoniae Kp342. The 19th European Congress on Clinical Microbiology and Infectious Diseases, Helsinki, Finland (16 – 19th May) Abstract no. O206

Findlay J, Hamouda A, Dancer S, Amyes SGB. (2010) Mechanisms of carbapenem resistance emerging during therapy in a strain of Klebsiella pneumoniae treated with meropenem. The 50th Interscience Conference on Antimicrobial Agents and Chemotherapy, Boston, USA (12 – 15th September) Abstract no. 2197

Evans BA, Hamouda A, Brown S, Findlay J, Amyes SGB. (2007) Eleven novel OXA-51-like enzymes from clinical isolates of Acinetobacter baumannii. Clinical Microbiology and Infection. 13: 1137-1138

Schneiders T, Findlay J, Amyes SGB. (2008) Efflux pumps in Acinetobacter baumannii. In Bergogne-Bérézin, E, Friedman H, Bendinelli M (Ed.) Acinetobacter biology and pathogenesis. (p105-127) Springer

Hamouda A, Findlay J, Al-Hassan L, Amyes SGB. (2010) The epidemiology of Acinetobacter baumannii of animal origin. 8th International Symposium on the Biology of Acinetobacter, Rome, Italy (1-3rd September) Abstract no. P33

Lopes BS, Hamouda A, Findlay J, Amyes SGB. (2011) The effect of frame-shift mutagen acriflavine on control of resistance genes in Acinetobacter baumannii. Journal of Medical Microbiology. 60: 211-215

Hamouda A, Findlay J, Amyes SGB. (2011) Carbapenems: do they have a future? Journal of Medical Microbiology. 60: 1229-1230

Hamouda A, Findlay J, Al-Hassan L, Amyes SGB. (2011) The epidemiology of Acinetobacter baumannii of animal origin. International Journal of Antimicrobial Agents. 38: 314-318

Klebsiella pneumoniae

antibiotic resistance

carbapenems

Klebsiella pneumoniae: a progression to multidrug resistance

Thesis or Dissertation

Doctoral

PhD Doctor of Philosophy