Proteomic analysis of a carbapenem-resistant Klebsiella pneumoniae strain in response to meropenem stress
Introduction
The worldwide emergence of Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae poses a great challenge to healthcare [1]. Expression of KPC-type β-lactamases confers decreased susceptibility to virtually all β-lactam antibiotics including cephalosporins and carbapenems [2]. High-level production of β-lactamases (carbapenemases) and porin deficiency are recognised as major causes of resistance development in K. pneumoniae [3], [4]. However, less attention has been paid towards other cellular proteins that might be equally important in the development of resistance.
The bacterial proteome is a dynamic entity that responds rapidly to external stimuli such as antibiotic stress [5]. The presence of antimicrobials in bacterial cells disturbs the internal harmony of the system and quickly cellular functions are diverted to revert the effect. Studying the proteomics of resistant bacteria under drug stress could identify novel strategies employed by bacteria to overcome the effects of antimicrobials.
Recent advances in proteomics and bioinformatics offer great potential in unravelling biological problems. Proteomic approaches have been used to elucidate the cellular responses of model micro-organisms such as Bacillus subtilis, Escherichia coli and Mycobacterium tuberculosis and many other micro-organisms to different antimicrobial agents [6], [7], [8], [9].
Since proteins are the functional entities of the cell, we investigated the effect of meropenem exposure on the proteome of a blaKPC-2-carrying multidrug-resistant clinical K. pneumoniae strain (NP6). The primary objective of this study was to identify proteins that were differentially expressed under antibiotic stress and that might play an important role in bacterial defence and survival mechanisms, besides KPC-2 carbapenemase. The soluble whole-cell proteomes of K. pneumoniae NP6 were studied in the presence and absence of meropenem by employing two-dimensional gel electrophoresis (2DE) coupled with mass spectrometry. To the best of our knowledge, this is the first study looking into differential protein expression of KPC-2 β-lactamase-producing K. pneumoniae in response to meropenem treatment. This study revealed that several cellular proteins are differentially expressed upon exposure to meropenem, thus providing a glimpse of the changes occurring in the cells following antibiotic treatment.
Section snippets
Bacterial strains and drug susceptibility testing
A clinical strain of K. pneumoniae (NP6), previously characterised in our laboratory, was used in this study [10]. Strain NP6 exhibited resistance against carbapenems [meropenem minimum inhibitory concentration (MIC) = 256 μg/mL] and was found to carry the KPC-2 β-lactamase resistance marker. Carbapenem-susceptible K. pneumoniae MTCC 432 (meropenem MIC = 0.125 mg/L) was procured from the Institute of Microbial Technology (IMTECH) (Chandigarh, India). Susceptibility testing for meropenem was
Results
The main focus of this study was to identify upregulated proteins in carbapenem-resistant K. pneumoniae NP6 grown in the presence and absence of meropenem. The MIC of meropenem for the resistant strain was 256 mg/L and that of the susceptible control strain MTCC 432 was 0.125 mg/L. Fig. 1 shows the 2DE profile of NP6 grown in the absence and presence of a subinhibitory concentration (0.5 × MIC) of meropenem. On comparing the two proteomic profiles, 16 spots were upregulated by ≥1.5-fold in the
Discussion
The emergence of resistance to carbapenems in bacteria has increased significantly in the past few decades. In this study, we sought to identify proteins playing a pivotal role in resistance mechanisms of K. pneumoniae NP6, besides KPC-type β-lactamase. To gain an insight into the post-treatment physiological changes occurring in bacteria, the whole-cell lysates of NP6 growing in exponential phase in the presence and absence of meropenem were analysed and compared. The bacterial culture was
Conclusion
In conclusion, the present study illustrates the proteins overexpressed upon exposure to a sublethal concentration of meropenem. In response to antibiotic treatment, bacterial cells rapidly changed their protein expression profile, perhaps to counter the effects of the antibiotic and to survive. These overexpressed proteins might have a cumulative effect on the physiology of bacteria, making them more resistant to meropenem. Such proteins and the mechanisms involving them can be further
Funding
The Department of Biotechnology is highly acknowledged for the DBT Award BT/HRD/NBA/34/01/2012 and grant BT/PR8281/BID/7/448/2013 to AUK. The Promotion of University Research and Scientific Excellence (DST-PURSE) Program Phase-II No. SR/PURSE Phase 2/9 is highly acknowledged for infrastructure support.
Competing interests
None declared.
Ethical approval
Not required.
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