Pharmacodynamics of Polymyxins and Their Combinations with Carbapenems against Multidrug-Resistant Klebsiella pneumoniae

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Emergence of antibiotic-resistant bacteria and dearth of new chemical agents in the antibiotics development pipeline present a major medical challenge. The Infectious Diseases Society of America (IDSA) launched the “Bad Bugs, No Drugs” campaign in 2004 to bring attention to the USA policy makers on this unmet medical need. In the 2000s, the global spread of Klebsiella pneumoniae carbapenemase and New Delhi metallo-β-lactamase-producing Enterobacteriaceae (mainly K. pneumoniae) significantly further reduced the choice of antibiotics in the clinic as these pathogens are usually resistant to almost all clinically available antibiotics except polymyxins. Polymyxins were discovered more than fifty years ago and had never been subjected to modern drug discovery procedures until recently. Two polymyxins are available for clinical use, polymyxin B and colistin (polymyxin E). Polymyxins were ignored from clinical practice from the 1970s due to toxicity and availability of ‘safer’ antibiotics. With no new antibiotic candidate against Gram-negative bacteria in near future, there is an urgent need to optimize the use and understand the mechanism of activity of polymyxins to prolong its therapeutic utility. In the first experimental chapter of this thesis, the antibacterial activity of colistin-doripenem combination regimens against MDR K. pneumoniae was examined in an in vitro one-compartment pharmacokinetic/pharmacodynamic (PK/PD) model. The colistin-doripenem combination at clinically achievable concentrations substantially increased bacterial killing against colistin-susceptible and -heteroresistant isolates at both low and high initial inocula. Emergence of colistin-resistant subpopulations in colistin-susceptible and -heteroresistant isolates was generally eliminated by combination regimens. In the second experimental chapter of the thesis, the mode of action of polymyxins was investigated. The activity of polymyxins and their analogues were examined for their ability to inhibit the type II NADH-quinone oxidoreductases (NDH-2) in the respiratory chains of Gram-negative bacteria. Polymyxin B and colistin inhibited the NDH-2 activity in a concentration-dependent manner using inner membrane preparations of K. pneumoniae (colistin-susceptible and resistant variants), Escherichia coli and Acinetobacter baumannii. These findings suggest that a novel secondary mode of action of polymyxins involves the inhibition of bacterial respiratory enzymes in the Gram-negative bacterial inner membrane. In the third experimental chapter, the surface components of polymyxin-susceptible and resistant variants of K. pneumoniae were examined by a number of biophysical tests. Comparing to the polymyxin-susceptible parent strain, the polymyxin-resistant variant displayed lower negative surface charges, greater outer membrane permeability and less sensitivity to the lytic action of lysozyme and sodium deoxycholate after colistin exposure. The binding affinity of polymyxin B and colistin to LPS purified from wild type was higher than the binding to LPS from the resistant variant. Taken together, a secondary mechanism of polymyxin resistance is believed due to diminished initial electrostatic contacts with the outer membrane that led to reduced killing activity. In the fourth experimental chapter of this thesis, the uptake of polymyxins by live K. pneumoniae cells was observed under time-lapse laser scanning confocal microscopy using a novel polymyxin-dansyl probe that possessed native antibacterial activity. The polymyxin probe initially accumulated in the outer membrane and subsequently penetrated the inner membrane and finally entered into the cytoplasm. These findings indicated this platform can be employed for the discovery of novel polymyxin-like lipopeptides with efficacy against polymyxin-resistant strains. Lastly, colistin-doripenem combinations were examined in a non-neutropaenic K. pneumoniae bacteremic mouse model to simultaneously examine bactericidal and endotoxin neutralization effects. Beside the susceptible reference isolate, the efficacy of colistin-carbapenem combination was evaluated for the first time against globally disseminated NDM-1-producer carbapenem-resistant isolate in an animal model. The combination therapy resulted in lower bacterial counts against both isolates, compared to colistin or doripenem monotherapy. Significant lower endotoxin level was observed in mice treated with colistin-doripenem combination therapy against the NDM-1-producer, compared to the control or any monotherapy groups. Against the NDM-1-producer this combination therapy led to significant lower TNF levels compared to the untreated control. These findings demonstrated that the colistin-doripenem combination is useful for the treatment of sepsis caused by NDM-1-producing K. pneumoniae. In summary, this thesis provides novel information on the optimal use and the mechanism of activity of polymyxins against K. pneumoniae. Colistin-doripenem combination was synergistic and able to suppress the emergence of polymyxin resistance in MDR K. pneumoniae infections. In non-neutropenic bacteraemic mice caused by an NDM-1-producer carbapenem-resistant K. pneumoniae, a significant reduction was evident in the bacterial load and endotoxin activity after treatment with colistin-doripenem combination. This study provides important information on the strategies to maximise the efficacy of polymyxins. This thesis is also the first to report the inhibition of NDH-2 respiratory enzymes in Gram-negative by polymyxins, supported by the microscopic observation of accumulation and penetration of the native polymyxin-like probe in the cell membrane. This secondary target of polymyxins is still vulnerable in polymyxin-resistant K. pneumoniae strains; therefore, it can be exploited for the development of new lipopeptide antibiotics targeting polymyxin-resistant bacteria.