Proteomic analysis of a carbapenem-resistant Klebsiella pneumoniae strain in response to meropenem stress

https://doi.org/10.1016/j.jgar.2016.12.010Get rights and content

Highlights

  • Proteomic analysis of multidrug-resistant clinical Klebsiella pneumoniae (NP6) under meropenem stress.

  • Strain NP6 harbours the KPC-2 β-lactamase resistance marker.

  • Upregulation of proteins involved in energy metabolism suggests their involvement in drug resistance.

  • Overexpressed LysM domain/BON superfamily protein might modulate the effect of meropenem.

  • Cumulative effect of these proteins might be involved in bacterial survival under drug pressure.

Abstract

Objectives

Antibiotic resistance has become a major problem in treating bacterial infections. The aim of this study was to elucidate the effects of meropenem on a blaKPC-2-harbouring multidrug-resistant clinical strain of Klebsiella pneumoniae through a proteomics approach in order to attain a deeper understanding of bacterial resistance strategies.

Methods

Analysis was performed by two-dimensional gel electrophoresis of whole-cell extracts of bacteria exposed to a sublethal concentration of meropenem compared with the untreated control. Differentially expressed proteins were identified by matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF).

Results

Based on Quantity One® software and MALDI-TOF analysis, 16 overexpressed proteins were identified in meropenem-treated bacteria. These proteins were primarily enzymes involved in defence against oxidative stress as well as glycolytic enzymes. LysM domain/BON superfamily protein was found overexpressed by >12-fold. STRING-10 was used to determine protein–protein interaction among the overexpressed proteins and to predict their functional associations. This study demonstrated that treatment with meropenem resulted in upregulation of various proteins involved in defence and repair mechanisms along with enzymes of energy metabolism.

Conclusions

These overexpressed proteins may play an important role in bacterial resistance mechanisms against carbapenems, however their role in resistance needs to be further validated. High expression of lysine M domain/BON superfamily protein may indicate its possible involvement in modulating the bacterial response to antibiotic stress, but its actual role requires more investigation. These findings may also help in the development of newer therapeutic agents or diagnostic markers against carbapenem resistance.

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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