Review on the Carbapenem Resistance Mechanisms of Klebsiella pneumoniae

During the past years, the emergence of multi-drug resistance Gram-negative bacilli (MDR-GNB), including the carbapenem resistant Klebsiella pneumoniae (CRKP) has increased leading to a significant threat to public health care. Recent advanced molecular methods have improved our knowledge on how antimicrobial resistance mechanisms develop and transferred among bacterial strains. The MDR pathogens, particularly CRKP, utilize various mechanisms of resistance such as antimicrobial agent degradation, modification of antimicrobial target and alteration of cell membrane permeability. Here, the emergence of CRKP and the major antibiotic resistance mechanisms employed by CRKP will be reviewed and described. Understanding such mechanisms can be essential to develop new antimicrobial drug and help with individual treatment decisions to use alternative options to carbapenem and β-lactam antibiotics.


INTRODUCTION
Klebsiella pneumoniae is an opportunistic significant Gram-negative pathogen responsible for various types of nosocomial and community-acquired infections such as pneumonia, liver abscess, sepsis and meningitis [1]. The prognosis and treatment of such infections became more challenging due to the ability of the bacteria to resist multiple antibiotics. This can be Review Article achieved by several antimicrobial resistance mechanisms that are employed by an emerged multi-drug resistant bacterial pathogens. An example of these resistance mechanisms include the production of hydrolyzing or modifying enzymes, the efflux transporter systems, and the decreased permeability of the cell membrane via loss of porins [2]. The main aim of this review is to explain the significance of the emergence of multi-drug resistant K. pneumoniae strains and to outline their major mechanisms of antimicrobial resistance, especially carbapenem resistance.

CARBAPENEM RESISTANCE MECHANISMS
There are three major antimicrobial resistance mechanisms against carbapenem that utilized by K. pneumoniae, These resistance mechanisms are classified as the following: 1) Production of carbapenem-hydrolyzing enzymes for degrading antibiotics.
3) Porin mutations to decrease permeability on the outer membrane. This international spread of KPCs isolates were associated with a single dominant strain, namely multilocus sequence type (ST258), that was reported to be responsible for nearly 70% of the USA outbreaks [13]. The dissemination of KPCs encoded genes, bla KPCs , can be mediated by molecular mechanisms such as mobility of small genetic materials called transposons (e.g. Tn3 type Tn4401 transposon). Additionally, the horizontal transfer of plasmids carrying bla KPCs gene is known as a major molecular resistance mechanism of KPC gene transmission via clonal spread [12,14].
The carbapenemases can be classified into four classes A, B, C and D based on their ambler class. The class A carbapenemases are identified as KPCs which have the ability to hydrolyze beta-lactams when their active sites contain serine [15]. In addition, the ambler class A (KPC) is commonly found in highly antimicrobial resistance K. pneumoniae strains. The class B enzymes cleave beta-lactam rings with zinc that acts as an essential cofactor. These enzymes known as metallo-betalactamases (MBLs) can resist beta-lactamase inhibitors [16]. The class C enzymes are known as AmpC enzymes that are chromosomally encoding cephalosporinases and these enzymes use serine as active site similar to class A [17]. The class D carbapenemases are also serine proteases that depend on serine to hydrolyze carbapenems. Oxacillinase-48 (OXA-48)-like enzymes belong to the ambler class D βlactamases in K. pneumoniae [2].

THE ENERGY-DEPENDENT EFFLUX PUMPS MECHANISM
Efflux pumps play an important role as antimicrobial resistance determinants that are conserved in all microorganisms [18,19]. It was firstly described the use of efflux pumps by Escherichia coli as defensive mechanisms to resist tetracycline antibiotic [20,21]. There are five main types of efflux pumps transporters in prokaryotes: the adenosine triphosphate (ATP)binding cassette ABC superfamily, the resistance-nodulation-division (RND) family [20] the small multidrug resistance (SMR) family [21], the major facilitator superfamily (MFS) [22], the multidrug and toxic compound extrusion (MATE) family [23]. The ABC, SMR, MFS and MATE families are known as major efflux transporters in both Gram-positive and Gram-negative organisms whereas the RND family are only common in Gram-negative bacteria [20,21]. The flavonide-responsive RND family of efflux transporters involves various members as shown in (Fig. 1). For instance, E. coli AcrAB-ToIC pump is a major member of RND family and present in other CRE strains including CRKP [24]. This pump known as a tripartite complex contributes to antibiotic resistance as it spans through the membranes of the bacteria (the periplasm, inner and outer membranes) in order to eject antibiotics out of the cell [2].
The AcrAB-ToIC multi-drug efflux pump in K. pneumoniae is formed by AcrA (a membrane fusion protein), AcrB (a cytoplasmic membrane protien) and TolC (an outer membrane protein). It is encoded by acrRAB operon that is negatively regulated by a dimeric protien AcrR repressor . Furthermore, the acrB protein is associated with the ToIC protein that is present in other gramnegative bacteria and is mainly responsible for the removal of numerous compounds from bacterial cells [25].

Fig. 1. Schematic diagram of the major efflux transporter system in K. pneumoniae. The resistance-nodulation-division (RND) family. Green circles represent antibiotic molecules
Along with carbapenem resistance, MDR-GN bacteria encoding the AcrAB-ToIC pump can lead to resistance to other antimicrobial classes such as tetracycline, fluoroquinolones, and macrolides [25].
Porin Mutations in the outer membrane: Porin mutations can decrease the permeability of the bacterial outer membrane as mechanism of antibiotic resistance. Additionally, OmpK35 and OmpK36 are major types of mutations in porin which are usually alone do not lead to carbapenem resistance in Enterobacteriaceae, however, these mutations in CTX-M and Amp-Cproducing Enterobacteriaceae often lead to carbapenem resistance [2,26]. The CTX-M enzyme belong to class A β-lactamases as type of penicillinase, while Amp-C is a class C βlactamases known as cephalosporinase. Both enzymes have a low level of carbapenem hydrolytic activity [2].
The low porin expression when is combined with the overexpression of hydrolytic β-lactamases can lead to "antibiotic trapping phenomenon" in which the carbapenem can be irreversibly bound by the degrading enzymes (trapped) rather than degraded [26]. The ompK35/36 porin variants isolated from KPC-producing K. pneumoniae in Italy which were associated with carbapenem resistance causing a significant potential threat to nosocomial settings [27]. Moreover, it was reported that ompK36 porin variant present in KPC-producing K. pneumoniae was linked with high level of carbapenem resistance and reduced response against carbapemenm-colistin treatment [28,29]. A previous multicenter study in the USA showed that both two porin mutations ompK35 and ompK36 were found in 84% and 34% of CRKP strains, respectively [30].

CONCLUSION
To summarize, CRKP strains utilize various mechanisms to resist broad spectrum antimicrobial agents including carbapenems. In this review, three major antimicrobial resistance mechanisms employed by MDR K. pneumoniae were described including hydrolytic extended spectrum β-lactamases, the efflux pump systems, and the loss of porins causing decreased permeability of the cell membrane. Understanding these mechanisms along with the significance of the emergence of carbapenem resistance strains can be essential to develop novel antimicrobial agents that can improve the prognosis of CRKP pathogens and help clinicians in treatment decisions in selected cases.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable. carbapenem-resistant Klebsiella pneumoniae: tracking molecular epidemiology and outcomes through a regional network. Antimicrob Agents Chemother. 2014;58 (7)